Dynamics of circulating lymphocytes responding to human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of children's and travelers' diarrhea, with no licensed vaccine. This study aimed to explore the role of cellular immunity in protection against human ETEC infection. Nine volunteers were experimentally infected with ETEC, of which six developed diarrhea. Lymphocytes were collected from peripheral blood buffy coats, before and 3, 5, 6, 7, 10, and 28 days after dose ingestion, and 34 phenotypic and functional markers were examined by mass cytometry. Thirty-three cell populations, derived by manually merging 139 cell clusters from the X-shift unsupervised clustering algorithm, were analyzed. Initially, the diarrhea group responded with increased CD56dim CD16+ natural killer cells, dendritic cells tended to rise, and mucosal-associated invariant T cells decreased. On day 5-7, an increase in plasmablasts was paralleled by a consistent rise in CD4+ Th17-like effector memory and regulatory cell subsets. CD4+ Th17-like central memory cells peaked on day 10. All Th17-like cell populations showed increased expression of activation, gut-homing, and proliferation markers. Interestingly, in the nondiarrhea group, these same CD4+ Th17-like cell populations expanded earlier, normalizing around day 7. Earlier development of these CD4+ Th17-like cell populations in the nondiarrhea group may suggest a recall response and a potential role in controlling ETEC infections.

Inflammation, the kynurenines, and mucosal injury during human experimental enterotoxigenic Escherichia coli infection.

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrhea in children and travelers, especially in low- and middle-income countries. ETEC is a non-invasive gut pathogen colonizing the small intestinal wall before secreting diarrhea-inducing enterotoxins. We sought to investigate the impact of ETEC infection on local and systemic host defenses by examining plasma markers of inflammation and mucosal injury as well as kynurenine pathway metabolites. Plasma samples from 21 volunteers experimentally infected with ETEC were collected before and 1, 2, 3, and 7 days after ingesting the ETEC dose, and grouped based on the level of intestinal ETEC proliferation: 14 volunteers experienced substantial proliferation (SP) and 7 had low proliferation (LP). Plasma markers of inflammation, kynurenine pathway metabolites, and related cofactors (vitamins B2 and B6) were quantified using targeted mass spectrometry, whereas ELISA was used to quantify the mucosal injury markers, regenerating islet-derived protein 3A (Reg3a), and intestinal fatty acid-binding protein 2 (iFABP). We observed increased concentrations of plasma C-reactive protein (CRP), serum amyloid A (SAA), neopterin, kynurenine/tryptophan ratio (KTR), and Reg3a in the SP group following dose ingestion. Vitamin B6 forms, pyridoxal 5'-phosphate and pyridoxal, decreased over time in the SP group. CRP, SAA, and pyridoxic acid ratio correlated with ETEC proliferation levels. The changes following experimental ETEC infection indicate that ETEC, despite causing a non-invasive infection, induces systemic inflammation and mucosal injury when proliferating substantially, even in cases without diarrhea. It is conceivable that ETEC infections, especially when repeated, contribute to negative health impacts on children in ETEC endemic areas.

Prevalence and assemblage of Giardia duodenalis in a case-control study of children under 5 years from Jimma, Southwest Ethiopia.

Giardia duodenalis is a common pathogenic intestinal protozoan parasite with high prevalence in developing countries, especially among children. The distribution of giardia assemblages among humans and their clinical relevance remains controversial. This study aimed to determine the prevalence and assemblage of Giardia among children under 5 years of age in Jimma, Southwest Ethiopia. Employing a case-control design, 606 children presenting with diarrhea at Jimma university medical center and Serbo Health Center were enrolled from December 2016 to July 2018 along with 617 matched controls without diarrhea. Giardia was detected and typed using real-time PCR. Univariate and multivariate regression analysis was performed. The total prevalence of Giardia was 41% (501/1223) and did not differ significantly between cases and controls (40% vs 42%). Prevalence increased by age, with the highest prevalence seen in children aged ≥ 25 months. Children without diarrhea with a history of diarrhea during the last month were more likely to be Giardia positive compared to children with no history diarrhea (OR 1.8 and 95%CI; 1.1-2.9). Regardless of current diarrhea symptoms, assemblage B predominated with 89%, followed by assemblage A (8%) and mixed infection assemblage A and B (3%). We report a high prevalence of Giardia by PCR detection in Jimma, Ethiopia, with assemblage B being predominant. There was a similar distribution of Giardia assemblages between children with and without diarrhea. Increasing age was a risk factor for Giardia infection. Community-based prevention and control strategies need to be employed to decrease the risk of giardia infection.

Transition to PCR diagnosis of cryptosporidiosis and giardiasis in the Norwegian healthcare system: could the increase in reported cases be due to higher sensitivity or a change in the testing algorithm?

Cryptosporidiosis has been a notifiable infection in Norway since 2012 and giardiasis since 1977. For both infections, there has been an increase in notified cases. We used a questionnaire to explore whether this may be associated with implementation of molecular diagnostic methods. We received responses from 14 of 16 laboratories, most of which had implemented molecular diagnostic methods for these parasites. Algorithms for testing had also been modified, and several laboratories now test more faecal samples than previously for both parasites. The increase in reported cases may reflect not only higher sensitivity of diagnostic methods, but also more sample testing.

A site assessment tool for inpatient controlled human infection models for enteric disease pathogens.

The use of the controlled human infection model to facilitate product development and to advance understanding of host-pathogen interactions is of increasing interest. While administering a virulent (or infective) organism to a susceptible host necessitates an ongoing evaluation of safety and ethical considerations, a central theme in conducting these studies in a safe and ethical manner that yields actionable data is their conduct in facilities well-suited to address their unique attributes. To that end, we have developed a framework for evaluating potential sites in which to conduct inpatient enteric controlled human infection model to ensure consistency and increase the likelihood of success.

Giardiasis treatment: an update with a focus on refractory disease.

PURPOSE OF REVIEW: Giardiasis remains a common cause of diarrhea and intestinal enteropathy globally. Here we give an overview of clinical treatment studies and discuss potential mechanisms and molecular targets for in-vitro testing of drug resistance. RECENT FINDINGS: Giardia is a cause of disease both in diarrheal and nondiarrheal cases. The prevalence of treatment refractory giardiasis is increasing. Recent studies reveal 5-nitroimidazole refractory infection occurs in up to 50% of cases. Mechanisms of drug resistance are not known. Placebo controlled studies of drug efficacy, taking the self-limiting course of giardiasis into account, has not been reported. No randomized controlled trials of treatment of refractory infection have been performed the last 25 years. Based on the clinical studies reported, combination treatment with a 5-nitroimidazole and a benzimidazole is more effective than repeated courses of 5-nitroimidazole or monotherapies in refractory cases. Quinacrine is effective in refractory cases, but potentially severe side effects limit its use. SUMMARY: A combination of a 5-nitroimidazole and albendazole or mebendazole, and quinacrine monotherapy, are rational choices in nitroimidazole refractory infections, but randomized controlled studies are needed. Further research into more recent clinical isolates is necessary to uncover mechanisms for the increase in metronidazole refractory giardiasis observed during the last decade.

The Intriguing Interaction of Escherichia coli with the Host Environment and Innovative Strategies To Interfere with Colonization: a Summary of the 2019 E. coli and the Mucosal Immune System Meeting.

The third E. coli and the Mucosal Immune System (ECMIS) meeting was held at Ghent University in Belgium from 2 to 5 June 2019. It brought together an international group of scientists interested in mechanisms of colonization, host response, and vaccine development. ECMIS distinguishes itself from related meetings on these enteropathogens by providing a greater emphasis on animal health and disease and covering a broad range of pathotypes, including enterohemorrhagic, enteropathogenic, enterotoxigenic, enteroaggregative, and extraintestinal pathogenic Escherichia coli As it is well established that the genus Shigella represents a subspecies of E. coli, these organisms along with related enteroinvasive E. coli are also included. In addition, Tannerella forsythia, a periodontal pathogen, was presented as an example of a pathogen which uses its surface glycans for mucosal interaction. This review summarizes several highlights from the 2019 meeting and major advances to our understanding of the biology of these pathogens and their impact on the host.

Plasma levels of guanylins are reduced in patients with Crohn's disease.

Background: Guanylin (GN) and uroguanylin (UGN) are endogenous ligands for the intestinal receptor guanylate cyclase C (GC-C), an important regulator of intestinal fluid homeostasis. Gene expression and protein levels of GN are suppressed in inflamed intestinal tissue from patients with inflammatory bowel disease (IBD), but knowledge about plasma levels of guanylins in these conditions is sparse. We aimed to investigate the fasting plasma levels of the prohormones proGN and proUGN in patients with Crohn's Disease (CD) and relate these to levels found in persons with other diarrheal conditions, as well as persons with normal bowel habits.Methods: Plasma from patients with CD, patients with Familial GUCY2C Diarrheal Disease (FGDS), diarrhea-predominant irritable bowel syndrome (IBS-D) and healthy controls (HC) was analyzed using ELISA assays.Results: Significantly lower fasting plasma levels of proguanylins were found in CD and FGDS patients, compared to HC. In CD patients, plasma proGN levels correlated negatively with Harvey Bradshaw Index and with number of stools/24 h.Conclusion: Our data indicate that diarrhea may be a determinant for levels of proGN in plasma, and should be further explored in studies of different diarrheal disorders.

Prolonged Duodenal Mucosal Lymphocyte Alterations in Patients With and Without Postinfectious Functional Gastrointestinal Disorders After Giardia Infection.

BACKGROUND: Persisting low-grade inflammation is suggested to play a role in postinfectious functional gastrointestinal disorders (PI-FGIDs). The present study examined alterations in duodenal mucosal lymphocytes during and after Giardia gastroenteritis in patients who did, or did not, develop PI-FGIDs. METHODS: Duodenal mucosal intraepithelial lymphocytes (IELs) and lamina propria CD3, CD4, CD8, and CD20 lymphocytes were quantified in 28 patients with chronic giardiasis (CG), 66 patients with persistent abdominal symptoms after acute Giardia infection (PI-FGID), 19 recovered controls (RCs), and 16 healthy volunteers (HCs). Associations with illness duration, abdominal symptoms, and histology grade were assessed. RESULTS: Duodenal CD4 IELs were significantly elevated in CG, then decreased, followed by an upward trend after 1 year in both the PI-FGID and RC groups. Duodenal lamina propria crypt CD4 T cells were decreased in CG, and stayed low for about 14 months before normalizing in both the PI-FGID and RC groups. Lamina propria CD20 cells were persistently elevated in all 3 Giardia-exposed groups. Biopsies with microscopic inflammation showed increased lamina propria CD20 levels. CONCLUSIONS: Duodenal mucosal lymphocyte alterations were prolonged after Giardia infection, but similar in patients who developed PI-FGID and recovered asymptomatic controls.

Identification of Conserved Candidate Vaccine Antigens in the Surface Proteome of Giardia lamblia.

Giardia lamblia, one of the most common protozoal infections of the human intestine, is an important worldwide cause of diarrheal disease, malabsorption, malnutrition, delayed cognitive development in children, and protracted postinfectious syndromes. Despite its medical importance, no human vaccine is available against giardiasis. A crude veterinary vaccine has been developed, and experimental vaccines based on expression of multiple variant-specific surface proteins have been reported, but poorly defined vaccine components and excessive antigen variability are problematic for pharmaceutical vaccine production. To expand the repertoire of antigen candidates for vaccines, we reasoned that surface proteins may provide an enriched source of such antigens since key host effectors, such as secretory IgA, can directly bind to such antigens in the intestinal lumen and interfere with epithelial attachment. Here, we have applied a proteomics approach to identify 23 novel surface antigens of G. lamblia that show >90% amino acid sequence identity between the two human-pathogenic genetic assemblages (A and B) of the parasite. Surface localization of a representative subset of these proteins was confirmed by immunostaining. Four selected proteins, uridine phosphorylase-like protein-1, protein 21.1 (GL50803_27925), α1-giardin, and α11-giardin, were subsequently produced in recombinant form and shown to be immunogenic in mice and G. lamblia-infected humans and confer protection against G. lamblia infection upon intranasal immunization in rodent models of giardiasis. These results demonstrate that identification of conserved surface antigens provides a powerful approach for overcoming a key rate-limiting step in the design and construction of an effective vaccine against giardiasis.

Prolonged and persistent diarrhoea is not restricted to children with acute malnutrition: an observational study in Ethiopia.

OBJECTIVES: To assess the prevalence of prolonged and persistent diarrhoea, to estimate their co-occurrence with acute malnutrition and association with demographic and clinical factors. METHODS: Case-control study where cases were children under 5 years of age with diarrhoea and controls were children without diarrhoea, frequency-matched weekly by age and district of residency. Controls for cases 0-11 months were recruited from vaccination rooms, and controls for cases 12-59 months were recruited by house visits using random locations in the catchment area of the study sites. Data were analysed by mixed model logistic regression. RESULTS: We enrolled 1134 cases and 946 controls. Among the cases, 967 (85%) had acute diarrhoea (AD), 129 (11%) had ProD and 36 (3.2%) had PD. More cases had acute malnutrition at enrolment (17% vs. 4%, P < 0.0001) and more were born prematurely (5.7% vs. 1.8%, P < 0.0001) than controls. About 75% of ProPD cases did not have acute malnutrition. Cases with AD and ProPD had different symptomatology, even beyond illness duration. CONCLUSIONS: ProPD is common among children presenting with diarrhoea and is not confined to children with acute malnutrition. There is an urgent need for studies assessing causes of ProPD with and without acute malnutrition to develop treatment guidelines for these conditions.

OBJECTIFS: Evaluer la prévalence des diarrhées prolongées et persistantes, estimer leur co-occurrence avec la malnutrition aiguë et leur association avec des facteurs démographiques et cliniques. MÉTHODES: Etude cas-témoins portant sur des enfants de moins de 5 ans souffrant de diarrhée et sur des témoins, des enfants sans diarrhée, appariées toutes les semaines, en fonction de l'âge et du district de résidence. Les témoins pour les cas de 0 à 11 mois ont été recrutés dans les salles de vaccination et les témoins pour les cas de 12 à 59 mois ont été recrutés au cours de visites à domicile en utilisant des endroits aléatoires dans la zone de recrutement des sites d'étude. Les données ont été analysées par la régression logistique de modèle mixte. RÉSULTATS: Nous avons inscrit 1134 cas et 946 témoins. Parmi les cas, 967 (85%) avaient une diarrhée aiguë (DA), 129 (11%) étaient atteints de diarrhées prolongée (ProD) et 36 (3,2%) de diarrhées persistante (DP). La malnutrition aiguë au moment de l'inscription était plus fréquente (17% contre 4%, P < 0,0001) et davantage étaient nés prématurément (5,7% contre 1,8%, P < 0,0001) par rapport aux témoins. 75% des cas de ProPD ne souffraient pas de malnutrition aiguë. Les cas de DA et de ProPD avaient une symptomatologie différente, même au-delà de la durée de la maladie. CONCLUSIONS: La ProPD est fréquente chez les enfants présentant une diarrhée et ne se limitait pas aux enfants souffrant de malnutrition aiguë. Il est urgent que des études évaluant les causes de ProPD avec et sans malnutrition aiguë développent des recommandations de traitement pour ces affections.

Prevalence of Irritable Bowel Syndrome and Chronic Fatigue 10 Years After Giardia Infection.

BACKGROUND & AIMS: Irritable bowel syndrome (IBS) is a complication that can follow gastrointestinal infection, but it is not clear if patients also develop chronic fatigue. We investigated the prevalence and odds ratio of IBS and chronic fatigue 10 years after an outbreak of Giardia lamblia, compared with a control cohort, and changes in prevalence over time. METHODS: We performed a prospective follow-up study of 1252 laboratory-confirmed cases of giardiasis (exposed), which developed in Bergen, Norway in 2004. Statistics Norway provided us with information from 2504 unexposed individuals from Bergen, matched by age and sex (controls). Questionnaires were mailed to participants 3, 6, and 10 years after the outbreak. Results from the 3- and 6-year follow-up analyses have been published previously. We report the 10-year data and changes in prevalence among time points, determined by logistic regression using generalized estimating equations. RESULTS: The prevalence of IBS 10 years after the outbreak was 43% (n = 248) among 576 exposed individuals and 14% (n = 94) among 685 controls (adjusted odds ratio for development of IBS in exposed individuals, 4.74; 95% CI, 3.61-6.23). At this time point, the prevalence of chronic fatigue was 26% (n = 153) among 587 exposed individuals and 11% (n = 73) among 692 controls (adjusted odds ratio, 3.01; 95% CI, 2.22-4.08). The prevalence of IBS among exposed persons did not change significantly from 6 years after infection (40%) to 10 years after infection (43%; adjusted odds ratio for the change 1.03; 95% CI, 0.87-1.22). However, the prevalence of chronic fatigue decreased from 31% at 6 years after infection to 26% at 10 years after infection (adjusted odds ratio for the change 0.74; 95% CI, 0.61-0.90). CONCLUSION: The prevalence of IBS did not change significantly from 6 years after an outbreak of Giardia lamblia infection in Norway to 10 years after. However, the prevalence of chronic fatigue decreased significantly from 6 to 10 years afterward. IBS and chronic fatigue were still associated with giardiasis 10 years after the outbreak.

Transient elevation of anti-transglutaminase and anti-endomysium antibodies in Giardia infection.

Markers of celiac disease (CeD) may be elevated in various conditions of intestinal inflammation or autoimmune disease. Recent reports argue that intestinal infection may induce development of CeD in susceptible individuals. Serum anti-tissue transglutaminase (tTG) and anti-endomysium antibodies (EMA) have been proposed in previous reports to be helpful in differentiating between giardiasis and CeD. In this report, we describe eight cases with elevated CeD serological markers and pathological duodenal histology during, or shortly after, Giardia infection. We present follow-up clinical and serological findings to determine which of these that were diagnosed with CeD. Serum levels of tTGand EMA did not discriminate well between patients where CeD was excluded, and those who were later diagnosed with CeD. The value of these serological CeD markers is discussed in relation to CeD diagnosis in cases with chronic or recent giardiasis.

Genetic and transcriptional analysis of inflammatory bowel disease-associated pathways in patients with GUCY2C-linked familial diarrhea.

OBJECTIVE: Activating mutations in the GUCY2C gene, which encodes the epithelial receptor guanylate cyclase C, cause diarrhea due to increased loss of sodium chloride to the intestinal lumen. Patients with familial GUCY2C diarrhea syndrome (FGDS) are predisposed to inflammatory bowel disease (IBD). We investigated whether genes in the guanylate cyclase C pathway are enriched for association with IBD and reversely whether genetic or transcriptional changes associated with IBD are found in FGDS patients. METHODS: (1) A set of 27 genes from the guanylate cyclase C pathway was tested for enrichment of association with IBD by Gene Set Enrichment Analysis, using genome-wide association summary statistics from 12,882 IBD patients and 21,770 controls. (2) We genotyped 163 known IBD risk loci and sequenced NOD2 in 22 patients with FGDS. Eight of them had concomitant Crohn's disease. (3) Global gene expression analysis was performed in ileal tissue from patients with FGDS, Crohn's disease and healthy individuals. RESULTS: The guanylate cyclase C gene set showed a significant enrichment of association in IBD genome-wide association data. Risk variants in NOD2 were found in 7/8 FGDS patients with concomitant Crohn's disease and in 2/14 FDGS patients without Crohn's disease. In ileal tissue, downregulation of metallothioneins characterized FGDS patients compared to healthy controls. CONCLUSIONS: Our results support a role of guanylate cyclase C signaling and disturbed electrolyte homeostasis in development of IBD. Furthermore, downregulation of metallothioneins in the ileal mucosa of FGDS patients may contribute to IBD development, possibly alongside effects from NOD2 risk variants.

Giardiasis: a diagnosis that should be considered regardless of the setting.

Although Giardia, the aetiological agent of giardiasis, is one of the most prevalent intestinal parasitic infections world-wide, for industrialised countries, it is mainly appreciated as an imported disease with the minimal local transmission. However, the current evidence challenges this perception; Giardia has relevance beyond the high prevalence areas. This infection may be asymptomatic or cause gastrointestinal complains and long-term sequelae, including irritable bowel syndrome, chronic fatigue and impaired child growth and cognitive development. Its detection and diagnosis present a challenge to physicians who may not be familiar with this infection. To improve interventions to control this parasitosis, it is necessary to maintain a high index of suspicion and remain vigilant in finding cases at risk for infection. A better understanding of the characteristics of populations importing infections alongside improved methods to reliably classify infections as imported or acquired locally will help to ensure early and accurate diagnosis. The evidence shows that public health problems like giardiasis are global issues that need to be addressed collectively by both high and low prevalence countries.

Longitudinal cohort study of serum antibody responses towards Giardia lamblia variant-specific surface proteins in a non-endemic area.

INTRODUCTION/AIMS: The long-term humoral immune response after a natural giardiasis infection is not well understood. The aim of this study was to evaluate longitudinal serum IgA and IgG/M responses towards conserved regions of two Giardia variant-specific surface proteins (VSP) and whether these responses differ between Giardia assemblages and durations of infection. METHODS: We recruited thirty Giardia-positive patients, mainly returning travellers, and eighteen healthy adults presumed to be Giardia unexposed. Blood samples were collected before treatment, and at 6 weeks, 6 months and 12 months after the infection cleared. We used a multiplex bead-based flow cytometry immunoassay to measure Giardia specific IgA and IgG/M responses targeting two recombinant antigens from G. lamblia VSP proteins 3 and 5 (VSP3 and VSP5). RESULTS: Serum levels of anti-VSP5 and anti-VSP3 IgA decreased rapidly to low levels after treatment but continued to be substantially higher than that of presumed unexposed controls even after 6 and 12 months. The IgG/M response decreased more gradually but remained significantly higher than presumed unexposed controls at all time points, except for anti-VSP3 at 12 months. There were no significant difference in responses for infections with assemblage A and assemblage B Giardia lamblia. Chronic infections (>8 weeks) were associated with a significantly lower anti-VSP5 IgG/M response. CONCLUSION: This study describes the kinetics of the humoral immune response against two Giardia VSP proteins over one year, and the considerable cross reactivity between the two human infective Giardia assemblages. Persons with chronic Giardia infection seem to have lower levels of VSP antibodies.

Comprehensive Analysis of Prevalence, Epidemiologic Characteristics, and Clinical Characteristics of Monoinfection and Coinfection in Diarrheal Diseases in Children in Tanzania.

The role of interactions between intestinal pathogens in diarrheal disease is uncertain. From August 2010 to July 2011, we collected stool samples from 723 children admitted with diarrhea (cases) to 3 major hospitals in Dar es Salaam, Tanzania, and from 564 nondiarrheic children (controls). We analyzed the samples for 17 pathogens and assessed interactions between coinfections in additive and multiplicative models. At least one pathogen was detected in 86.9% of the cases and 62.8%, of the controls. Prevalence of coinfections was 58.1% in cases and 40.4% in controls. Rotavirus, norovirus genogroup II, Cryptosporidium, and Shigella species/enteroinvasive Escherichia coli were significantly associated with diarrhea both as monoinfections and as coinfections. In the multiplicative interaction model, we found 2 significant positive interactions: rotavirus + Giardia (odds ratio (OR) = 23.91, 95% confidence interval (CI): 1.21, 470.14) and norovirus GII + enteroaggregative E. coli (OR = 3.06, 95% CI: 1.17, 7.98). One significant negative interaction was found between norovirus GII + typical enteropathogenic E. coli (OR = 0.09, 95% CI: 0.01, 0.95). In multivariate analysis, risk factors for death were presence of blood in stool and severe dehydration. In conclusion, coinfections are frequent, and the pathogenicity of each organism appears to be enhanced by some coinfections and weakened by others. Severity of diarrhea was not affected by coinfections.

Giardia-specific cellular immune responses in post-giardiasis chronic fatigue syndrome.

BACKGROUND: The role of pathogen specific cellular immune responses against the eliciting pathogen in development of post-infectious chronic fatigue syndrome (PI-CFS) is not known and such studies are difficult to perform. The aim of this study was to evaluate specific anti-Giardia cellular immunity in cases that developed CFS after Giardia infection compared to cases that recovered well. Patients reporting chronic fatigue in a questionnaire study three years after a Giardia outbreak were clinically evaluated five years after the outbreak and grouped according to Fukuda criteria for CFS and idiopathic chronic fatigue. Giardia specific immune responses were evaluated in 39 of these patients by proliferation assay, T cell activation and cytokine release analysis. 20 Giardia exposed non-fatigued individuals and 10 healthy unexposed individuals were recruited as controls. RESULTS: Patients were clinically classified into CFS (n = 15), idiopathic chronic fatigue (n = 5), fatigue from other causes (n = 9) and recovered from fatigue (n = 10). There were statistically significant antigen specific differences between these Giardia exposed groups and unexposed controls. However, we did not find differences between the Giardia exposed fatigue classification groups with regard to CD4 T cell activation, proliferation or cytokine levels in 6 days cultured PBMCs. Interestingly, sCD40L was increased in patients with PI-CFS and other persons with fatigue after Giardia infection compared to the non-fatigued group, and correlated well with fatigue levels at the time of sampling. CONCLUSION: Our data show antigen specific cellular immune responses in the groups previously exposed to Giardia and increased sCD40L in fatigued patients.

Genetic variation in potential Giardia vaccine candidates cyst wall protein 2 and α1-giardin.

Giardia is a prevalent intestinal parasitic infection. The trophozoite structural protein a1-giardin (a1-g) and the cyst protein cyst wall protein 2 (CWP2) have shown promise as Giardia vaccine antigen candidates in murine models. The present study assesses the genetic diversity of a1-g and CWP2 between and within assemblages A and B in human clinical isolates. a1-g and CWP2 sequences were acquired from 15 Norwegian isolates by PCR amplification and 20 sequences from German cultured isolates by whole genome sequencing. Sequences were aligned to reference genomes from assemblage A2 and B to identify genetic variance. Genetic diversity was found between assemblage A and B reference sequences for both a1-g (90.8% nucleotide identity) and CWP2 (82.5% nucleotide identity). However, for a1-g, this translated into only 3 amino acid (aa) substitutions, while for CWP2 there were 41 aa substitutions, and also one aa deletion. Genetic diversity within assemblage B was larger; nucleotide identity 92.0% for a1-g and 94.3% for CWP2, than within assemblage A (nucleotide identity 99.0% for a1-g and 99.7% for CWP2). For CWP2, the diversity on both nucleotide and protein level was higher in the C-terminal end. Predicted antigenic epitopes were not affected for a1-g, but partially for CWP2. Despite genetic diversity in a1-g, we found aa sequence, characteristics, and antigenicity to be well preserved. CWP2 showed more aa variance and potential antigenic differences. Several CWP2 antigens might be necessary in a future Giardia vaccine to provide cross protection against both Giardia assemblages infecting humans.

A Cross-Sectional Study of the Prevalence of Gastrointestinal Symptoms and Pathology in Patients With Common Variable Immunodeficiency.

OBJECTIVES: The objective of this study was to study the prevalence of gastrointestinal (GI) symptoms and histopathology in patients with common variable immunodeficiency (CVID) as well as linking the findings to GI infections and markers of systemic immune activation. METHODS: In this cross-sectional study, we addressed GI symptoms in 103 patients and GI histopathological findings in 53 patients who underwent upper and lower endoscopic examination. The most frequent histopathological findings were linked to GI symptoms, B-cell phenotype, and markers of systemic immune activation (soluble (s)CD14, sCD25, and sCD163). Microarray analysis compared "celiac-like disease" in CVID to celiac disease. Screening for selected bacterial and viral infections in fecal samples and gut mucosal biopsies was performed. RESULTS: The main findings of this study were as follows: most common GI symptoms were bloating (34%), pain (30%), and diarrhea (26%). The most frequent histopathological findings were increased intraepithelial lymphocytes in the descending part of the duodenum, i.e., "celiac-like disease" (46% of patients), decreased numbers of plasma cells in GI tract mucosa (62%), and lymphoid hyperplasia (38%), none of which were associated with GI symptoms. Reduced plasma cells in GI mucosa were associated with B-cell phenotypic characteristics of CVID, and increased serum levels of sCD14 (P=0.025), sCD25 (P=0.01), and sCD163 (P=0.04). Microarray analyses distinguished between CVID patients with "celiac-like disease" and celiac disease. Positive tests for bacterial and viral infections were scarce both in fecal samples and gut mucosal biopsies, including PCR test for norovirus in biopsy specimens (0 positive tests). CONCLUSIONS: In conclusion, GI pathology is common in CVID, but does not necessarily cause symptoms. However, reduced plasma cells in GI mucosa were linked to systemic immune activation, "celiac-like disease" in CVID and true celiac disease appear to be different disease entities, as assessed by gene expression, and infections (including norovirus) are rarely a cause of the CVID enteropathy.