The diagnosis and clinical importance of Giardiasis

P arasitologyAS published in CLI September 2005The diagnosis and clinical importanceof Giardiasisby Dr T. MankGlobally Giardia lamblia is one of the most important non-viral causesof human diarrhoea, with infections occurring not only in developingcountries but also in the developed world. The ingestion ofcysts does not usually result in clinical illness, but Giardia infectioncan produce a broad spectrum of gastrointestinal symptoms whichcan persist for long periods if left untreated. This article discusses thebiology and epidemiology of the parasite, and considers the varioustechniques that can be used for its diagnosis. Microscopic examinationof stool samples is still the mainstay for routine diagnosis despitethe fact that progress has been made in developing and validatingnon-morphologically based diagnostic tests and the proven utility ofEIAs.Giardia is a ubiquitous enteric protozoan parasite that affects humans, domesticanimals and wildlife throughout the world. The parasite is a flagellate,which was discovered by the inventor of the microscope, Antoni vanLeeuwenhoek. In a letter written to Hooke at the British Royal Society ofParasitology in 1681 he accurately described the motile trophozoite of G. lamblia,which he observed in a sample of his own stool as "very prettily movinganimacules, some rather larger, others somewhat smaller than a blood corpuscleand all of one and the same structure …." "their bodies were somewhatlonger than broad, and their belly, which was flatlike, furnished with sundrylittle paws….". The descriptive information of this historic letter was notwidely distributed and it was Vilem Lambl who comprehensively described thetrophozoites, which he found in the stools of paediatric patients in Prague in1859. His published observations were accompanied by beautiful drawings ofthe organism based on his many microscopic observations.Figure 2. Trophozoite of Giardia stained with Giemsa.The life cycleThe life cycle of Giardia is well-known and comprises two developmental stages; thetrophozoite and the cyst [Figure 1]. The most common location of the former stageis in the crypts within the duodenum of the host (e.g. human) where trophozoiteslive closely applied to the mucosa. There, binary fission repeatedly takes place, theeventual result of which is the establishment of the protozoan, often in enormousnumbers. The binucleate trophozoite is usually described as being teardrop-shapedwith the posterior end being pointed; it measures 10-20 µm [Figures 2 and 3]. Theanterior portion of the ventral surface of the organism is modified to form a suckingdisc, which serves to attach the organism to the mucosa. The transformation oftrophozoites into cysts takes place in the host intestinal tract, when the trophozoites,together with the faecal mass, move down through the colon. The cysts, which areshed into the environment by the host, may be either round or oval, and they containfour nuclei [Figure 4]. The cyst represents the resting stage of the organism; itsrigid outer wall protects the parasite against changes in environmental temperature,dehydration, and such disinfectants as chlorine, all of which would destroy thetrophozoite. The cyst is also the infective stage; the cycle continues when a suitablehost ingests the cyst. Stomach acidity and other factors trigger the excystationprocess, which usually takes places in the new host’s small intestine.Although various criteria, including host specificity, differing body dimensions, variationsin structure, and molecular tools have all been used to differentiate species ofGiardia, there is still considerable debate over the appropriate classification andnomenclature regarding this group of organisms. However, five species of Giardia arecurrently recognised on the basis of parasite morphology and host occurrence. Theseare G. agilis (amphibians), G. ardeae (birds), G. duodenalis (most mammals includinghumans), G.microti (voles and muskrats) and G. muris (rodents) [Figure 5].Most species of Giardia are host-adapted, with the exception of Giardia duodenalis(syn. G. lamblia, G. intestinalis) which seems to have a much broader host rangeinfecting many mammalian species. Despite disagreement concerning the names"duodenalis", "intestinalis", and "lamblia" all three continue to be used to describethis organism although the term Giardia lamblia is predominantly used in humanmedicineFigure 1. The life cycle of Giardia lambliaClinical features in human infectionsThe natural course of giardiasis is often very mild; in most cases, the ingestion ofcysts will not result in any clinical illness. On the other hand, Giardia infection can

P arasitologyAs published in CLI September 2005Figure 3. Scanning electron micrograph of a Giardia lambliatrophozoite from a biopsy of human jejunum.sometimes produce abroad spectrum of gastrointestinalsymptoms inaddition to diarrhoea.The clinical syndrome ofacute giardiasis has beenwell characterised, especiallyin travellers. Themost prominent symptomof the infection isprotracted diarrhoea. Itcan be mild and producesemisolid stools, or it canbe intensive and debilitatingwhen the stoolsbecome watery and voluminous.Untreated, thistype of diarrhoea may last weeks or months, although it may vary in intensity, withexacerbations and remissions.In the majority of healthy individuals the infection is self-limiting, a proportion(estimated at 30-50%) of infected patients, however, will go on to chronic giardiasis,characterised by steatorrhoea accompanied by a classic malabsorption syndromewith substantial weight loss, general debility and fatigue. Chronic infectionin early childhood is associated with poor cognitive function and failure to thrive.The factors determining the variability in clinical outcome in giardiasis are poorlyunderstood. Host factors, such as immune status, nutritional status and age, as wellas differences in virulence and pathogenicity of Giardia strains are recognised asimportant factors determining the severity of infection. The recent application ofmolecular techniques to Giardia lamblia isolates has revealed high levels of geneticdiversity within this species. Currently, there are six recognised variants orassemblages (assemblage A, B, C, D, E and F), each having a varying degree of hostspecificity. Several studies on the correlation between clinical presentation andgenotypes have been performed. However due to the conflicting results reportedin the literature, it is still not completely clear what the relation is between symptomsand infection with different genotypes, in particular those belonging toassemblages A and B, which are so far the only genotypes known to cause humandisease.Unfortunately in contrast to the case of infections with Entamoeba, where it waspossible to define two distinct genotypes that are now named E. histolytica and E.dispar, applying such a dichotomy is not (yet) possible with Giardia infections.Worldwide co-operation, including the exchanging of the various genotypes andstandardisation of methods, will eventually reveal some answers to the remainingquestions. By genotyping the different strains it will become possible to study thevariety of associated symptoms, although host factors, e.g. host immune responseto infection, will also have to be taken into account.Diagnosis in human infectionsProgress has been made in developing and validating non-morphologically baseddiagnostic tests for intestinal parasite antigens. Immunofluorescence microscopy(IF), enzyme linked immunosorbent assays (ELISA), parasite DNA polymerasechain reaction - restriction fragment length polymorphism [PCR-RFLP], and realtime PCR [RT-PCR]) have all been utilised for Giardia diagnosis. However,microscopy is still the cornerstone and gold standard for detecting intestinal parasitesin stool samples in both clinical and veterinary diagnostic parasitology laboratories.Traditionally fresh or preserved stool samples are microscopically examineddirectly or with (permanent) staining, with or without concentration.Unfortunately, the sensitivity of this conventional ovum-and-parasite (O&P)examination on a single stool sample for G. lamblia is less than optimal. It wasrecently determined by Cartwright to be only 74% [1]. The poor sensitivity of asingle parasitological stool examination for diagnosing giardiasis is mainly due tothe variable excretion or low-level shedding of the parasite in both symptomaticand asymptomatic patients. Furthermore, symptoms can occur before intact parasitesare detected in the stool, hence repeatedexaminations are necessary until morphologicalforms are seen, as is well described and recommendedin many parasitology textbooks. To overcomesampling issues, the Triple-Faeces-Test (TFT)was recently developed in the Netherlands as an allround test for the laboratory diagnosis of intestinalparasites. The advantages of both fixatives, permanentstaining methods and multiple sampling arecombined in this test [2].Figure 4. Cyst stage of Giardia,stained with iodine, from a stoolspecimen.Since the early nine-ties enzyme immunosorbentassays (EIAs) for detecting Giardia-specific antigensin stool samples have become commerciallyavailable. A number of clinical evaluations ofGiardia EIAs have been reported in the literature;the sensitivity of EIA has been found to be eithersimilar, or in most cases slightly superior to single,as well as to multiple, microscopic stool examinations[3-7]. Due to these findings, and due to thefact that Giardia lamblia is by far the most commonlyfound enteric pathogen in general practice, EIA is an attractive alternative toconventional O&P examination. However, in the case of a negative test result andpersistent symptoms, a conventional parasitological stool examination, includinga permanent stained smear from a preserved stool sample, should also be performed.Serological approaches to the diagnosis of giardiasis have been developed and havebeen proven to be most useful in epidemiological surveys [8-10]. PCR assays forspecific genera/genotypes of intestinal parasites are useful for surveys, but not forthe clinical diagnostic laboratory. Stools must be screened for a variety ofpathogens and the cost of different PCR assays would be too high. RT-PCR mayhave a role in diagnostic and reference laboratories, as several targets could becombined in one multiplex RT-PCR assay, allowing the simultaneous detection ofmultiple infections such as E. histolytica, G. lamblia and Cryptosporidium speciesand genotypes infectious to humans.With the use of the new approaches in routine parasitological stool examinations,a substantial increase in sensitivity and specificity in the laboratory diagnosis ofintestinal protozoan infections can be achieved. Nevertheless it is still extremelyimportantto familiarise physicians with the clinical relevance and epidemiology of theseinfections.Physicians should include a parasitological stool examination in their diagnosticwork-up of patients with persistent (lasting longer than 1 week) or intermittentdiarrhoea. This is indicated because of the frequency of protozoal diarrhoea ingeneral practice, and the negligible diagnostic value of symptoms and otheranamnestic data from patients infected with Giardia lamblia or other intestinalprotozoa, as well as the fact that most intestinal protozoal infections can be treated.EpidemiologyGiardia lamblia is recognised as a major cause of diarrheal illness in humans andlivestock. Worldwide it is one of the most important non-viral infectious agentscausing diarrhoeal illness in humans [11-12]. The parasite is not restricted topeople living in the developing countries, where sanitation is frequently non-existentand people are forced to drink unfiltered surface water, but also occursamong people living in developed, market economies where public hygiene isgood and water supplies are purified and piped.The infection is endemic in all regions of the world, and also occurs in sporadicepidemic bursts. Exact figures on incidence and prevalence depend of the populationexamined. In the Netherlands, the prevalence of the infection variesbetween 2% and 14%, being high in patients who consult their general practitionerwith persistent diarrhoea and low (2%) in asymptomatic subjects [13-15].Survival in fresh water has enabled Giardia to achieve the reputation of being themost common cause of epidemic waterborne diarrheal disease. The first record-

P arasitologyAs published in CLI September 2005ed waterborne outbreak of giardiasis involved travelers to St. Petersburg, Russia.Since then many epidemics have been well characterised in both North Americaand Europe, and they have usually been associated with inadequate water treatment.High rates of infection have also been reported for hikers and campers inthe USA. Because some of these areas were remote from human habitation,infected wild animals, especially beavers, are suspected of being a host or reservoirfor Giardia lamblia [16-17].Direct person-to-person spread by faecal-oral transmission is another majormechanism by which the disease is transmitted. The organism tends to be foundmore frequently in children or in groups that live in close quarters. Outbreaks ofgiardiasis are well recognised in day care centres, residential institutions andschools. Many infections are asymptomatic but spread of cysts can occur toother members of the family or to other residents. Transmission of the parasitealso occurs during sexual activity, in particular as a result of intimate oro-analcontact.The contribution of foodborne transmission of giardiasis has not been wellcharacterised. However, there have been a number of reports where food hasclearly been identified as the source in several outbreaks of giardiasis. In mostcases an infected food handler has been implicated, presumably transmittingcysts to freshly prepared food [18].TreatmentNitro-imidazoles (metronidazole and tinidazole) and albendazole are the drugsof choice for giardiasis, but lack of treatment compliance and side effects canresult in treatment failures. For treatment of special patient groups e.g., pregnantand immunocompromised patients, alternative drugs, such as paromomycin,and/or prolonged or combination therapy may sometimes be necessary.References1. Cartwright CP. Utility of multiple-stool-specimen ova and parasite examinations in ahigh-prevalence setting. Journal of Clinical Microbiology 1999; 37: 2408-2411.2. Gool T, Mank TG. Triple faeces test: an effective tool for detection of intestinal parasitesin routine clinical practice Eur J Clin Microbio Infec Dis 2003; 22: 284-290.3. Mank TG et al. Sensitivity of microscopy versus enzyme immunoassay in the laboratorydiagnosis of Giardiasis. European Journal of Clinical Microbiology 1997; 16: 615-619.4. Garcia LS, Shimizu RY. Evaluation of nine immunoassay kits (enzyme immunoassayand direct fluorescence) for detection of Giardia lamblia and Cryptosporidium parvumin human fecal specimens. Journal of Clinical Microbiology 1997; 35: 1526-1529.5. Rosoff JD et al. Stool diagnosis of Giardiasis using a commercially available enzymeimmunoassay to detect Giardia-specific antigen 65. Journal of Clinical Microbiology 1989; 27:1997-2002.6. Scheffer EA,Van Etta LL. Evaluation of a rapid commercial enzyme immunoassay for detectionof Giardia lamblia in formalin-preserved stool specimens. Journal of ClinicalMicrobiology 1994; 32: 1807-1808.7. Zimmerman SK, Needham CA. Comparison of conventional stool concentration and preserved-smearmethods with Merifluor Cryptosporidium/Giardia direct immunofluorescenceassay and ProSpect Giardia EZ microplate assay for detection of Giardia lamblia. Journal ofClinical Microbiology 1995;33: 1942-1943.8. Isaac-Renton JL et al.Epidemic and endemic seroprevalenceof antibodies toCryptosporidium and Giardiain residents of three communitieswith different drinkingwater supplies. Am J TropMed Hyg 1999; 60: 578-583.9. Isaac-Renton JL et al.A secondcommunity outbreak ofwaterborne giardiasis inCanada and serological investigationof patients. Trans RSoc Trop Med Hyg 1994; 88:395-399.10. Miotti PG et al. Age-relatedrate of seropositivity ofantibody to Giardia lamblia in four diverse populations. J Clin Microbiol 1986; 24: 972-975.11. Meyer EA. 1990 Taxonomy and nomenclature. In: EA Meyer (ed.), Giardiasis.,Amsterdam:Elsevier Holland, 1999, p 51-60.12. Thompson RCA et al. Giardia- from molecules to disease and beyond. Parasitology today1993; 9: 313-315.13. Mank TG et al. Comparison of fresh versus sodium acetate acetic acid formalin preservedstool specimens for diagnosis of intestinal protozoal infections. European Journal of ClinicalMicrobiology and Infectious Diseases 1995; 14: 1076-1081.14. Mank TG et al. Persistent diarrhoea in a general practice population in the Netherlands,prevalence of protozoal and other intestinal infections. In: Proceedings of the IXthInternational Congress of Parasitology, Chiba, Japan,pp. 803-807.15. Wit Mas DE et al. Gastroenteritis in sentinel general practices, the Netherlands. EmergingInfectious Diseases 2001; 7: 82-91.16. Dykes AC et al. Municipal waterborne giardiasis. An epidemiologic investigation. AnnIntern Med 1980; 93: 165-170.17. Healy GR. Giardiasis in perspective: the evidence of animals as a source of human Giardiainfections, p 305-313. In: EA Meyer (ed.), Giardiasis. Human Parasitic Diseases, vol. 3. NewYork: Elsevier Biomedical Press, 1990.18. Adam RD. The biology of Giardia spp. Microbiological Reviews 1991; 55: 706-732.The authorTheo G Mank, Ph.D.Medical ParasitologistDepartment of ParasitologyPublic Health Laboratory HaarlemBoerhaavelaan 262035 RC HaarlemThe NetherlandsTel.: +31 23 5307800Fax: +31 23 5307805E-mail: t.mank@streeklabhaarlem.nlFigure 5. Circular lesions on the mucosa of a mouse smallintestine resulting from attachment of Giardia muris.