ARRANGEMENT OF REGULATIONS Regulations : PART 1—GENERAL POLICIES PROCEDURES AND DEFINITIONS PART 2—PERSONNEL LICENSING PART 3—APPROVED TRAINING ORGANIZATIONS PART 4—AIRCRAFT REGISTRATION AND MARKING PART 5—AIRWORTHINESS PART 6—APPROVED MAINTENANCE ORGANIZATION PART 7—INSTRUMENTS AND EQUIPMENT PART 8—OPERATIONS PART 9—AIR OPERATOR CERTIFICATION AND ADMINISTRATION
Microsoft word - giardiasis phd printed version.docGiardiasis
with emphasis on treatment and post-infectious manifestations National Centre for Tropical Infectious Diseases Department of Medicine Haukeland University Hospital Institute of Medicine University of Bergen, Norway Department of Public Health and Primary Health Care University of Bergen Water, is taught by thirst. Land - by the Oceans passed. Transport - by throe - Peace - by its battles told - Love, by Memorial Mold - Birds, by the Snow. Emily Dickinson, 1830-1886
I. ACKNOWLEDGEMENTS . 4
II. LIST OF PAPERS . 5
ABBREVIATIONS . 6
BACKGROUND . 7
Giardiasis . 7
Parasitology . 7 Epidemiology . 8 Pathogenesis . 13 Clinical presentation . 14 Laboratory diagnostic methods . 17 V. AIMS OF THE STUDY . 26
SUMMARY OF PAPERS . 27
Paper 1 . 27
Paper 2 . 28
Paper 3 . 29
Paper 4 . 30
VII. MAIN RESULTS AND DISCUSSION . 31
VIII. PROPOSALS FOR FUTURE STUDIES . 37
CONCLUSIONS . 38
X. REFERENCES . 39
PAPER I-IV . 55
XII. APPENDIX . 91
Questionnaire used in paper 3 and 4 .91
This study was initiated during the Bergen Giardia outbreak in 2004, and has been carried out at National Centre for Tropical Infectious Diseases, Department of Medicine, Haukeland University Hospital, who has also funded this work. I am grateful to this institution for providing facilities and for the financial support. I want to thank my main supervisor and mentor, Professor Nina Langeland for giving me the opportunity to work with global health, which has always been my main interest. I want to thank her for encouraging me to start, and inspiring me to continue, on this scientific project, for excellent and always constructive support and supervision. And I want to thank my co-supervisor Professor Guri Rørtveit, who by providing constructive criticism and valuable advices has been an important contributor to this work. I am also most grateful to my other co-authors Kurt Hanevik, Geir Egil Eide, Knut-Arne Wensaas, Lucy Robertson, Trygve Hausken and Elisabeth Astrup Strand for a fruitful collaboration during this period. I thank Unit for Infectious Diseases for laying the groundwork for clinical research, and I warmly thank my colleges for creating a positive working environment which makes it a privilege and pleasure to work at this Unit. A special thank to Axel Schreiner and Bjørn Myrvang who have inspired and supported my interest in tropical infectious diseases, and patiently shared their knowledge in the field with My husband Kjartan has encouraged me and supported me during challenging periods of this work, my son Johannes has helped me with the figures in the manuscript and my youngest son Bjørn has asked me thought-provoking questions regarding the time it takes to write a paper and whether it is worthwhile. My excuse for being absent minded for a period, is that I hope my children learn the importance and privilege of seeking sound knowledge and education, like my parents taught me once. I am most grateful to my family. II. List of papers
1. Mørch K, Hanevik K, Robertson LJ, Strand EA, Langeland N: Treatment ladder and genetic characterisation of parasites in refractory giardiasis after an outbreak in Norway. Journal of Infection 2008; 56: 268 - 273. 2. Hanevik K, Mørch K, Eide GE, Langeland N, Hausken T: Effects of albendazole/metronidazole or tetracycline/folate treatments on persisting symptoms after Giardia infection: A randomised open clinical trial. Scand J Infect Dis. 2008; 40 (6-7): 517 - 22. 3. Mørch K, Hanevik K, Rortveit G, Wensaas KA, Langeland N: High rate of fatigue and abdominal symptoms 2 years after an outbreak of giardiasis. Trans R Soc Trop Med Hyg. 2009; 103: 530 - 532. 4. Mørch K, Hanevik K, Rortveit G, Wensaas KA, Eide GE, Hausken T, Langeland N: Severity of Giardia infection associated with post-infectious fatigue and abdominal symptoms two years after. BMC Infectious Diseases 2009, 9:206. III. Abbreviations
Bid = Two times daily E. coli = Escherichia coli EIA = Enzyme immunoassay ELISA = Enzyme-linked immunosorbent assay ETEC = Enterotoxigenic E. coli G. = Giardia HIV = Human immunodeficiency virus Ig = Immunoglobuline IFA = Immunofluorescence assay IIF = Indirect immunofluorescence PCR = Polymerase chain reaction MSIS = Norwegian Surveillance System for Communicable Diseases Syn = Synonymous Tid = Three times daily VSP = Variant surface protein WHO = World Health Organisation IV. Background
Giardia duodenalis (syn. G. lamblia, G. intestinalis) is a single cell parasite, inhabiting the small intestine. Like Plasmodium species causing malaria by infecting red blood cells, the genus Giardia belongs to the family protozoans . The name lamblia has its origin from Vilem Lambl who described the trophozoite in humans in 1859, and the cyst form was discovered by Grassi twenty years later; however, Antony van Leeuwenhoek described the parasite in his own stool as early as in the 17th century . Six different Giardia species, characterised by differences in morphology and host specificity, have been described since 1952 [1, 3]; G. duodenalis infects humans as well as other primates, dogs, cats, livestock, rodents and some other wild animals, G. agilis infects amphibians, G. psittaci and G. ardeae birds and G. microti and G muris rodents. A revision of taxonomy is under debate, since genetic studies and sequencing of the Giardia genome have revealed several genotypic groupings/assemblages that could be classified as new species, including assemblage A and B in G. duodenalis, which are proposed to be classified as G. duodenalis (= assemblage A) and G. enterica (= assemblage B) [1, 4]. In addition, the following assemblages infecting animals have been proposed to be classified as species: Assemblage C/D (infecting dogs and other canids): G. canis, assemblage F (cats): G. cati, assemblage E (cattle and other hoofed livestock): G. bovis, and assemblage G (rats) G. simondi. The mechanism for host specificity is not known . Giardia has three morphologic forms; cysts, excyzoites and trophozoites . Cysts are responsible for faecal-oral transmission, and are able to survive for a long period in the environment, especially in cold water in which experimental studies have shown survival for up to two months . In the upper part of the small intestine, they release excyzoites containing four nuclei, which attach to the intestinal wall and rapidly divide into four trophozoites . Trophozoites cause disease in the small intestine where they multiply by simple binary fission, though there is some evidence also of sexual reproduction . The trophozoites have a characteristic duplication of organelles; four pairs of flagella enabling them to move, two identical nuclei, two median bodies and a ventral sucking disc which enables it to attach to the intestinal surface (Figure 1) . Trophozoites may be found in fresh faeces, but usually encyst, triggered by bile salts  or cholesterol depletion and micelle destruction , before being excreted in the stool. Figure 1. Giardia trophozoite showing ventral disc, flagellae, nuclei and median bodies.
Diarrhoea is probably the most common infectious disease worldwide, and following lower respiratory infections the second leading cause of death due to infections; in 2004 WHO reported an incidence of 4.6 billion episodes and 2.2 million deaths due to diarrhoea per year, of these 1.8 million deaths in developing countries . The most common etiologic agents are species among the viruses rotavirus, calicivirus, astrovirus and enteric adenovirus; the bacteria E. coli, Shigella, Salmonella, Campylobacter and Vibrio cholera, and the parasites Giardia, Entamoeba histolytica, Cryptosporidium, Cyclospora and Isospora . Infections transmitted faecal-orally are more easily spread under conditions associated with poverty; such as decreased access to clean water, inappropriate sewage disposal, poor hygiene, crowding, close contact to farm animals and low educational level. A predominance of bacteria and parasites as diarrhoeal pathogens in the South, and viruses in the North, reflects this situation . A prevalence of 200 million cases of giardiasis in tropical countries has been estimated by WHO . However, decreased access to reliable diagnostic tools in socioeconomic underdeveloped areas makes it difficult to assess the aetiology of diarrhoea in clinical practice, and reporting systems are insufficient. Such estimates are also limited by the fact that few case-control studies have been performed in developing countries, and the clinical studies that are available show that prevalence varies greatly between and within countries. Serologic studies show a high infection rate, and that people are commonly infected with Giardia during childhood, in developing countries. In a national serologic survey in Mexico, 55% (1914/3461) of the samples were positive for anti-Giardia IgG, and seropositivity increased with age . In a study comparing populations in developing and developed areas, anti-Giardia IgG was found in 44% (27/62), 48% (16/33) and 46% (12/26) of adults in an Apache Indian reservation in Arizona, in Panama and Peru respectively, while significantly fewer (18%, 7/41) adults in Baltimore were seropositive . In Baltimore, seropositivity was low throughout childhood, children in Panama achieved adult levels of antibodies between nine and 20 years, Apache Indians by eight years of age, whereas 46% of Peruvian children were seropositive already by the age of six months . Similar high prevalence was found in a cohort with low socioeconomic standard in Egypt, where 37% were anti-Giardia IgG positive . Despite such high prevalence, giardiasis is not commonly associated with acute diarrhoea in hospital based studies. In different reviews, rotavirus and E. coli spp. are the most common causes of acute diarrhoea in both developing and developed countries [10, 15, 16]. In a study from India among children hospitalised due to diarrhoea, and children admitted to hospital for causes other than diarrhoea included as controls (n = 158 cases, n = 99 controls), the following pathogens were detected among cases and controls respectively: Rotavirus 43% and 10%, enterophatogenic E. coli 16% and 2%, norovirus 16% and 7%, and Cryptosporidium spp. 15% and 2%, while other agents were less prevalent, among these Giardia only 0.6% and 0% . This study also demonstrated that molecular techniques increased the detection rate and changed the picture, compared to conventional diagnostic Such low prevalence among hospitalised children is supported by other case-control studies, using children admitted for reasons other than diarrhoea as controls. In a study from Nigeria (n = 215 cases, n = 100 controls) Giardia was identified in only one case, and in none of the controls . In a large study from Bangladesh (n = 2534 cases, n = 1229 controls), Giardia was inversely associated with diarrhoea (8% versus 18%) . A significantly higher prevalence among controls (23%) than cases (14%) was also found in a study from Thailand These findings emphasises that when interpreting the clinical relevance of Giardia, it must be taken into account that identifying the parasite may be confounding rather than causal, since many cases are asymptomatic. In an uncontrolled study of 529 hospitalised children with diarrhoea from a rural area in Mozambique, Giardia was detected in 3%; however, 36% of the 74% cases who had malaria, which commonly causes diarrhoea, also had an enteropathogen (not specified) isolated. Both low level malaria parasitaemia and enteropathogens may be either without clinical relevance, or etiologic agents, in patients with diarrhoea. This illustrates the importance of case-control studies. A review of 33 mainly population based cross-sectional prevalence studies from Asia showed that prevalence varied greatly, also within countries . Studies from Nepal, which is considered one of the poorest countries in the world, reported prevalence between 2% (elderly home) and 73% (children), and Thailand, which generally has a higher socioeconomic status, reported prevalence between 1% in a study from North Thailand  and 37% in a study among orphans in Pathum Thani Province . These serologic and clinical studies show that giardiasis is highly prevalent in many areas, but may not lead to severe acute diarrhoea with dehydration needing hospitalisation in most cases. The impact of giardiasis on the high mortality of diarrhoeal infections globally is not known, however the fact that Giardia in 2004 was included in the WHO's "Neglected disease initiative" may hopefully strengthen the focus on this poverty related disease . In Western countries, Giardia infection usually occurs after travel to endemic areas, during waterborne outbreaks, or through person to person spread in institutions or between risk groups such as homosexual men. Common causes of acute travellers diarrhoea are enterotoxigenic E. coli (ETEC), Campylobacter, Shigella and Salmonella spp, while persistent diarrhoea more often are caused by protozoans (Giardia, Cryptosporidium, Entamoeba histolytica, Cyclospora, Isospora and microsporidia spp.) . Among 17 228 travellers visiting tropical medicine clinics in Europe, 4% had acute diarrhoea due to Giardia, most commonly after travel to South-Central Asia (11%), in a recent report from the GeoSentinel Surveillance Network . In a study among 328 travellers and foreign residents with diarrhoea in Nepal, Giardia was diagnosed in 12%, and was more likely to occur in cases with diarrhoea lasting more than two weeks (27%) than in acute diarrhoea In Norway, laboratory confirmed cases of giardiasis are notifiable to the Norwegian Surveillance System for Communicable Diseases (MSIS). About 200-300 reported Giardia cases have been infected abroad each year since 1990 (Table 1) . Of diarrhoeal infections in Norway registered by MSIS in 2008, 270 were caused by Giardia, 2875 by Campylobacter, four by Diphtheria, 1941 by Salmonella, 134 by Shigella and 50 by Yersinia Approximately 50 cases each year of Giardia infections acquired in Norway, have been registered by MSIS since 1980 (Table 1) . However, in 2004 as many as 621 cases were registered, due to a large waterborne outbreak in Bergen in the period from August to December . Leaking sewage pipes into the city's water source, Svartediket (Figure 2), during a period of heavy rainfall, combined with insufficient water treatment, was identified as the source of the outbreak, and approximately 1 300 laboratory confirmed cases were registered. Based on the excessive number of metronidazole prescriptions made in the period, and taking into account that half of cases may be asymptomatic, more than 5000 cases have probably been infected during this outbreak . This is the largest waterborne outbreak, and the first Giardia outbreak, registered in Norway. A total of 72 waterborne outbreaks, infecting 10 616 cases, have been registered in the period 1988 to 2002, 26% of these were caused by Campylobacter, 18% by norovirus and 46% had unknown aetiology . Waterborne outbreaks are the most important route of Giardia transmission in Western countries. Several factors contribute to this; experimental studies have shown that cysts may survive for up to two months in cold water , conventional water treatment methods may not eliminate the parasites , and contamination of a community water supply has the potential to affect a great number of people. Before the Bergen outbreak in 2004, the largest outbreak registered in Europe had been at the ski resort Sälen in Sweden in 1986, affecting >1400 people . Waterborne outbreaks of protozoan parasites reported worldwide in the period 1955 to 2003 have recently been extensively reviewed, and Giardia accounted for 132 (41%) of these . Only outbreaks in Western countries have been reported, probably due to resource limitations and insufficient outbreak investigations in developing countries. Zoonotic transmission has been reported in giardiasis , however, the impact of such transmission is not clear. Beavers were the likely source of an outbreak in USA in 1986 , but animal sources in outbreaks are not commonly reported . Clinical case definitions are useful if parasitological testing must be avoided when laboratories experience capacity problems during large outbreaks. During a waterborne outbreak in Colorado, a Giardia case definition included illness lasting seven days or more combined with two or more of the symptoms diarrhoea, flatulence, foul-smelling stools, nausea, abdominal cramps or excessive tiredness . The sensitivity and specificity of this definition was 88% and 73% respectively. Figure 2. Svartediket.
Smaller outbreaks due to other routes of transmission have been well described from Western countries, although rarely. Raw vegetables and fruits contaminated with cysts from water or from an infected food handler are typical sources in food-borne infections [36-42]. Shellfish may accumulate pathogens from infected water and be a potential source of infection, and Giardia cysts have been identified in both oysters and mussels [43, 44]. Several outbreaks have been reported from child day care centres [45-48]. Transmission may also occur in swimming and wading pools [42, 49, 50]. Other groups at risk include homosexual men [51- 55] and persons living in institutions [47, 56]. Table 1. Giardia cases in Norway reported to MSIS and place of infection in the period
1980 1990 2000 2003
Both parasite and host factors seem to be involved in the pathophysiological processes causing diarrhoea, maldigestion and malabsorption in giardiasis, although incompletely In vitro studies on human samples have shown that Giardia attach by its adhesive ventral disc to the microvillus brush border of the intestinal epithelium, and cause barrier dysfunction by disrupting tight junctions and inducing epithelial apoptosis [57-59]. Further have experimental studies shown that activated CD8 T lymphocytes produce cytokines responsible for shortening of epithelial microvilli, which lead to malabsorption of electrolytes, nutrients and water as well as inhibition of the digestive enzymes lipase, protease and disaccharidase . Disaccharidase insufficiency, and consequently failure in splitting and absorbing milk lactose, causes osmotic diarrhoea characteristic for temporary lactose intolerance commonly seen in giardiasis. Bacterial overgrowth in the small intestine may also play a part in the pathogenesis of the disease . In clinical studies, inflammation and villous shortening in duodenal biopsies varies from 4% to 87% [62, 63], and why there is such a high variability in mucosal reactions, as well as in clinical manifestations, is not known. Genotypes and mixed infections have been proposed to be responsible for disease variability , but results from studies of the association between genotypes and severity of disease are not conclusive. An experimental study by Nash et al showed an association between strain variation and infectivity , and it seems that infection with a genotype less prevalent in a community induce more severe symptoms, however, both assemblage A and B have been associated with different symptom patterns in studies from different populations [19, 66-76]. The host defence mechanisms are complex. Experimental studies recently reviewed [77-79] have shown that natural barrier mechanisms (mucus, peristalsis, proteases, lipases, bile salts, intestinal microbiota and paneth cells), innate immune responses (nitric oxide, reactive oxygen species, lactoferrin, defensins, phagocytes, mast cells and dendritic cells) and adaptive immune responses, both cell-mediated as described above and humoral, are involved although incompletely understood. Although Giardia is not an invasive parasite, it induces a humoral immune reaction with production of immunoglobulines (IgG, IgM and IgA) [31, 65, 79, 80]. Clinical studies support that adaptive immune response play a role; children in endemic areas and non-immune travellers seem to have a higher risk for symptomatic disease than those who have been exposed for a longer period of time , and hypo- gammaglobulinemia is associated with chronic infection . Proteins on the surface of the parasite, variant surface proteins (VSPs), are major immuno-reactive proteins , and are also responsible for an important defence mechanism by enabling the parasite to undergo variation of its surface proteins and thereby evade the host immune response [83, 84]. HIV infection does not seem to be associated with more severe disease [85, 86]. Interestingly HIV infection stimulates the production of CD8 T-lymphocytes in the gut , and these lymphocytes are probably essential in the immune reaction against the Giardia parasite, as described above. 4. Clinical presentation
Experimental studies create optimal situations to study the course of an infection, although ethical considerations obviously limit the use of this method. Despite the questionable method, results from two experimental studies in humans have been reported, and are commonly used as references to the natural course of giardiasis. In 1953, Rendtorff reported results from four experiments in a controlled study of prison volunteers experimentally infected with Giardia cysts . Of all cases receiving cysts, 53% (21/40) became infected. Risk for infection was associated with infectious dose: All 13 cases who received from 100 to 1 million cysts, compared to 36% (8/22) who received 10 or 25 cysts, became infected, while only one cyst was not infectious in any cases. Persistent infection after at least 129 and 132 days was found in 15% of infected cases (2/14), while 85% (12/14) spontaneously cleared the infection within 5-41 days (mean 18, median 13 days). Asymptomatic infection was found in 40% (6/15), while frequent and loose stools lasting from two to four days was observed in 60% (9/15) in these experiments . The variation in infectivity and clinical presentation reported by Rendtorff was observed in another experimental study in 1987 . Nash et al infected 15 healthy volunteers with two different Giardia strains, GS/M and Isr. Of cases infected with GS/M, 100% (10/10) became infected, and 50% of these became ill after a prepatent period (the time from inoculation until parasites are detected in stool) of 7.5 days (mean). Severity of symptoms varied; one volunteer had diarrhoea, flatulence, abdominal pain, anorexia, vomiting, abdominal cramps, headache, malaise and abdominal gurgling for six days, three volunteers had loose stools and milder symptoms, while one volunteer had fever and headache but no diarrhoea. Interestingly none of the volunteers inoculated with the Isr strain became infected; suggesting that degree of infectivity may be strain dependent. Both of these experimental studies are limited by few cases, and that they included adult males only. However, symptoms during acute infection in larger cohorts in developed countries have been studied during outbreaks, and these reports support that symptoms are variable and that the majority of infected cases remain asymptomatic. Among laboratory confirmed cases during an outbreak in New Hampshire in 1980, the following symptoms were recorded (n = 213): Diarrhoea 86%, abdominal cramps 81%, anorexia 65%, flatulence 58%, abdominal distension 55% and weight loss 53%. Duration of symptoms was 10 days (mean) and 13% were hospitalised. A community survey revealed that 76% of the city residents had an asymptomatic and self limiting infection during this During the Bergen outbreak in 2004, 137 laboratory confirmed cases were interviewed during the early phase of the epidemic, and among these 90% reported diarrhoea, nausea, stomach pain, flatulence and foul smelling stools, 83% reported weight loss (mean 5 kg, range 1-23 kg), 36% vomiting, 17% reported fever and 7% were hospitalised . Giardiasis seems to be self limiting in most cases, but a striking feature of the parasite is its ability to induce chronic infection, symptomatic or asymptomatic, if not treated. Rendtorff reported chronic infection in 15% (2/14) of experimentally infected and untreated cases . A Nordic meta-analysis reported Giardia infection in 6% of symptomatic cases (one or more of the symptoms vomiting, gastroenteritis, diarrhoea and abdominal pain/cramps/discomfort), and 3% of asymptomatic cases, in the population, supporting that chronic giardiasis is prevalent in non-endemic countries . Chronic infection may present with symptoms similar to irritable bowel syndrome (IBS); a condition characterised by abdominal pain or discomfort, associated with altered bowel habits, lasting for more than 12 weeks . Among 137 cases with symptoms of dyspepsia or IBS who satisfied the Rome II criteria , 7% had giardiasis in one report from Italy . Malabsorption in chronic giardiasis may cause chronic diarrhoea and steatorrhea, weight loss and nutrient and vitamin deficiencies . Vitamin B12 malabsorption, and anaemia due to Vitamin B12 and folate deficiencies, has been reported [94-96]. Vitamin A deficiency, a potentially severe condition since such deficiency is an important cause of blindness in developing countries, has also been documented in giardiasis; a significant improvement of vitamin A absorption was demonstrated after anti-Giardia treatment in one study . Osmotic diarrhoea due to lactose malabsorption is common in giardiasis, and may persist for weeks after eradication of the parasite . Fat and carhohydrate malabsorption has been documented in controlled clinical studies , but the role of amino acid malabsorption is unclear. Although there have been casuistic reports on severe protein loss and hypoalbuminaemia , no association between giardiasis and protein loosing enteropathy was found in a study in Gambian children . In a case-control study from India malabsorption syndrome was caused by Giardia in 24% (12/50) of adult cases compared to 8% (4/50) of healthy controls . In children the difference was not significant (16% versus 6%) which also illustrate the problem in interpreting the finding of Giardia in patients from endemic areas since the infection may be In developing countries where frequent re-infections are common , giardiasis contributes together with other infectious agents to malnutrition. Considering its high prevalence and the parasites' ability to induce chronic infection and malabsorption, one would suspect an impact on growth in children. Since first reported in 1921, several studies have shown an association between severe giardiasis and such impairment [103-105]. More than one episode of giardiasis per year during infancy was associated with poor cognitive function at 9 years of age in 239 Peruvian children . However, in another longitudinal study of 220 Peruvian children, no significant association between Giardia and nutritional status or diarrhoea was found . Also studies in asymptomatic children have shown diverging impact on growth. In one study from Brazil (n = 597) asymptomatic giardiasis was significantly associated with impeded growth , while studies from day care centres in western countries have not found this association , suggesting a difference in disease susceptibility between healthy and malnourished children. Rare extra-intestinal manifestations have been reported, probably due to immune mechanisms since Giardia is not invasive. These reports have been of giardiasis associated with reactive arthritis and synovitis [110, 111], urticaria and pruritis , uveitis  and allergy [114, 5. Laboratory diagnostic methods
Giardiasis is diagnosed by examining stool samples by light microscopy, immunofluorescence assay (IFA), enzyme immunoassay (EIA) or polymerase chain reaction (PCR) methods. Analyses of serum or duodenal aspirates may also be performed. Light microscopy is a labour intensive method, but has the advantage of detecting additional parasites if present. Stool is concentrated and examined directly (wet-mount preparation) or permanently stained (trichrome) for cysts or trophozoites (detection of trophozoites requires fresh stool). Sensitivity is poor when only a single sample is analysed, particularly if there are few cysts or quality of microscopy is insufficient, but sporadic samples in an infected person may also be negative due to intermittent excretion of cysts; in one report of 91 cases all with three samples analysed, one, two or all three stool samples were microscopy positive in only 23%, 22% and 55% respectively . Sensitivity increases when multiple samples collected from separate defecations are analysed; sensitivity of 73%, 81% and 85% from analysis of one, two or three samples respectively have been reported (n=73) . Sensitivity of microscopy increases when samples are stained with a specific anti-Giardia antibody coupled to a fluorescent compound (IFA). Cysts show fluorescence when examined under ultraviolet light in a fluorescence microscope. High sensitivity (92%-100%) and specificity (100%) has been reported with this method [118-120]. EIA also uses antibodies to detect Giardia specific antigen in stool, and these tests are less time consuming since the result is read after only few minutes. Several commercial rapid antigen tests are available, and high sensitivity (80%-99%) and specificity (>99%) has been reported [118, 121-124]. However, EIA was significantly less sensitive (61%) than microscopy in a study from the Bergen outbreak . Reduced sensitivity of EIAs was associated with low cyst numbers and single samples in these studies. PCR is a sensitive and specific method also when there are few cysts [119, 124, 126]. In one study comparing PCR and IFA, sensitivity was 97% and 92% respectively (no significant difference) and specificity was 100% for both . PCR is mainly used in research, and during the Bergen outbreak PCR was used to genotype and sub-genotype the Giardia parasite and thereby elucidate host and parasite factors , but the method has limited use in routine diagnosis. Detection of trophozoites in duodenal aspirate was previously considered a sensitive test when stool microscopy was negative, but studies supporting this were based on few cases, as shown in a review by Goka et al . Larger studies have shown very low benefit from this test. In one study only 44% (32/73) of Giardia positive cases had positive duodenal aspirates, compared to 85% stool microscopy positive . This finding was supported by a study from the Bergen outbreak were only 10% (4/40) of Giardia positive cases had trophozoites in duodenal biopsies . Both indirect immunofluorescence (IIF) and enzyme-linked immunosorbent assay (ELISA) may be used in detecting specific anti-Giardia antibodies [128, 129]. Serologic methods have been used in epidemiologic studies as described previously, but not widely in routine diagnostic laboratories. In endemic areas, detection of Giardia antibodies may be due to previous exposure, as reported in a study from Bangladesh where no association between sero-positivity and positive stool microscopy was found . However, in areas with no previous exposure, serologic tests are more specific in diagnosing acute infection. In a study among 352 exposed cases during the outbreak in Sälen in Sweden, IgG and/or IgA antibodies were detected in 68% of Giardia positive cases, in 22% of Giardia negative cases and only among 10% of healthy controls . A significant increase in sensitivity was observed when serum was collected more than three weeks after infection. Also in a controlled study from Cuba, serology was strongly associated with positive microscopy, although sero-positivity increased with age which might have been due to previous exposure . 6. Treatment
Six classes of drugs are effective against the Giardia parasite: Quinacrine has been used since the 1930s, nitroimidazoles, furazolidone and paromomycin since the 1960s, benzimidazoles since the 1980s and the last 10 years nitazoxanide has been reported to be effective against giardiasis . Results from controlled clinical studies of efficacy, based on parasitological response, are presented in table 2 (modified from table in press by our group ). Recommendations of effective dosages based on these studies, as well as mechanisms of action and reported adverse effects, are presented in table 3 (modified from tables published and submitted by our group [133, 134]). Pharmacological aspects in anti-Giardia drugs have also recently been reviewed by others [132, 135, 136]. Efficacy reported in the clinical studies are not directly comparable due to variations in the populations studied (age, endemicity), time until follow-up, number of stool samples examined, dosage of drugs and duration of treatment. In studies from endemic areas, relapse rates may be overestimated if duration of follow-up is too long, due to high risk of re- infections, as reported in a study from Peru where 98% of children became re-infected within 6 months after effective treatment . However, relapses may be underestimated if follow- up time is too short. In one study from a non-endemic area, 43% (6/14) of relapses were diagnosed after more than four weeks . Per protocol rather than intention to treat analyses were presented in most of these studies, which potentially also may overestimate Metronidazole has for many years been the drug of choice for giardiasis. However, single dose treatment with tinidazole, ornidazole or secnidazole (nitroimidazoles with long half lives) seems to have similar efficacy and fewer side effects. Short course treatment with metronidazole and other classes of anti-Giardia drugs are less effective. Nitazoxanide and benzimidazoles are well tolerated, and have the advantage of also being active against helminth infections which are endemic in developing countries [138-142], although benzimidazole treatment has low efficacy in some studies. Quinacrine is highly effective in most studies, but is not recommended as first line treatment due to potentially severe side effects. The aminoglycoside paromomycin seems to be less effective than other drugs, although few reports are available. Available documentation of risks during pregnancy varies for different anti-Giardia drugs. Of the nitroimidazoles, metronidazole has been best studied. Metronidazole has shown carcinogenic and teratogenic effect in animal studies, and although carcinogenic effect has not been reported in humans, it raises concern about safety during pregnancy [143-146]. In a meta-analysis including 1336 women exposed during first trimester , and in two studies including 228 and 1041 pregnant women respectively [148, 149], no association with birth defects were found. However, malformations possibly associated with metronidazole during first trimester has been reported, although rarely [146, 150]. Based on these reports, metronidazole during pregnancy is controversial, although often recommended during second and third trimester, weighted against indication and availability of alternative drugs, since the possible risk is low . Nitroimidazoles should be avoided during first trimester. Benzimidazoles have been widely used and recommended by WHO after first trimester in treatment programs among pregnant women, in order to reduce hookworm induced anaemia [151, 152]. In controlled studies evaluating birth defects following these programs, significant risk for teratogenic effects were not found [153-155]. However, the dosages used in anti-helminth programs are lower than recommended dosages for giardiasis, and experimental studies have suggested teratogenic effects . One controlled clinical study found higher risk of major birth defects (odds ratio 1.66), although not significant, among women who had used mebendazole during first trimester . Based on clinical reports on safety during second and third trimester, benzimidazoles could be used during this period, but should be avoided during first trimester. Due to mutagenic effects in animal studies, and lack of clinical studies in humans on teratogenic effects from furazolidone, quinacrine and nitazoxanide, these drugs should be avoided throughout pregnancy [157, 158]. Paromomycin is the only anti-Giardia drug not contraindicated during first trimester , since it is poorly absorbed and therefore has negligible systemic effect. Studies on treatment refractory giardiasis are limited. In one study, albendazole in combination with metronidazole was effective in nine out of 10 metronidazole resistant cases . Quinacrine combined with metronidazole or tinidazole was effective in five of six treatment refractory cases in one study, four of these were immunosuppressed . Such synergistic effect of combining metronidazole and quinacrine has also been reported in vitro . One report showed that nitazoxanide was effective in a patient suffering from HIV and metronidazole/albendazole resistant giardiasis, and the Giardia isolate from the patient showed the same resistance pattern in vitro and in a mouse model . Clinical resistance has been demonstrated for all drugs as described above, however, several factors may influence clinical response other than drug resistance, like inadequate immune response, compliance problems, reduced quality of drugs or impaired absorption due to vomiting and diarrhoea. Laboratory studies have the advantage of eliminating these factors, and in vitro resistance has been reported in all classes of drugs [164, 165]. Different laboratory methods have been used; in vivo animal studies and in vitro tests of viability, or molecular characteristics, of cultured human Giardia isolates or laboratory induced resistant clones. Although mechanisms of drug resistance are not completely understood, molecular studies have elucidated some characteristics. The enzyme pyruvat:ferredoxin oxidoreductase (PFOR) was downregulated in metronidazole resistant but not in furazolidone resistant Giardia strains, which is consistent with the role this enzyme has in activating metronidazole, while furazolidone probably is activated by nicotinamide adenine dinucleotide (NADH) and not by PFOR . In a molecular study of genes potentially involved in resistance, laboratory induced nitazoxanide resistant and metronidazole resistant Giardia clones were compared to Giardia WB C6 wild type, and in addition to slightly reduced expression of pyruvat oxidoreductase (POR) and nitroreductase (NR), a gene encoding a major variant surface protein (VSP) was significantly reduced in both resistant clones, indicating that the parasites ability to evade host immune response by variable expression of surface proteins may be a target for these drugs . The nitazoxanide resistant strain showed cross resistance to metronidazole, while the metronidazole resistant strain was sensitive to nitazoxanide in this study . Cross resistance found in vitro between other anti-Giardia drugs has also been reported; furazolidone resistant strains induced more easily quinacrine resistance in one study , and albendazole resistance was more readily induced in a furazolidone resistant strain in one Drug susceptibility testing is not routinely used, although relatively simple systems for resistance testing are commercially available and could be used in surveillance of drug resistance in giardiasis . Other drugs have been studied experimentally, and have recently also been reviewed by others [132, 171]. The following compounds have shown anti-Giardia effect in vitro or in animal studies: Bithionol, dichlorophene and hexachlorophene , pyrimethamine and chloroquine , the tricyclic antidepressant drug chlorimipramine , sodium fusidate which also has the advantage of not being terotegenic , ronidazole, satranidazole, fexinidazole, flunidazole and nimorazole (5-nitroimidazoles), nitrofurantoin and niridazole , mefloquine, doxycycline and rifampin , ivermectin , fenbendazole , ciprofloxacin , bismuth subcitrate , thiosemicarbazone , new benzimidazoles [183-185], ethyl-phenylcarbamates , menadione which also killed cysts , the naturally derived saturated fatty acid dodecanoid (lauric) acid , nocodazole and colchicine , silymarin , new thiazolides , disulfiram  and azithromycin Controlled clinical trials of experimental drugs against Giardia have shown some promising results. Bacitracin zinc had anti-Giardia effect both in vitro  and in vivo; one clinical study reported 95% (20/21) efficacy . In a study from Cuba, chloroquine 10mg bid for five days cured 86% , and chloroquine has also shown anti-Giardia effect in vitro [173, 197]. D-propranolol has demonstrated inhibition of growth and motility of the protozoan in vitro , and clinical effect in a metronidazole resistant case has been reported . The bee glue preparation propolis inhibited growth in vitro in one report , and in a clinical study treatment for 20 days cured 80% . Ozone have shown anti-Giardia effect in vitro in different studies [202, 203], and in a clinical trial ozonized sunflower oil cured 64% . glycoside
; Ns, not stated. yil ; qd, four times day ole, quinfamide or both. dose; bid, twice dail iven in this table. g , if other is not stated. e patients from the adult n; A, adults; C, children; sd, sing n of follow-up in da ), duration in day , with 11 cases in the nita rst, second and third tre in combination with Praz divided into three doses.
Of 275 cases with intestinal parasitic infections, Abbreviations: D, da Response assessed b Uncontrolled stud Placebo controlled stud Table 3. Drugs active against Giardia infection; mechanisms of action, recommended
dosages and side effects (modified from [133, 134]). Mechanism
Metronidazole Reductive activation of nitro GI discomfort, metallic taste, group, by ferredoxin and the disulphiram-like effects. Headache, 500 mg sd3 x 10d Anti-parasitic effect: vertigo, insomnia, irritability, neuropathy, seizures. Rash. efficacy low when -Produce toxic radicals Tinidazole Reddish-brown urine. Transient -Inhibit trophozoite elevation of transaminases. than 5 days, the Leukopenia. Pancreatitis, hepatitis, Ornidazole cholangitis (rare). effective as single Metronidazole less tolerated than dose due to longer Secnidazole the other 5-nitromidazole Nitrofuran derivatives
Possibly reductive activation Nausea, vomiting, diarrhoea. by the enzyme NADH Haemolytic anemia in neonates and given to neonates oxidase, and production of toxic nitro radicals which in G6PD-deficiency. Disulphiram- or breastfeeding damage the parasites like activity. Interaction with MAO functional organelles inhibitors. Brownish urine. risk of haemolytic including its DNA. Albendazole Inhibits cytoskeleton Usually well tolerated. Nausea, polymerization and impaire vomiting, diarrhoea, epigastric against helminths. glucose uptake by binding to the parasites -tubulin Mebendazole Usually well tolerated. Transient (Optimal dose and duration refractory cases in combination with metronidazole. Quinacrine Not fully understood. Potentially severe side effects. Possibly inhibition of Vomiting, bitter taste, nausea, nuclein acid cyntesis by binding to DNA, or headache. Yellow discoloration of refractory cases, decreased oxygen skin, urine or sclerae (reversible). consumption due to Urticaria, exfoliative dermatitis, combination with interference with the enzyme NADH oxidase. exacerbation of psoriasis. Haemolysis in G6PD-deficiency. Paromomycin Interaction with 50S and 30 Usually well tolerated. Regarded as safe S ribosomal subunits leads to Gastrointestinal discomfort. misreading of mRNA, and thereby inhibits the parasites protein synthesis. 5-nitrothiazolyl derivatives
Nitazoxanide Not fully understood. Anti- Usually well tolerated. Abdominal parasitic effect after pain, diarrhoea, vomiting, against helminths reductive activation. Inhibition of parasite headache, yellowish urine. and some bacterial nitroreductase G1NR-1. enteric infections. Effective in metronidazole resistant infection. Abbreviations: GI, gastrointestinal; Sd, single dose; PFOR, Pyruvate ferredoxin oxidoreductase; G6PD, Glucose-6-phosphat-dehydrogenase; NADH, Nicotinamide adenine dinucleotide; DNA, Deoxyribonucleic acid; mRNA, messenger ribonucleic acid; bid, twice daily; tid, three times daily; qd, four times daily; d, days; g, gram. 1Based on results from clinical studies (Table 2). 2mg/kg/day, divided into three doses. 3One day duration of sd if not other is stated. 4mg/kg. 5mg/kg/day, divided into four doses. V. Aims of the study
To evaluate efficacy of a treatment ladder, and genetic characteristics, in treatment refractory giardiasis after an outbreak in Bergen, Norway in 2004. To investigate if cases with persistent abdominal symptoms after Giardia infection, and no detectable Giardia parasites in stool, suffered from chronic, cryptic giardiasis. To investigate the prevalence of fatigue and abdominal symptoms, and factors associated with such symptoms, two years after the Giardia outbreak. VI. Summary of papers
A. Paper 1
Metronidazole is the only drug licensed against Giardia infection in Norway. The objectives of this study were to evaluate the efficacy of three different anti-Giardia treatment regimens in cases who had not responded to metronidazole treatment, and to compare genetic characteristics of the parasites. This was a clinical observational study among cases who had been infected with Giardia during the outbreak in Bergen in autumn 2004, and who experienced chronic, treatment refractory infection. Among 1268 laboratory confirmed cases registered during the outbreak in August-December 2004, 120 cases were referred to our out patient clinic due to protracted abdominal symptoms. Of these, 42 cases still had Giardia cysts in stool samples after mean 2.2 (range 1-3) courses of metronidazole, and 38 of these were treated according to a standardised treatment ladder in the period between January 2005 and December 2006. All patients were treated with albendazole 400 mg bid in combination with metronidazole 250 mg bid for one week. Those who did not respond to this regimen were treated with paromomycin 500 mg tid for one week. Those who failed on both these regimens were treated with quinacrine 100 mg tid in combination with metronidazole 750 mg tid for three weeks. Treatment efficacy was based on parasitological response defined as seven microscopy negative stool samples up to four weeks after the end of treatment. Clinical symptoms and adverse events were evaluated at baseline and four weeks after treatment. Giardia isolates were available from 45% of cases (17/38) for characterisation by PCR and sequencing at the gdh and -giardin genes respectively. Albendazole in combination with metronidazole was effective in 79% (30/38), paromomycin was effective in 50% (3/6), and quinacrine in combination with metronidazole was effective in 100% (3/3) of the cases. Discoloration of skin, confusion, nightmares, dizziness and nausea were recorded in the quinacrine/metronidazole group, and mild hair loss was reported by one patient in the albendazole/metronidazole group. Except for the episode of mild hair loss, no unexpected or severe side effects were recorded in any of the groups. Sequencing of PCR products revealed that all cases had Giardia cysts of genotype gd-ber3 at the gdh gene and BG-ber2 at the -giardin gene, while previously published sequence profiles from the peak of the outbreak were more heterogenous. In this study, albendazole and quinacrine, both in combinations with metronidazole, were effective against metronidazole refractory giardiasis, while paromomycin seemed to be less effective. Particular sub-genotypes may be associated with treatment refractory infection in B. Paper 2
The aim of this study was to evaluate if Giardia negative patients, referred to our outpatient clinic due to protracted abdominal symptoms after the Bergen outbreak in 2004, suffered from cryptic and metronidazole refractory chronic giardiasis. This was a prospective randomised open clinical study. The included patients had been exposed to contaminated water and had clinical giardiasis during the outbreak, and after treatment with one to three courses of metronidazole for five to ten days, all cases had more than three microscopy negative stool samples and one negative faecal antigen test. Based on reports on synergistic anti-Giardia effect of metronidazole and albendazole combination treatment as described in paper 1, patients in one arm were treated with albendazole 400 mg bid and metronidazole 250 mg tid for seven days (A/M). Based on the hypothesis that an illness similar to post-infectious tropical sprue may be one possible explanation for protracted abdominal symptoms and weight loss, patients in the other arm received tetracycline 250 mg tid and folic acid 5 mg once daily for 28 days (T/F). Abdominal symptoms were reported by the patients on a written questionnaire at baseline, at the end of treatment, one month after treatment and finally one year after treatment. The primary endpoint was global improvement of symptoms one month and one year after treatment. Secondary endpoints were improvement of nausea, bloating, abdominal pain, diarrhoea, constipation and anorexia recorded on a scale from zero to ten, and changes in blood inflammation and malabsorption parameters. Symptom scores were analysed regarding time and treatment using mixed linear modelling. A total of 25 cases were included in the study. Blood tests taken at baseline and after one month were not significantly changed. At the end of treatment, total symptom score improved in both groups, although significantly only in the T/F group, while bloating decreased significantly in both groups at this point. One month after treatment, 23% (3/13) in the T/F group and 8% (1/12) in the A/M group reported global symptom improvement. However, after one year total symptom scores were unchanged from baseline in both groups. C. Paper 3
The objective of this study was to evaluate the prevalence of fatigue and abdominal symptoms among cases who had been infected during the Bergen outbreak in 2004, two years after. Inclusion criteria was laboratory confirmed giardiasis during the Bergen outbreak. All 1262 Giardia–positive cases registered in the period of October 2004 to June 2005, received a mailed questionnaire in August 2006 (Appendix). The following questions regarding fatigue and abdominal symptoms, respectively, were used in the statistical analyses: "Do you have abdominal symptoms now that you did not have prior to the Giardia infection?" (no/unsure/yes, dichotomized into no/unsure vs. yes) and "Do you have problems with fatigue?" (less or same as usual/more than usual/much more than usual). The last two answer options were defined as fatigue. The association between fatigue and abdominal symptoms, gender and age, was investigated by simple and multiple ordinal logistic regression analyses using SPSS. Among the 1017 (81%) respondents, 64% were women and the median age was 31 years, compared to 61% and median 30 years among all 1262 cases. Fatigue and abdominal symptoms was reported by 41% (419/1017) and 38% (389/1017), respectively, and 25% (253/1017) reported both symptoms. Increasing age was significantly associated with fatigue (p < 0.001) in all analyses. Female gender was significantly associated with fatigue in the simple (p = 0.038) but not in the multiple regression analyses. Neither age nor gender was significantly associated with abdominal symptoms. A significant association between fatigue and abdominal symptoms (p < 0.001) were found in all analyses, and neither gender nor age interacted with this A high level of post-infectious fatigue and abdominal symptoms, not previously reported in giardiasis, were found in this study. D. Paper 4
This paper describes risk factors associated with post-Giardia fatigue and abdominal symptoms two years after the Bergen outbreak. Inclusion criteria and questionnaire (Appendix) used were the same as described in paper 3, and in this study the data were further analysed with respect to risk factors. Number of treatment courses, delayed education and sick leave were used as indices of protracted and severe Giardia infection in the statistical analyses, and the sub-cohort of treatment resistant cases described in paper 1 were also included in these analyses. Previous abdominal problems, symptoms during infection, age and gender were also evaluated as possible risk factors. Simple and multiple ordinal logistic regression analyses were used to investigate the association between these possible risk factors (explanatory variables) and degree of abdominal symptoms and fatigue (response variables). More than one course of anti-Giardia treatment and delayed education, were significantly associated with both fatigue and abdominal symptoms in all analyses. In the multiple regression analysis, female gender, bloating at the time of infection and treatment refractory infection were associated with abdominal symptoms. Age, previous abdominal problems without seeking health care, malaise at the time of infection and sick leave were associated with fatigue in the multiple regression analysis. Indices of protracted and severe Giardia infection were associated with post-infectious fatigue and abdominal symptoms in this study. VII. Main results and discussion
Paper 1. Treatment-ladder and genetic characterisation of parasites in refractory
giardiasis after an outbreak in Norway
The paper describes high efficacy of combination treatment of albendazole or quinacrine in combination with metronidazole, respectively, and less efficacy of paromomycin, in metronidazole refractory chronic Giardia infection. Synergistic effect of metronidazole and albendazole has been reported from a randomised trial of metronidazole resistant cases , and our study is in line with this finding. Randomised trials on quinacrine and paromomycin in treatment refractory giardiasis have not been performed, although a case series among resistant cases successfully treated with quinacrine combined with nitroimidazole support our findings . This study was an observational study using a treatment-ladder, while a randomised study design would have strengthened our findings. Metronidazole is the only anti-Giardia drug licensed in Norway, and delays in availability were different for the different drugs. A randomised study would have delayed treatment in patients with bothersome symptoms, and was therefore not performed for ethical reasons. The three regimens can not be compared, since patients treated in the late steps in the ladder had clinically more multi-resistant parasites than patients treated in the first step. The efficacy of paromomycin and quinacrine/metronidazole should also be interpreted with care due to low number of cases. In order to study genetic characteristics of the parasites, two gene sequences were analysed and the parasites were identical at these sequences, which was different from the picture during the peak of the outbreak, where 10 different sub-genotypes were described . This leads to the hypothesis that the sub-genotypes found in our study may have been responsible for more virulent or resistant infection. The genes involved in metronidazole resistance are not fully characterised. Results from molecular studies show that such resistance may be mediated by altered gene expression, possibly involving reduced expression of the genes encoding for PFOR and VSPs , however, this remains to be further investigated. The sub-genotypes characterised in the present study do not fully explain metronidazole resistance, since these sub-genotypes were also found in cases diagnosed subsequent to the outbreak who responded to metronidazole treatment . However, if these strains were more virulent, resistant or infective than other strains, they may have persisted longer in the environment, and thereby infected cases subsequent to the outbreak and also induced chronic infection. The host-parasite interactions in giardiasis are not fully understood, as previously discussed, and other factors explaining chronic infection in this cohort are probably also involved. This is underlined by the fact that one of the more resistant cases in this study, who finally responded to quinacrine/metronidazole, had clinically significant IgA deficiency, an immunological disorder known to predispose to chronic giardiasis . Paper 2. Effects of albendazole/metronidazole or tetracycline/folate treatments on
persisting symptoms after Giardia infection: A randomized open clinical trial
After the Bergen outbreak all patients referred to our out patient clinic with chronic infection, diagnosed by detection of cysts in stool, were successfully treated as described above. However, approximately half the cases referred due to protracted abdominal symptoms did not have detectable parasites in stool. Chronic infection and fluctuation in cyst excretion is well described in giardiasis , and could be a possible explanation for symptoms in these Diarrhoea and weight loss were common during the outbreak , which are characteristic symptoms of malabsorption. Important differential diagnoses to malabsorption in addition to giardiasis are Cryptosporidium infection, celiac disease, lymphoma and tropical sprue . Cryptosporidium had been excluded by negative faecal antigen tests, and anti-endomysial and anti-tissue transglutaminase antibodies as well as duodenal biopsies did not reveal celiac disease or lymphoma as etiologic agents . Tropical sprue is a syndrome of undefined aetiology, characterised by small intestinal mucosa damage and often severe malabsorption of vitamine B12, folate and fat, following an episode of acute diarrhoea . The name refers to that the condition is geographically restricted to specific areas in the tropics; it is much more common in Asia than in South America and Africa. Antibiotics may be effective, which support an infectious aetiology. The cases in our cohort had not travelled abroad, but the hypothesis was that a similar condition, involving bacterial agents, could explain the This study described that treatment directed against chronic giardiasis and tropical sprue, among cases with persistent abdominal symptoms following giardiasis, had no effect on symptoms one year after treatment. The temporary symptom reduction reported may have been due to anti-inflammatory effect, or effect on gut microbial flora, of both treatments. The difference in temporary symptom reduction seen between the groups may be explained by the longer duration of T/F treatment. It is not known if reporting of symptoms were influenced by cases expecting to improve during treatment, which would have been elucidated if a placebo group had been included. The reason for not including a placebo group was mainly that extending to three arms would have been more resource and time consuming in a clinical setting, and would have delayed treatment in the symptomatic group. Taking these limitations into account, the lack of change in symptoms after one year, leads to the conclusion that chronic giardiasis, or a tropical sprue-like illness, did not explain symptoms in this cohort. Paper 3. High rate of fatigue and abdominal symptoms 2 years after an outbreak of
During the Bergen outbreak, protracted symptoms after infection, among Giardia negative patients, were reported both in primary health care and among cases referred to our outpatient clinic [62, 245]. Of those referred, all Giardia positive cases had been successfully treated as described in paper 1, while the majority were Giardia negative but still complained of IBS- like symptoms and fatigue . Chronic infection could hypothetically explain protracted symptoms , but this was excluded among referred cases as described in paper 2. Extensive work-up among all referred cases, including stool culture and microscopy, endoscopy and blood tests, did not reveal the cause of IBS-like symptoms and fatigue, and on this background the present study of post-infectious symptoms among all cases was initiated. We reported fatigue in 41% and IBS-like symptoms in 38% of patients two years after the outbreak, among all laboratory confirmed cases. Such complications are not previously reported after giardiasis, but post-infectious fatigue and IBS are well described complications after other infections [246, 247], which support this finding. However, the prevalence of such symptoms is high in the general population, and both age and gender are possible risk factors; in Norwegian population studies, fatigue has been reported by 22% , and IBS by 10% . It is therefore an important methodological limitation in the present study that an age and sex matched control group was not included. Another limitation is the recording of symptoms, especially fatigue which was recorded by one question only (Appendix). Validated questionnaires have been developed for chronic fatigue , and Rome diagnostic criteria are widely used for IBS . Use of validated questionnaires would have strengthened our findings. However, these forms are extensive, and the high response rate (81%), which is a strength of our study, is probably partly explained by the short two-page questionnaire used. It is likely that abdominal symptoms recorded in our study are similar to IBS, since the questions used have been used in recording severity of IBS previously , and 81% in a sub-group of cases from the Bergen outbreak fulfilled the Rome II criteria for IBS . The inclusion criteria may have created a selection bias. Only laboratory confirmed cases were included, to ensure that only Giardia cases were investigated. However, this selects cases who visited their doctor due to symptoms during giardiasis, and could exclude cases with milder symptoms and cases with a less care seeking behaviour. In a large case-control study (n = 4388) among fatigue syndrome and IBS patients, both these patient groups consulted their GP more often, and received more often sickness certificates, than controls within three years prior to diagnosis . Many cases will have experienced a self-limiting infection, and if they hesitated in visiting their doctor they may have become well without treatment. More women were registered during and subsequent to the outbreak , and this could reflect that women seek medical care earlier, and that more men experienced a self- limiting infection, although water drinking habits has also been suggested to explain this Co-morbidities were not registered in our study, and it is possible that patients with co- morbidities would visit their doctors earlier than healthy, young individuals. A strong association between psychiatric disorders and chronic fatigue has been reported in population based studies , and other conditions prior to diagnosis have also been found more frequently in chronic fatigue compared to controls . These possible confounding factors could have been elucidated if we had recorded information on co-morbidity and previous health in our study. The possibility that any economical loss could be compensated by the health system creates a risk for over-reporting of symptoms. This could have been elucidated by using application for compensation as explanatory variable in the analyses, but this was not recorded in this study. Finally, recall bias is an obvious limitation, which also has been discussed in the paper. Taking these limitations into account, our finding of a high level of fatigue and IBS-like symptoms, which often lead to pronounced symptoms and reduced quality of life among young individuals, should lead to further investigations on post-infectious complications in Paper 4. Severity of Giardia infection associated with post-infectious fatigue and
Severity of infection has been reported as an important risk factor for both post-infectious fatigue and IBS following different infections [246, 255]. To study if this was the case also in giardiasis, we defined factors that hypothetically could indicate severity (number of treatment courses, treatment refractory infection, delay in education progress and sick leave), and used these as explanatory variables in the statistical analyses. We found a significant association between several of these indices of severity and fatigue and abdominal symptoms. The factors defined as indices of severity have several limitations. Several treatment courses may have been a sign of treatment refractory and protracted infection, but also a sign of PI- IBS if stools were not controlled at follow-up, which often was the case during the outbreak due to reduced laboratory capacity. However, it is likely that patients experienced a change in symptom pattern when the parasites were cleared, especially disappearance of the foul smell, and then additional treatment courses may not have been requested. A further limitation of several treatment courses as an index of protracted infection is that some cases have been infected for several months before they received treatment, due to late detection of the outbreak , while those who became ill when the outbreak was well known from the media may have received treatment immediately. Nevertheless, the finding of several treatment courses as a risk factor was supported by that laboratory confirmed treatment refractory and chronic infection in a sub-group (paper 1) was associated with post-infectious abdominal symptoms as well. These findings suggest that treatment resistant parasites may have been more virulent and caused more severe infection during this outbreak. Both delays in education progress and sick leave may have been caused by post-infectious complications rather than protracted and severe infection, and this has been discussed. However, it is probable that cases were unable to work or study due to severe symptoms during infection, while the post-infectious complications developed more slowly or fluctuated, which also is our clinical impression from referred cases. If sick leave or delayed education progress were due to giardiasis, they are good indices of severity of infection, since many cases experience mild symptoms during infection which would not influence their work Some questions were designed in a way that did not include all respondents (Appendix), and it is not clear whether these limitations in the questionnaire have influenced the outcome of the multiple regression analyses. The category "not recovered" was included in the categorical variable "Treatment courses" to avoid loosing cases, although this category could reflect the response variables. However, when the analyses were performed without this variable, other risk factors remained significant. Frequent re-infections in endemic regions , and the fact that giardiasis most often is self- limiting , have led to recommendations of not to treat in many cases . However, if our findings are causal, early detection and treatment of Giardia infection could be important also in order to avoid protracted infection, and thereby prevent post-infectious fatigue and abdominal symptoms. VIII. Proposals for future studies
x Molecular studies on resistance in treatment refractory cases. x Prospective, randomised studies on combination treatment in refractory giardiasis. x Controlled, long term follow up studies using validated questionnaires on prevalence of post-infectious fatigue and IBS. x Prospective, controlled studies on fatigue and IBS after severe versus mild or asymptomatic infections, using validated questionnaires, clinical evaluation and intestinal biopsies evaluating mucosa damage. x Clinical, epidemiologic and molecular studies in collaboration with centres in endemic countries to evaluate manifestations among cases experiencing frequent re-infections and co-infections compared to non-immune cases in western countries. IX. Conclusions
Metronidazole in combination with albendazole or quinacrine, were effective and safe treatments in a cohort of metronidazole refractory giardiases, while paromomycin was effective in only 50%; however, efficacy from the different treatment regimens can not be compared due to limitations in the design of the study. The Giardia parasites causing metronidazole refractory infection were closely related compared to the sub-genotypes at the beginning of the outbreak, indicating that parasite factors may have been partly responsible for treatment resistance during this outbreak. Persistent abdominal symptoms in a cohort of Giardia-negative cases after the outbreak were not due to chronic Giardia infection or to a tropical sprue-like infection. Post-infectious fatigue and IBS-like symptoms, not previously described in giardiasis, were found in 41% and 38%, respectively, among all laboratory confirmed cases during the Bergen outbreak two years after clearing the Giardia infection. A strong association between fatigue and IBS-like symptoms suggest that these symptoms may be manifestations of the same post-infectious condition following giardiasis. Indices of severe and protracted Giardia infection were associated with post-infectious fatigue and IBS-like symptoms two years after. X. References
Monis PT, Caccio SM, Thompson RC: Variation in Giardia: towards a taxonomic
revision of the genus. Trends Parasitol 2009, 25(2):93-100.
Hill DR NT: Intestinal Flagellate and Ciliate Infections, vol. 2, Second edn:
Studies on the cytology and life history of a Giardia from a laboratory
rat. Univ Calif Publ Zool 1952, 57:53-146.
Morrison HG, McArthur AG, Gillin FD, Aley SB, Adam RD, Olsen GJ, Best AA,
Cande WZ, Chen F, Cipriano MJ et al: Genomic minimalism in the early diverging
intestinal parasite Giardia lamblia. Science 2007, 317(5846):1921-1926.
Bernander R, Palm JE, Svard SG: Genome ploidy in different stages of the Giardia
lamblia life cycle. Cell Microbiol 2001, 3(1):55-62.
Bingham AK, Jarroll EL, Jr., Meyer EA, Radulescu S: Giardia sp.: physical factors
of excystation in vitro, and excystation vs eosin exclusion as determinants of
viability. Exp Parasitol 1979, 47(2):284-291.
Gillin FD, Reiner DS, Gault MJ, Douglas H, Das S, Wunderlich A, Sauch JF:
Encystation and expression of cyst antigens by Giardia lamblia in vitro. Science
Lujan HD, Mowatt MR, Byrd LG, Nash TE: Cholesterol starvation induces
differentiation of the intestinal parasite Giardia lamblia. Proc Natl Acad Sci U S A
The global burden of disease update: 2004 update.
O'Ryan M, Prado V, Pickering LK: A millennium update on pediatric diarrheal
illness in the developing world. Semin Pediatr Infect Dis 2005, 16(2):125-136.
The World Health Report 1996. Fighting Disease Fostering Development.
Cedillo-Rivera R, Leal YA, Yepez-Mulia L, Gomez-Delgado A, Ortega-Pierres G,
Tapia-Conyer R, Munoz O: Seroepidemiology of giardiasis in Mexico. Am J Trop
Med Hyg 2009, 80(1):6-10.
Miotti PG, Gilman RH, Santosham M, Ryder RW, Yolken RH: Age-related rate of
seropositivity of antibody to Giardia lamblia in four diverse populations. J Clin
Microbiol 1986, 24(6):972-975.
Abdel Fattah SM, Maklad KA, Gadallah MA: Age-related rate of seropositivity of
antibody to Giardia lamblia in different age groups in Cairo. J Egypt Soc
Parasitol 1991, 21(3):707-713.
Guerrant RL, Hughes JM, Lima NL, Crane J: Diarrhea in developed and developing
countries: magnitude, special settings, and etiologies. Rev Infect Dis 1990, 12
Thapar N, Sanderson IR: Diarrhoea in children: an interface between developing
and developed countries. Lancet 2004, 363(9409):641-653.
Ajjampur SS, Rajendran P, Ramani S, Banerjee I, Monica B, Sankaran P, Rosario V,
Arumugam R, Sarkar R, Ward H et al: Closing the diarrhoea diagnostic gap in
Indian children by the application of molecular techniques. J Med Microbiol 2008,
Ogunsanya TI, Rotimi VO, Adenuga A: A study of the aetiological agents of
childhood diarrhoea in Lagos, Nigeria. J Med Microbiol 1994, 40(1):10-14.
Haque R, Roy S, Kabir M, Stroup SE, Mondal D, Houpt ER: Giardia assemblage A
infection and diarrhea in Bangladesh. J Infect Dis 2005, 192(12):2171-2173.
Wongstitwilairoong B, Srijan A, Serichantalergs O, Fukuda CD, McDaniel P,
Bodhidatta L, Mason CJ: Intestinal parasitic infections among pre-school children
in Sangkhlaburi, Thailand. Am J Trop Med Hyg 2007, 76(2):345-350.
Dib HH, Lu SQ, Wen SF: Prevalence of Giardia lamblia with or without diarrhea
in South East, South East Asia and the Far East. Parasitol Res 2008, 103(2):239-
Saksirisampant W, Prownebon J, Kulkumthorn M, Yenthakam S, Janpla S,
Nuchprayoon S: Prevalence of intestinal parasitic infections among school
children in the central region of Thailand. J Med Assoc Thai 2006, 89(11):1928-
Saksirisampant W, Nuchprayoon S, Wiwanitkit V, Yenthakam S, Ampavasiri A:
Intestinal parasitic infestations among children in an orphanage in Pathum
Thani province. J Med Assoc Thai 2003, 86 Suppl 2:S263-270.
Savioli L, Smith H, Thompson A: Giardia and Cryptosporidium join the
'Neglected Diseases Initiative'. Trends Parasitol 2006, 22(5):203-208.
Traveler's diarrhea due to intestinal protozoa. Clin Infect Dis 2001,
Gautret P, Schlagenhauf P, Gaudart J, Castelli F, Brouqui P, von Sonnenburg F,
Loutan L, Parola P: Multicenter EuroTravNet/GeoSentinel study of travel-related
infectious diseases in Europe. Emerg Infect Dis 2009, 15(11):1783-1790.
Taylor DN, Houston R, Shlim DR, Bhaibulaya M, Ungar BL, Echeverria P: Etiology
of diarrhea among travelers and foreign residents in Nepal. JAMA 1988,
Year statistic Norwegian Surveillance System for Communicable Diseases
(http://wwwfhino/) 2008. Nygard K, Schimmer B, Sobstad O, Walde A, Tveit I, Langeland N, Hausken T,
Aavitsland P: A large community outbreak of waterborne giardiasis-delayed
detection in a non-endemic urban area. BMC Public Health 2006, 6:141.
Nygard K, Gondrosen B, Lund V: [Water-borne disease outbreaks in Norway].
Tidsskr Nor Laegeforen 2003, 123(23):3410-3413.
Ljungstrom I, Castor B: Immune response to Giardia lamblia in a water-borne
outbreak of giardiasis in Sweden. J Med Microbiol 1992, 36(5):347-352.
Karanis P, Kourenti C, Smith H: Waterborne transmission of protozoan parasites:
a worldwide review of outbreaks and lessons learnt. J Water Health 2007, 5(1):1-
Traub RJ, Monis PT, Robertson I, Irwin P, Mencke N, Thompson RC:
Epidemiological and molecular evidence supports the zoonotic transmission of
Giardia among humans and dogs living in the same community. Parasitology
2004, 128(Pt 3):253-262.
Birkhead G, Janoff EN, Vogt RL, Smith PD: Elevated levels of immunoglobulin A
to Giardia lamblia during a waterborne outbreak of gastroenteritis. J Clin
Microbiol 1989, 27(8):1707-1710.
Hopkins RS, Juranek DD: Acute giardiasis: an improved clinical case definition for
epidemiologic studies. Am J Epidemiol 1991, 133(4):402-407.
Robertson LJ, Gjerde B: Occurrence of parasites on fruits and vegetables in
Norway. J Food Prot 2001, 64(11):1793-1798.
Robertson LJ, Greig JD, Gjerde B, Fazil A: The potential for acquiring
cryptosporidiosis or giardiosis from consumption of mung bean sprouts in
Norway: a preliminary step-wise risk assessment. Int J Food Microbiol 2005,
Mintz ED, Hudson-Wragg M, Mshar P, Cartter ML, Hadler JL: Foodborne giardiasis
in a corporate office setting. J Infect Dis 1993, 167(1):250-253.
Mohammed Mahdy AK, Lim YA, Surin J, Wan KL, Al-Mekhlafi MS: Risk factors
for endemic giardiasis: highlighting the possible association of contaminated
water and food. Trans R Soc Trop Med Hyg 2008, 102(5):465-470.
Petersen LR, Cartter ML, Hadler JL: A food-borne outbreak of Giardia lamblia. J
Infect Dis 1988, 157(4):846-848.
Quick R, Paugh K, Addiss D, Kobayashi J, Baron R: Restaurant-associated
outbreak of giardiasis. J Infect Dis 1992, 166(3):673-676.
Stuart JM, Orr HJ, Warburton FG, Jeyakanth S, Pugh C, Morris I, Sarangi J, Nichols
G: Risk factors for sporadic giardiasis: a case-control study in southwestern
England. Emerg Infect Dis 2003, 9(2):229-233.
Gomez-Couso H, Mendez-Hermida F, Castro-Hermida JA, Ares-Mazas E: Giardia in
shellfish-farming areas: detection in mussels, river water and waste waters. Vet
Parasitol 2005, 133(1):13-18.
Schets FM, van den Berg HH, Engels GB, Lodder WJ, de Roda Husman AM:
Cryptosporidium and Giardia in commercial and non-commercial oysters
(Crassostrea gigas) and water from the Oosterschelde, The Netherlands. Int J
Food Microbiol 2007, 113(2):189-194.
Steketee RW, Reid S, Cheng T, Stoebig JS, Harrington RG, Davis JP: Recurrent
outbreaks of giardiasis in a child day care center, Wisconsin. Am J Public Health
Nunez FA, Lopez JL, de la Cruz AM, Finlay CM: [Risk factors for Giardia lamblia
infection in children in daycare centers in Havana, Cuba]. Cad Saude Publica
White KE, Hedberg CW, Edmonson LM, Jones DB, Osterholm MT, MacDonald KL:
An outbreak of giardiasis in a nursing home with evidence for multiple modes of
transmission. J Infect Dis 1989, 160(2):298-304.
Svenungsson B, Velicko I, Petersson I, De Jong B, Andersson Y, Lebbad M:
[Giardiasis as differential diagnosis in diarrhea outbreaks in child day centers.
Written hygienic guidelines and adequate testing can reduce the transmission].
Lakartidningen 2007, 104(7):500-503.
Katz DE, Heisey-Grove D, Beach M, Dicker RC, Matyas BT: Prolonged outbreak of
giardiasis with two modes of transmission. Epidemiol Infect 2006, 134(5):935-941.
Porter JD, Ragazzoni HP, Buchanon JD, Waskin HA, Juranek DD, Parkin WE:
Giardia transmission in a swimming pool. Am J Public Health 1988, 78(6):659-662.
Esfandiari A, Swartz J, Teklehaimanot S: Clustering of giardiosis among AIDS
patients in Los Angeles County. Cell Mol Biol (Noisy-le-grand) 1997, 43(7):1077-
Harris JR, Morton RS: Sexual transmission of intestinal parasites. Br J Vener Dis
Keystone JS, Keystone DL, Proctor EM: Intestinal parasitic infections in
homosexual men: prevalence, symptoms and factors in transmission. Can Med
Assoc J 1980, 123(6):512-514.
Pakianathan MR, McMillan A: Intestinal protozoa in homosexual men in
Edinburgh. Int J STD AIDS 1999, 10(12):780-784.
Peters CS, Sable R, Janda WM, Chittom AL, Kocka FE: Prevalence of enteric
parasites in homosexual patients attending an outpatient clinic. J Clin Microbiol
Naiman HL, Sekla L, Albritton WL: Giardiasis and other intestinal parasitic
infections in a Manitoba residential school for the mentally retarded. Can Med
Assoc J 1980, 122(2):185-188.
Buret AG, Mitchell K, Muench DG, Scott KG: Giardia lamblia disrupts tight
junctional ZO-1 and increases permeability in non-transformed human small
intestinal epithelial monolayers: effects of epidermal growth factor. Parasitology
2002, 125(Pt 1):11-19.
Troeger H, Epple HJ, Schneider T, Wahnschaffe U, Ullrich R, Burchard GD, Jelinek
T, Zeitz M, Fromm M, Schulzke JD: Effect of chronic Giardia lamblia infection on
epithelial transport and barrier function in human duodenum. Gut 2007,
Chin AC, Teoh DA, Scott KG, Meddings JB, Macnaughton WK, Buret AG: Strain-
dependent induction of enterocyte apoptosis by Giardia lamblia disrupts
epithelial barrier function in a caspase-3-dependent manner. Infect Immun 2002,
Scott KG, Yu LC, Buret AG: Role of CD8+ and CD4+ T lymphocytes in jejunal
mucosal injury during murine giardiasis. Infect Immun 2004, 72(6):3536-3542.
Tandon BN, Tandon RK, Satpathy BK, Shriniwas: Mechanism of malabsorption in
giardiasis: a study of bacterial flora and bile salt deconjugation in upper
jejunum. Gut 1977, 18(3):176-181.
Hanevik K, Hausken T, Morken MH, Strand EA, Morch K, Coll P, Helgeland L,
Langeland N: Persisting symptoms and duodenal inflammation related to Giardia
duodenalis infection. J Infect 2007, 55(6):524-530.
Oberhuber G, Kastner N, Stolte M: Giardiasis: a histologic analysis of 567 cases.
Scand J Gastroenterol 1997, 32(1):48-51.
Pathophysiology of enteric infections with Giardia duodenalius.
Parasite 2008, 15(3):261-265.
Nash TE, Herrington DA, Losonsky GA, Levine MM: Experimental human
infections with Giardia lamblia. J Infect Dis 1987, 156(6):974-984.
Paintlia AS, Descoteaux S, Spencer B, Chakraborti A, Ganguly NK, Mahajan RC,
Samuelson J: Giardia lamblia groups A and B among young adults in India. Clin
Infect Dis 1998, 26(1):190-191.
Homan WL, Mank TG: Human giardiasis: genotype linked differences in clinical
symptomatology. Int J Parasitol 2001, 31(8):822-826.
Read C, Walters J, Robertson ID, Thompson RC: Correlation between genotype of
Giardia duodenalis and diarrhoea. Int J Parasitol 2002, 32(2):229-231.
Aydin AF, Besirbellioglu BA, Avci IY, Tanyuksel M, Araz E, Pahsa A:
Classification of Giardia duodenalis parasites in Turkey into groups A and B
using restriction fragment length polymorphism. Diagn Microbiol Infect Dis 2004,
Almeida AA, Delgado ML, Soares SC, Castro AO, Moreira MJ, Mendonca CM,
Canada NB, Da Costa JM: Genotype analysis of Giardia isolated from
asymptomatic children in northern Portugal. J Eukaryot Microbiol 2006, 53 Suppl
Gelanew T, Lalle M, Hailu A, Pozio E, Caccio SM: Molecular characterization of
human isolates of Giardia duodenalis from Ethiopia. Acta Trop 2007, 102(2):92-
Sahagun J, Clavel A, Goni P, Seral C, Llorente MT, Castillo FJ, Capilla S, Arias A,
Gomez-Lus R: Correlation between the presence of symptoms and the Giardia
duodenalis genotype. Eur J Clin Microbiol Infect Dis 2008, 27(1):81-83.
Pelayo L, Nunez FA, Rojas L, Furuseth Hansen E, Gjerde B, Wilke H, Mulder B,
Robertson L: Giardia infections in Cuban children: the genotypes circulating in a
rural population. Ann Trop Med Parasitol 2008, 102(7):585-595.
Kohli A, Bushen OY, Pinkerton RC, Houpt E, Newman RD, Sears CL, Lima AA,
Guerrant RL: Giardia duodenalis assemblage, clinical presentation and markers
of intestinal inflammation in Brazilian children. Trans R Soc Trop Med Hyg 2008,
Ajjampur SS, Sankaran P, Kannan A, Sathyakumar K, Sarkar R, Gladstone BP, Kang
G: Giardia duodenalis assemblages associated with diarrhea in children in South
India identified by PCR-RFLP. Am J Trop Med Hyg 2009, 80(1):16-19.
Haque R, Mondal D, Karim A, Molla IH, Rahim A, Faruque AS, Ahmad N,
Kirkpatrick BD, Houpt E, Snider C et al: Prospective case-control study of the
association between common enteric protozoal parasites and diarrhea in
Bangladesh. Clin Infect Dis 2009, 48(9):1191-1197.
Roxstrom-Lindquist K, Palm D, Reiner D, Ringqvist E, Svard SG: Giardia
immunity--an update. Trends Parasitol 2006, 22(1):26-31.
Muller N, von Allmen N: Recent insights into the mucosal reactions associated
with Giardia lamblia infections. Int J Parasitol 2005, 35(13):1339-1347.
Immune response to Giardia duodenalis. Clin Microbiol Rev 2000,
13(1):35-54, table of contents.
Tellez A, Palm D, Weiland M, Aleman J, Winiecka-Krusnell J, Linder E, Svard S:
Secretory antibodies against Giardia intestinalis in lactating Nicaraguan women.
Parasite Immunol 2005, 27(5):163-169.
Istre GR, Dunlop TS, Gaspard GB, Hopkins RS: Waterborne giardiasis at a
mountain resort: evidence for acquired immunity. Am J Public Health 1984,
Oksenhendler E, Gerard L, Fieschi C, Malphettes M, Mouillot G, Jaussaud R, Viallard
JF, Gardembas M, Galicier L, Schleinitz N et al: Infections in 252 patients with
common variable immunodeficiency. Clin Infect Dis 2008, 46(10):1547-1554.
Surface antigenic variation in Giardia lamblia. Mol Microbiol 2002,
Bruderer T, Papanastasiou P, Castro R, Kohler P: Variant cysteine-rich surface
proteins of Giardia isolates from human and animal sources. Infect Immun 1993,
Escobedo AA, Nunez FA: Prevalence of intestinal parasites in Cuban acquired
immunodeficiency syndrome (AIDS) patients. Acta Trop 1999, 72(1):125-130.
Manatsathit S, Tansupasawasdikul S, Wanachiwanawin D, Setawarin S, Suwanagool
P, Prakasvejakit S, Leelakusolwong S, Eampokalap B, Kachintorn U: Causes of
chronic diarrhea in patients with AIDS in Thailand: a prospective clinical and
microbiological study. J Gastroenterol 1996, 31(4):533-537.
Brenchley JM, Price DA, Schacker TW, Asher TE, Silvestri G, Rao S, Kazzaz Z,
Bornstein E, Lambotte O, Altmann D et al: Microbial translocation is a cause of
systemic immune activation in chronic HIV infection. Nat Med 2006, 12(12):1365-
The experimental transmission of human intestinal protozoan
parasites. II. Giardia lamblia cysts given in capsules. Am J Hyg 1954, 59(2):209-
Lopez CE, Dykes AC, Juranek DD, Sinclair SP, Conn JM, Christie RW, Lippy EC,
Schultz MG, Mires MH: Waterborne giardiasis: a communitywide outbreak of
disease and a high rate of asymptomatic infection. Am J Epidemiol 1980,
Horman A, Korpela H, Sutinen J, Wedel H, Hanninen ML: Meta-analysis in
assessment of the prevalence and annual incidence of Giardia spp. and
Cryptosporidium spp. infections in humans in the Nordic countries. Int J
Parasitol 2004, 34(12):1337-1346.
Thompson WG, Longstreth GF, Drossman DA, Heaton KW, Irvine EJ, Muller-Lissner
SA: Functional bowel disorders and functional abdominal pain. Gut 1999, 45
Grazioli B, Matera G, Laratta C, Schipani G, Guarnieri G, Spiniello E, Imeneo M,
Amorosi A, Foca A, Luzza F: Giardia lamblia infection in patients with irritable
bowel syndrome and dyspepsia: a prospective study. World J Gastroenterol 2006,
Tropical malabsorption. Semin Gastrointest Dis 2002, 13(4):221-231.
Olivares JL, Fernandez R, Fleta J, Ruiz MY, Clavel A: Vitamin B12 and folic acid in
children with intestinal parasitic infection. J Am Coll Nutr 2002, 21(2):109-113.
Hjelt K, Paerregaard A, Krasilnikoff PA: Giardiasis: haematological status and the
absorption of vitamin B12 and folic acid. Acta Paediatr 1992, 81(1):29-34.
Wright SG, Tomkins AM, Ridley DS: Giardiasis: clinical and therapeutic aspects.
Gut 1977, 18(5):343-350.
Mahalanabis D, Simpson TW, Chakraborty ML, Ganguli C, Bhattacharjee AK,
Mukherjee KL: Malabsorption of water miscible vitamin A in children with
giardiasis and ascariasis. Am J Clin Nutr 1979, 32(2):313-318.
Pettoello Mantovani M, Guandalini S, Ecuba P, Corvino C, di Martino L: Lactose
malabsorption in children with symptomatic Giardia lamblia infection: feasibility
of yogurt supplementation. J Pediatr Gastroenterol Nutr 1989, 9(3):295-300.
Korman SH, Bar-Oz B, Mandelberg A, Matoth I: Giardiasis with protein-losing
enteropathy: diagnosis by fecal alpha 1-antitrypsin determination. J Pediatr
Gastroenterol Nutr 1990, 10(2):249-252.
Sullivan PB, Lunn PG, Northrop-Clewes CA, Farthing MJ: Parasitic infection of the
gut and protein-losing enteropathy. J Pediatr Gastroenterol Nutr 1992, 15(4):404-
Behera B, Mirdha BR, Makharia GK, Bhatnagar S, Dattagupta S, Samantaray JC:
Parasites in patients with malabsorption syndrome: a clinical study in children
and adults. Dig Dis Sci 2008, 53(3):672-679.
Gilman RH, Marquis GS, Miranda E, Vestegui M, Martinez H: Rapid reinfection by
Giardia lamblia after treatment in a hyperendemic Third World community.
Lancet 1988, 1(8581):343-345.
Giardiasis. Gastroenterol Clin North Am 1996, 25(3):493-515.
Farthing MJ, Mata L, Urrutia JJ, Kronmal RA: Natural history of Giardia infection
of infants and children in rural Guatemala and its impact on physical growth. Am
J Clin Nutr 1986, 43(3):395-405.
Cole TJ, Parkin JM: Infection and its effect on the growth of young children: a
comparison of The Gambia and Uganda. Trans R Soc Trop Med Hyg 1977,
Berkman DS, Lescano AG, Gilman RH, Lopez SL, Black MM: Effects of stunting,
diarrhoeal disease, and parasitic infection during infancy on cognition in late
childhood: a follow-up study. Lancet 2002, 359(9306):564-571.
Hollm-Delgado MG, Gilman RH, Bern C, Cabrera L, Sterling CR, Black RE,
Checkley W: Lack of an adverse effect of Giardia intestinalis infection on the
health of Peruvian children. Am J Epidemiol 2008, 168(6):647-655.
Prado MS, Cairncross S, Strina A, Barreto ML, Oliveira-Assis AM, Rego S:
Asymptomatic giardiasis and growth in young children; a longitudinal study in
Salvador, Brazil. Parasitology 2005, 131(Pt 1):51-56.
Pickering LK, Woodward WE, DuPont HL, Sullivan P: Occurrence of Giardia
lamblia in children in day care centers. J Pediatr 1984, 104(4):522-526.
Shaw RA, Stevens MB: The reactive arthritis of giardiasis. A case report. Jama
Letts M, Davidson D, Lalonde F: Synovitis secondary to giardiasis in children. Am
J Orthop 1998, 27(6):451-454.
Prieto-Lastra L, Perez-Pimiento A, Gonzalez-Sanchez LA, Iglesias-Cadarso A:
[Chronic urticaria and angioedema in Giardia lamblia infection]. Med Clin (Barc)
Gelfer S, Scharf J, Zonis S, Mertzbach D: [Acute uveitis associated with Giardia
lamblia infection]. Harefuah 1984, 107(3-4):75-76.
Di Prisco MC, Hagel I, Lynch NR, Jimenez JC, Rojas R, Gil M, Mata E: Association
between giardiasis and allergy. Ann Allergy Asthma Immunol 1998, 81(3):261-265.
Hanevik K, Dizdar V, Langeland N, Hausken T: Development of functional
gastrointestinal disorders after Giardia lamblia infection. BMC Gastroenterol
Utility of multiple-stool-specimen ova and parasite examinations
in a high-prevalence setting. J Clin Microbiol 1999, 37(8):2408-2411.
Goka AK, Rolston DD, Mathan VI, Farthing MJ: The relative merits of faecal and
duodenal juice microscopy in the diagnosis of giardiasis. Trans R Soc Trop Med
Hyg 1990, 84(1):66-67.
Garcia LS, Shimizu RY: Evaluation of nine immunoassay kits (enzyme
immunoassay and direct fluorescence) for detection of Giardia lamblia and
Cryptosporidium parvum in human fecal specimens. J Clin Microbiol 1997,
Nantavisai K, Mungthin M, Tan-ariya P, Rangsin R, Naaglor T, Leelayoova S:
Evaluation of the sensitivities of DNA extraction and PCR methods for detection
of Giardia duodenalis in stool specimens. J Clin Microbiol 2007, 45(2):581-583.
Zimmerman SK, Needham CA: Comparison of conventional stool concentration
and preserved-smear methods with Merifluor Cryptosporidium/Giardia Direct
Immunofluorescence Assay and ProSpecT Giardia EZ Microplate Assay for
detection of Giardia lamblia. J Clin Microbiol 1995, 33(7):1942-1943.
Garcia LS, Shimizu RY, Novak S, Carroll M, Chan F: Commercial assay for
detection of Giardia lamblia and Cryptosporidium parvum antigens in human
fecal specimens by rapid solid-phase qualitative immunochromatography. J Clin
Microbiol 2003, 41(1):209-212.
Hanson KL, Cartwright CP: Use of an enzyme immunoassay does not eliminate the
need to analyze multiple stool specimens for sensitive detection of Giardia
lamblia. J Clin Microbiol 2001, 39(2):474-477.
Johnston SP, Ballard MM, Beach MJ, Causer L, Wilkins PP: Evaluation of three
commercial assays for detection of Giardia and Cryptosporidium organisms in
fecal specimens. J Clin Microbiol 2003, 41(2):623-626.
Schuurman T, Lankamp P, van Belkum A, Kooistra-Smid M, van Zwet A:
Comparison of microscopy, real-time PCR and a rapid immunoassay for the
detection of Giardia lamblia in human stool specimens. Clin Microbiol Infect 2007,
Strand EA, Robertson LJ, Hanevik K, Alvsvag JO, Morch K, Langeland N:
Sensitivity of a Giardia antigen test in persistent giardiasis following an extensive
outbreak. Clin Microbiol Infect 2008, 14(11):1069-1071.
Miller KM, Sterling CR: Sensitivity of nested PCR in the detection of low numbers
of Giardia lamblia cysts. Appl Environ Microbiol 2007, 73(18):5949-5950.
Robertson LJ, Forberg T, Hermansen L, Gjerde BK, Langeland N: Molecular
characterisation of Giardia isolates from clinical infections following a
waterborne outbreak. J Infect 2007, 55(1):79-88.
Visvesvara GS, Smith PD, Healy GR, Brown WR: An immunofluorescence test to
detect serum antibodies to Giardia lamblia. Ann Intern Med 1980, 93(6):802-805.
Smith PD, Gillin FD, Brown WR, Nash TE: IgG antibody to Giardia lamblia
detected by enzyme-linked immunosorbent assay. Gastroenterology 1981,
Gilman RH, Brown KH, Visvesvara GS, Mondal G, Greenberg B, Sack RB, Brandt F,
Khan MU: Epidemiology and serology of Giardia lamblia in a developing
country: Bangladesh. Trans R Soc Trop Med Hyg 1985, 79(4):469-473.
Rojas L, Torres DR, Mediola BJ, Finlay CM: Detection of specific anti-giardia
serum antibody by an immunofluorescence test in children with clinical
giardiasis. Am J Trop Med Hyg 1989, 40(5):477-479.
Escobedo AA, Cimerman S: Giardiasis: a pharmacotherapy review. Expert Opin
Pharmacother 2007, 8(12):1885-1902.
Escobedo A, Almirall P, Robertson L, Franco R, Hanevik K, Mørch K, Cimerman S:
Giardiasis: the ever-present threat of a neglected disease. Infectious Disorders -
Drug Targets 2010. In press.
Robertson L HK, Escobedo AA, Mørch K, Langeland N.: Giardiasis - why do the
symptoms sometimes never stop? Trends Parasitol 2010, 26(2):75-82.
135. Rossignol JF: Cryptosporidium and Giardia: Treatment options and prospects
for new drugs. Exp Parasitol 2009.
Raether W, Hanel H: Nitroheterocyclic drugs with broad spectrum activity.
Parasitol Res 2003, 90 Supp 1:S19-39.
Jokipii L, Jokipii AM: Comparison of four dosage schedules in the treatment of
giardiasis with metronidazole. Infection 1978, 6(2):92-94.
Albonico M, Mathema P, Montresor A, Khakurel B, Reggi V, Pandey S, Savioli L:
Comparative study of the quality and efficacy of originator and generic
albendazole for mass treatment of soil-transmitted nematode infections in Nepal.
Trans R Soc Trop Med Hyg 2007, 101(5):454-460.
Nontasut P, Singhasivanon V, Prarinyanuparp V, Chiamratana B, Sanguankiat S,
Dekumyoy P, Setasuban P: Effect of single-dose albendazole and single-dose
mebendazole on Necator americanus. Southeast Asian J Trop Med Public Health
Sirivichayakul C, Pojjaroen-Anant C, Wisetsing P, Praevanit R, Chanthavanich P,
Limkittikul K: The effectiveness of 3, 5 or 7 days of albendazole for the treatment
of Trichuris trichiura infection. Ann Trop Med Parasitol 2003, 97(8):847-853.
Davila-Gutierrez CE, Vasquez C, Trujillo-Hernandez B, Huerta M: Nitazoxanide
compared with quinfamide and mebendazole in the treatment of helminthic
infections and intestinal protozoa in children. Am J Trop Med Hyg 2002, 66(3):251-
Juan JO, Lopez Chegne N, Gargala G, Favennec L: Comparative clinical studies of
nitazoxanide, albendazole and praziquantel in the treatment of ascariasis,
trichuriasis and hymenolepiasis in children from Peru. Trans R Soc Trop Med Hyg
Palermo AM, Reynoso AS, Lopez Nigro M, Carballo MA, Mudry MD: Teratogenic
evaluation of metronidazole and ornidazole using Drosophila melanogaster as an
experimental model. Birth Defects Res A Clin Mol Teratol 2004, 70(4):157-162.
Mudry MD, Martinez-Flores I, Palermo AM, Carballo MA, Egozcue J, Garcia Caldes
M: Embryolethality induced by metronidazole (MTZ) in Rattus norvegicus.
Teratog Carcinog Mutagen 2001, 21(3):197-205.
Tiboni GM, Marotta F, Castigliego AP: Teratogenic effects in mouse fetuses
subjected to the concurrent in utero exposure to miconazole and metronidazole.
Reprod Toxicol 2008, 26(3-4):254-261.
Gardner TB, Hill DR: Treatment of giardiasis. Clin Microbiol Rev 2001, 14(1):114-
Burtin P, Taddio A, Ariburnu O, Einarson TR, Koren G: Safety of metronidazole in
pregnancy: a meta-analysis. Am J Obstet Gynecol 1995, 172(2 Pt 1):525-529.
Diav-Citrin O, Shechtman S, Gotteiner T, Arnon J, Ornoy A: Pregnancy outcome
after gestational exposure to metronidazole: a prospective controlled cohort
study. Teratology 2001, 63(5):186-192.
Czeizel AE, Rockenbauer M: A population based case-control teratologic study of
oral metronidazole treatment during pregnancy. Br J Obstet Gynaecol 1998,
Cantu JM, Garcia-Cruz D: Midline facial defect as a teratogenic effect of
metronidazole. Birth Defects Orig Artic Ser 1982, 18(3 Pt A):85-88.
Larocque R, Casapia M, Gotuzzo E, MacLean JD, Soto JC, Rahme E, Gyorkos TW: A
double-blind randomized controlled trial of antenatal mebendazole to reduce low
birthweight in a hookworm-endemic area of Peru. Trop Med Int Health 2006,
Christian P, Khatry SK, West KP, Jr.: Antenatal anthelmintic treatment,
birthweight, and infant survival in rural Nepal. Lancet 2004, 364(9438):981-983.
Gyorkos TW, Larocque R, Casapia M, Gotuzzo E: Lack of risk of adverse birth
outcomes after deworming in pregnant women. Pediatr Infect Dis J 2006,
Ndyomugyenyi R, Kabatereine N, Olsen A, Magnussen P: Efficacy of ivermectin
and albendazole alone and in combination for treatment of soil-transmitted
helminths in pregnancy and adverse events: a randomized open label controlled
intervention trial in Masindi district, western Uganda. Am J Trop Med Hyg 2008,
de Silva NR, Sirisena JL, Gunasekera DP, Ismail MM, de Silva HJ: Effect of
mebendazole therapy during pregnancy on birth outcome. Lancet 1999,
Whittaker SG, Faustman EM: Effects of benzimidazole analogs on cultures of
differentiating rodent embryonic cells. Toxicol Appl Pharmacol 1992, 113(1):144-
Jackson D, Robson JM: The action of furazolidone on pregnancy. J Endocrinol
Cancel AM, Dillberger JE, Kelly CM, Bolte HF, Creasy DM, Sokal DC: A lifetime
cancer bioassay of quinacrine administered into the uterine horns of female rats.
Regul Toxicol Pharmacol 2009.
Kreutner AK, Del Bene VE, Amstey MS: Giardiasis in pregnancy. Am J Obstet
Gynecol 1981, 140(8):895-901.
Cacopardo B, Patamia I, Bonaccorso V, Di Paola O, Bonforte S, Brancati G:
[Synergic effect of albendazole plus metronidazole association in the treatment of
metronidazole-resistant giardiasis]. Clin Ter 1995, 146(12):761-767.
Nash TE, Ohl CA, Thomas E, Subramanian G, Keiser P, Moore TA: Treatment of
patients with refractory giardiasis. Clin Infect Dis 2001, 33(1):22-28.
Smith PD, Gillin FD, Spira WM, Nash TE: Chronic giardiasis: studies on drug
sensitivity, toxin production, and host immune response. Gastroenterology 1982,
Abboud P, Lemee V, Gargala G, Brasseur P, Ballet JJ, Borsa-Lebas F, Caron F,
Favennec L: Successful treatment of metronidazole- and albendazole-resistant
giardiasis with nitazoxanide in a patient with acquired immunodeficiency
syndrome. Clin Infect Dis 2001, 32(12):1792-1794.
Upcroft P, Upcroft JA: Drug targets and mechanisms of resistance in the
anaerobic protozoa. Clin Microbiol Rev 2001, 14(1):150-164.
Drug resistance in Giardia: clinical versus laboratory isolates. Drug
Resist Updat 1998, 1(3):166-168.
Townson SM, Upcroft JA, Upcroft P: Characterisation and purification of
pyruvate:ferredoxin oxidoreductase from Giardia duodenalis. Mol Biochem
Parasitol 1996, 79(2):183-193.
Muller J, Sterk M, Hemphill A, Muller N: Characterization of Giardia lamblia WB
C6 clones resistant to nitazoxanide and to metronidazole. J Antimicrob Chemother
Upcroft JA, Campbell RW, Upcroft P: Quinacrine-resistant Giardia duodenalis.
Parasitology 1996, 112 ( Pt 3):309-313.
Upcroft J, Mitchell R, Chen N, Upcroft P: Albendazole resistance in Giardia is
correlated with cytoskeletal changes but not with a mutation at amino acid 200 in
beta-tubulin. Microb Drug Resist 1996, 2(3):303-308.
Upcroft JA, Upcroft P: Drug susceptibility testing of anaerobic protozoa.
Antimicrob Agents Chemother 2001, 45(6):1810-1814.
Busatti HG, Santos JF, Gomes MA: The old and new therapeutic approaches to the
treatment of giardiasis: Where are we? Biologics 2009, 3:273-287.
Takeuchi T, Kobayashi S, Tanabe M, Fujiwara T: In vitro inhibition of Giardia
lamblia and Trichomonas vaginalis growth by bithionol, dichlorophene, and
hexachlorophene. Antimicrob Agents Chemother 1985, 27(1):65-70.
Gordts B, Hemelhof W, Asselman C, Butzler JP: In vitro susceptibilities of 25
Giardia lamblia isolates of human origin to six commonly used antiprotozoal
agents. Antimicrob Agents Chemother 1985, 28(3):378-380.
Weinbach EC, Costa JL, Wieder SC: Antidepressant drugs suppress growth of the
human pathogenic protozoan Giardia lamblia. Res Commun Chem Pathol
Pharmacol 1985, 47(1):145-148.
Farthing MJ, Inge PM: Antigiardial activity of the bile salt-like antibiotic sodium
fusidate. J Antimicrob Chemother 1986, 17(2):165-171.
Boreham PF, Phillips RE, Shepherd RW: The activity of drugs against Giardia
intestinalis in neonatal mice. J Antimicrob Chemother 1986, 18(3):393-398.
Crouch AA, Seow WK, Thong YH: Effect of twenty-three chemotherapeutic
agents on the adherence and growth of Giardia lamblia in vitro. Trans R Soc Trop
Med Hyg 1986, 80(6):893-896.
Youssef MY, Essa MM, Sadaka HA, Eissa MM, Rizk AM: Effect of ivermectin on
combined intestinal protozoal infection (giardiasis and cryptosporidiosis). J Egypt
Soc Parasitol 1996, 26(3):543-553.
O'Handley RM, Olson ME, McAllister TA, Morck DW, Jelinski M, Royan G, Cheng
KJ: Efficacy of fenbendazole for treatment of giardiasis in calves. Am J Vet Res
Sousa MC, Poiares-da-Silva J: The cytotoxic effects of ciprofloxacin in Giardia
lamblia trophozoites. Toxicol In Vitro 2001, 15(4-5):297-301.
Sousa MC, Poiares-Da-Silva J: Cytotoxicity induced by bismuth subcitrate in
Giardia lamblia trophozoites. Toxicology in Vitro 1999, 13(4-5):591-598.
Bharti N, Husain K, Garza MTG, Cruz-Vega DE, Castro-Garza J, Mata-Cardenas BD,
Naqvi F, Azam A: Synthesis and in vitro antiprotozoal activity of 5-
nitrothiophene-2-carboxaldehyde thiosemicarbazone derivatives. Bioorganic &
Medicinal Chemistry Letters 2002, 12(23):3475-3478.
Valdez J, Cedillo R, Hernandez-Campos A, Yepez L, Hernandez-Luis F, Navarrete-
Vazquez G, Tapia A, Cortes R, Hernandez M, Castillo R: Synthesis and antiparasitic
activity of 1H-benzimidazole derivatives. Bioorganic & Medicinal Chemistry
Letters 2002, 12(16):2221-2224.
Andrzejewska M, Yepez-Mulia L, Tapia A, Cedillo-Rivera R, Laudy AE, Starosciak
BJ, Kazimierczuk Z: Synthesis, and antiprotozoal and antibacterial activities of S-
substituted 4,6-dibromo- and 4,6-dichloro-2-mercaptobenzimidazoles. European
Journal of Pharmaceutical Sciences 2004, 21(2-3):323-329.
Andrzejewska M, Yepez-Mulia L, Cedillo-Rivera R, Tapia A, Vilpo L, Vilpo J,
Kazimierczuk Z: Synthesis, antiprotozoal and anticancer activity of substituted 2-
trifluoromethyl- and 2-pentafluoroethylbenzimidazoles. European Journal of
Medicinal Chemistry 2002, 37(12):973-978.
Jimenez-Cardoso E, Flores-Luna A, Angeles E, Martinez P, Lopez-Castanares R,
Castaneda-Hernandez G, Perez-Urizar J: In vitro antigiardial activity of IRE-6A
and IRE-7B, two ethyl-phenylcarbamate derivatives. Revista De Investigacion
Clinica 2003, 55(4):444-447.
Paget T, Maroulis S, Mitchell A, Edwards MR, Jarroll EL, Lloyd D: Menadione kills
trophozoltes and cysts of Giardia intestinalis. Microbiology-Sgm 2004, 150:1231-
Rayan P, Stenzel D, McDonnell PA: The effects of saturated fatty acids on Giardia
duodenalis trophozoites in vitro. Parasitology Research 2005, 97(3):191-200.
Mariante RM, Vancini RG, Melo AL, Benchimol M: Giardia lamblia: Evaluation of
the in vitro effects of nocodazole and colchicine on trophozoites. Experimental
Parasitology 2005, 110(1):62-72.
Chon SK, Kim NS: Evaluation of silymarin in the treatment on asymptomatic
Giardia infections in dogs. Parasitology Research 2005, 97(6):445-451.
Muller J, Ruhle G, Muller N, Rossignol JF, Hemphill A: In vitro effects of
thiazolides on Giardia lamblia WB clone C6 cultured axenically and in coculture
with Caco2 cells. Antimicrobial Agents and Chemotherapy 2006, 50(1):162-170.
Nash T, Rice WG: Efficacies of zinc-finger-active drugs against Giardia lamblia.
Antimicrobial Agents and Chemotherapy 1998, 42(6):1488-1492.
Crouch AA, Seow WK, Whitman LM, Thong YH: Sensitivity in vitro of Giardia
intestinalis to dyadic combinations of azithromycin, doxycycline, mefloquine,
tinidazole and furazolidone. Trans R Soc Trop Med Hyg 1990, 84(2):246-248.
Andrews BJ, Mylvaganam H, Yule A: Sensitivity of Trichomonas vaginalis,
Tritrichomonas foetus and Giardia intestinalis to bacitracin and its zinc salt in
vitro. Trans R Soc Trop Med Hyg 1994, 88(6):704-706.
Andrews BJ, Panitescu D, Jipa GH, Vasile-Bugarin AC, Vasiliu RP, Ronnevig JR:
Chemotherapy for giardiasis: randomized clinical trial of bacitracin, bacitracin
zinc, and a combination of bacitracin zinc with neomycin. Am J Trop Med Hyg
Escobedo AA, Nunez FA, Moreira I, Vega E, Pareja A, Almirall P: Comparison of
chloroquine, albendazole and tinidazole in the treatment of children with
giardiasis. Ann Trop Med Parasitol 2003, 97(4):367-371.
Baveja UK, Bhatia VN, Warhurst DC: Giardia lamblia: in-vitro sensitivity to some
chemotherapeutic agents. J Commun Dis 1998, 30(2):79-84.
Farthing MJ, Inge PM, Pearson RM: Effect of D-propranolol on growth and
motility of flagellate protozoa. J Antimicrob Chemother 1987, 20(4):519-522.
Popovic O, Jojic N, Milutinovic-Djuric S, Djurdjevic D, Milovic V: Propranolol for
metronidazole-resistant giardiasis. Ital J Gastroenterol 1991, 23(6):359.
Torres D HI, Palacios E: Efecto de un extracto alcoholico de propoleos sobre el
crecimiento de Giardia lamblia in vitro. REv Cubana Cienc Vet 1990, 21:15-20.
Nunez FA EA, Finlay CM: Eficacia de various esquemas de tratamiento para la
infection por Giardia lamblia ninos. Rev Panam Infectol 2004, 6:17-20.
Hernandez F, Hernandez D, Zamora Z, Diaz M, Ancheta O, Rodriguez S, Torres D:
Giardia duodenalis: effects of an ozonized sunflower oil product (Oleozon) on in
vitro trophozoites. Exp Parasitol 2009, 121(3):208-212.
Wickramanayake GB, Rubin AJ, Sproul OJ: Inactivation of Giardia lamblia cysts
with ozone. Appl Environ Microbiol 1984, 48(3):671-672.
Amoroto F, Gonzales et al: Eficacia del Aceite ozonizado (Oleozon) en el
tratamiento de la giardiasis. Ensayo clinico Fase III, aleatorizado, abierto y
controlado. Rev Cubana Farmacol 2002, 36 (Suppl.):173-175.
Sabchareon A, Chongsuphajaisiddhi T, Attanath P: Treatment of giardiasis in
children with quinacrine, metronidazole, tinidazole and ornidazole. Southeast
Asian J Trop Med Public Health 1980, 11(2):280-284.
Rastegar-Lari A, Salek-Moghaddam A: Single-dose secnidazole versus 10-day
metronidazole therapy of giardiasis in Iranian children. J Trop Pediatr 1996,
Bassily S, Farid Z, el-Masry NA, Mikhail EM: Treatment of intestinal E. histolytica
and G. lamblia with metronidazole, tinidazole and ornidazole: a comparative
study. J Trop Med Hyg 1987, 90(1):9-12.
Nigam P, Kapoor KK, Kumar A, Sarkari NB, Gupta AK: Clinical profile of
giardiasis and comparison of its therapeutic response to metronidazole and
tinidazole. J Assoc Physicians India 1991, 39(8):613-615.
Jokipii AM, Jokipii L: Comparative evaluation of two dosages of tinidazole in the
treatment of giardiasis. Am J Trop Med Hyg 1978, 27(4):758-761.
Single-dose tinidazole for the treatment of giardiasis. Antimicrob
Agents Chemother 1985, 27(2):227-229.
Di Prisco MC, Jimenez JC, Rodriguez N, Costa V, Villamizar J, Silvera A, Carrillo M,
Lira C, Zerpa E, Lopez Y: Clinical trial with Secnidazole in a single dose in
Venezuelan children infected by Giardia intestinalis. Invest Clin 2000, 41(3):179-
Jokipii L, Jokipii AM: Treatment of giardiasis: comparative evaluation of
ornidazole and tinidazole as a single oral dose. Gastroenterology 1982, 83(2):399-
Ozbilgin A, Ertan P, Yereli K, Tamay AT, Kurt O, Degerli K, Balcioglu IC, Ok UZ,
Onag A: Giardiasis treatment in Turkish children with a single dose of
ornidazole. Scand J Infect Dis 2002, 34(12):918-920.
Oren B, Schgurensky E, Ephros M, Tamir I, Raz R: Single-dose ornidazole versus
seven-day metronidazole therapy of giardiasis in Kibbutzim children in Israel.
Eur J Clin Microbiol Infect Dis 1991, 10(11):963-965.
Cimerman B, Camilo Coura L, JM CS, Gurvitz R, Rocha RS, Bandeira S, Cimerman
S, Katz N: Evaluation of Secnidazole Gel and Tinidazole Suspension in the
Treatment of Giardiasis in Children. Braz J Infect Dis 1997, 1(5):241-247.
Hall A, Nahar Q: Albendazole as a treatment for infections with Giardia
duodenalis in children in Bangladesh. Trans R Soc Trop Med Hyg 1993, 87(1):84-
Dutta AK, Phadke MA, Bagade AC, Joshi V, Gazder A, Biswas TK, Gill HH, Jagota
SC: A randomised multicentre study to compare the safety and efficacy of
albendazole and metronidazole in the treatment of giardiasis in children. Indian J
Pediatr 1994, 61(6):689-693.
Baqai R, Zuberi SJ, Qureshi H, Ahmed W, Hafiz S: Efficacy of albendazole in
giardiasis. East Mediterr Health J 2001, 7(4-5):787-790.
Yereli K, Balcioglu IC, Ertan P, Limoncu E, Onag A: Albendazole as an alternative
therapeutic agent for childhood giardiasis in Turkey. Clin Microbiol Infect 2004,
Karabay O, Tamer A, Gunduz H, Kayas D, Arinc H, Celebi H: Albendazole versus
metronidazole treatment of adult giardiasis: An open randomized clinical study.
World J Gastroenterol 2004, 10(8):1215-1217.
Pengsaa K, Sirivichayakul C, Pojjaroen-anant C, Nimnual S, Wisetsing P:
Albendazole treatment for Giardia intestinalis infections in school children.
Southeast Asian J Trop Med Public Health 1999, 30(1):78-83.
Kollaritsch H, Jeschko E, Wiedermann G: Albendazole is highly effective against
cutaneous larva migrans but not against Giardia infection: results of an open
pilot trial in travellers returning from the tropics. Trans R Soc Trop Med Hyg
Escobedo AA, Canete R, Gonzalez ME, Pareja A, Cimerman S, Almirall P: A
randomized trial comparing mebendazole and secnidazole for the treatment of
giardiasis. Ann Trop Med Parasitol 2003, 97(5):499-504.
Pengsaa K, Limkittikul K, Pojjaroen-anant C, Lapphra K, Sirivichayakul C, Wisetsing
P, Nantha-aree P, Chanthavanich P: Single-dose therapy for giardiasis in school-age
children. Southeast Asian J Trop Med Public Health 2002, 33(4):711-717.
Bulut BU, Gulnar SB, Aysev D: Alternative treatment protocols in giardiasis: a
pilot study. Scand J Infect Dis 1996, 28(5):493-495.
Sadjjadi SM, Alborzi AW, Mostovfi H: Comparative clinical trial of mebendazole
and metronidazole in giardiasis of children. J Trop Pediatr 2001, 47(3):176-178.
al-Waili NS, al-Waili BH, Saloom KY: Therapeutic use of mebendazole in giardial
infections. Trans R Soc Trop Med Hyg 1988, 82(3):438.
Gascon J, Moreno A, Valls ME, Miro JM, Corachan M: Failure of mebendazole
treatment in Giardia lamblia infection. Trans R Soc Trop Med Hyg 1989,
di Martino L, Nocerino A, Mantovani MP: Mebendazole in giardial infections:
confirmation of the failure of this treatment. Trans R Soc Trop Med Hyg 1991,
Canete R, Escobedo AA, Gonzalez ME, Almirall P, Cantelar N: A randomized,
controlled, open-label trial of a single day of mebendazole versus a single dose of
tinidazole in the treatment of giardiasis in children. Curr Med Res Opin 2006,
Canete R, Escobedo AA, Gonzalez ME, Almirall P: Randomized clinical study of
five days apostrophe therapy with mebendazole compared to quinacrine in the
treatment of symptomatic giardiasis in children. World J Gastroenterol 2006,
Giardiasis in infancy and childhood: a prospective study of 160 cases
with comparison of quinacrine (Atabrine) and metronidazole (Flagyl). Am J Trop
Med Hyg 1979, 28(1):19-23.
Craft JC, Murphy T, Nelson JD: Furazolidone and quinacrine. Comparative study
of therapy for giardiasis in children. Am J Dis Child 1981, 135(2):164-166.
Levi GC, de Avila CA, Amato Neto V: Efficacy of various drugs for treatment of
giardiasis. A comparative study. Am J Trop Med Hyg 1977, 26(3):564-565.
Murphy TV, Nelson JD: Five v ten days' therapy with furazolidone for giardiasis.
Am J Dis Child 1983, 137(3):267-270.
236. Quiros-Buelna Furazolidone and metronidazole for treatment of giardiasis in
children. Scand J Gastroenterol Suppl 1989, 169:65-69.
Carter CH, Bayles A, Thompson PE: Effects of paromomycin sulfate in man
against Entamoeba histolytica and other intestinal protozoa. Am J Trop Med Hyg
Rodriguez-Garcia R, Rodriguez-Guzman LM, Cruz del Castillo AH: [Effectiveness
and safety of mebendazole compared to nitazoxanide in the treatment of Giardia
lamblia in children]. Rev Gastroenterol Mex 1999, 64(3):122-126.
Escobedo AA, Alvarez G, Gonzalez ME, Almirall P, Canete R, Cimerman S, Ruiz A,
Perez R: The treatment of giardiasis in children: single-dose tinidazole compared
with 3 days of nitazoxanide. Ann Trop Med Parasitol 2008, 102(3):199-207.
Rossignol JF, Ayoub A, Ayers MS: Treatment of diarrhea caused by Giardia
intestinalis and Entamoeba histolytica or E. dispar: a randomized, double-blind,
placebo-controlled study of nitazoxanide. J Infect Dis 2001, 184(3):381-384.
Ortiz JJ, Ayoub A, Gargala G, Chegne NL, Favennec L: Randomized clinical study
of nitazoxanide compared to metronidazole in the treatment of symptomatic
giardiasis in children from Northern Peru. Aliment Pharmacol Ther 2001,
Diaz E, Mondragon J, Ramirez E, Bernal R: Epidemiology and control of intestinal
parasites with nitazoxanide in children in Mexico. Am J Trop Med Hyg 2003,
Romero Cabello R, Guerrero LR, Munoz Garcia MR, Geyne Cruz A: Nitazoxanide
for the treatment of intestinal protozoan and helminthic infections in Mexico.
Trans R Soc Trop Med Hyg 1997, 91(6):701-703.
Robertson LJ, Hermansen L, Gjerde BK, Strand E, Alvsvag JO, Langeland N:
Application of genotyping during an extensive outbreak of waterborne giardiasis
in Bergen, Norway, during autumn and winter 2004. Appl Environ Microbiol 2006,
Wensaas KA, Langeland N, Rortveit G: Prevalence of recurring symptoms after
infection with Giardia lamblia in a non-endemic area. Scand J Prim Health Care
Hickie I, Davenport T, Wakefield D, Vollmer-Conna U, Cameron B, Vernon SD,
Reeves WC, Lloyd A: Post-infective and chronic fatigue syndromes precipitated
by viral and non-viral pathogens: prospective cohort study. Bmj 2006,
Halvorson HA, Schlett CD, Riddle MS: Postinfectious irritable bowel syndrome--a
meta-analysis. Am J Gastroenterol 2006, 101(8):1894-1899; quiz 1942.
Loge JH, Ekeberg O, Kaasa S: Fatigue in the general Norwegian population:
normative data and associations. J Psychosom Res 1998, 45(1 Spec No):53-65.
Vandvik PO, Lydersen S, Farup PG: Prevalence, comorbidity and impact of
irritable bowel syndrome in Norway. Scand J Gastroenterol 2006, 41(6):650-656.
Chalder T, Berelowitz G, Pawlikowska T, Watts L, Wessely S, Wright D, Wallace EP:
Development of a fatigue scale. J Psychosom Res 1993, 37(2):147-153.
Kane SV, Sandborn WJ, Rufo PA, Zholudev A, Boone J, Lyerly D, Camilleri M,
Hanauer SB: Fecal lactoferrin is a sensitive and specific marker in identifying
intestinal inflammation. Am J Gastroenterol 2003, 98(6):1309-1314.
Hamilton WT, Gallagher AM, Thomas JM, White PD: Risk markers for both
chronic fatigue and irritable bowel syndromes: a prospective case-control study
in primary care. Psychol Med 2009, 39(11):1913-1921.
Robertson LJ, Forberg T, Hermansen L, Gjerde BK, Langeland N: Demographics of
Giardia infections in Bergen, Norway, subsequent to a waterborne outbreak.
Scand J Infect Dis 2008, 40(2):189-192.
Wessely S, Chalder T, Hirsch S, Pawlikowska T, Wallace P, Wright DJ:
Postinfectious fatigue: prospective cohort study in primary care. Lancet 1995,
Spiller R, Garsed K: Postinfectious irritable bowel syndrome. Gastroenterology
XI. Paper I-IV
scientific report Mps1 promotes rapid centromere accumulationof Aurora BMaike S. van der Waal1*, Adrian T. Saurin1,2*, Martijn J.M. Vromans1, Mathijs Vleugel1,2,Claudia Wurzenberger3, Daniel W. Gerlich3w, Rene´ H. Medema4, Geert J.P.L. Kops1,2 & Susanne M.A. Lens1+1Department of Medical Oncology, 2Department of Molecular Cancer Research, University Medical Center Utrecht, Utrecht,The Netherlands, 3Institute of Biochemistry, Department of Biology, Swiss Federal Institute of Technology, Zu¨rich, Switzerland,and 4Division of Cell Biology, Netherlands Cancer Institute, Amsterdam, The Netherlands