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In this case, we present an uncommon gastrointestinal infection in an immunocompromised patient that was solely diagnosed because of close collaboration between treating physicians and microbiologists. The patient is a 42-year-old male who underwent heart transplantation 5 years earlier. He presented with fever, weight loss, diarrhoea and tiredness. Initial investigations could not elucidate the aetiology of his symptoms. The patient was referred to the department of infectious diseases for further evaluation. Serology for Yersinia species was ordered and the result was suggestive for the possibility of a Yersinia species infection. Close collaboration between treating physicians and microbiologists followed and led to additional investigations, which revealed the diagnosis of a Yersinia pseudotuberculosis infection with extensive lesions in the gastrointestinal tract. Treatment with ciprofloxacin resulted in complete resolution of symptoms and healing of the gastrointestinal lesions. In conclusion, this case underlines the need for a multidisciplinary approach to complex patients of which symptoms have yet to be understood.

Mycoplasma genitalium is a sexually transmitted urogenital pathogen, and infection can result in serious symptoms. As M. genitalium is rather difficult to culture, infections are usually detected by molecular methods. Unfortunately, there has recently been a significant increase in resistance to azithromycin and moxifloxacin used for the treatment of M. genitalium infections. The increased resistance to (often empirically prescribed) M. genitalium treatments has resulted in frequent therapy failures and stresses the need for routine detection of antimicrobial resistance. In M. genitalium, antimicrobial resistance is almost always the result of DNA mutations and thus can easily be detected by molecular techniques. Regrettably, many microbiology laboratories do not use molecular techniques for the detection of bacterial antimicrobial resistance. As molecular tests are becoming available for M. genitalium, both for the establishment of infection and the detection of antimicrobial resistance, it is now more important to ensure that knowledge on the resistance mechanisms is transferred from the laboratory to the clinician. This review will provide a brief summary of the current status of antimicrobial resistance, its molecular mechanisms and the impact on the current status of M. genitalium treatment. (C) 2020 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

The implementation of polymerase chain reaction (PCR)-based diagnostics of intestinal protozoa has led to higher sensitivity and (subtype) specificity, more convenient sampling, and the possibility for high-throughput screening. PCR for routine detection of human intestinal protozoa in fecal samples is used by an increasing number of clinical laboratories. This paper discusses the recent developments in the diagnosis of intestinal protozoa, with an emphasis on PCR-based diagnostics. Although many reviews have described the technical aspects of PCR-based diagnostics, this review focuses on the clinical consequences that result from the shift from microscopic toward PCR-based diagnostics. Despite its undisputed superiority, the use of PCR comes with challenges that clinicians should be aware of.

In Japan and Australia, multidrug-resistant Mycoplasma genitalium infections are reported with increasing frequency. Although macrolide-resistant M. genitalium strains are common in Europe and North America, fluoroquinolone-resistant strains are still exceptional. However, an increase of multidrug-resistant M. genitalium in Europe and America is to be expected. The aim of this paper is to increase awareness on the rising number of multidrug-resistant M. genitalium strains. Here, one of the first cases of infection with a genetically proven multidrug-resistant M. genitalium strain in Europe is described. The patient was a native Dutch 47-year-old male patient with urethritis. Mycoplasma genitalium was detected, but treatment failed with azithromycin, doxycycline and moxifloxacin. A urogenital sample was used to determine the sequence of the 23S rRNA, gyrA, gyrB and parC genes. The sample contained an A2059G single nucleotide polymorphism (SNP) in the 23S rRNA gene and an SNP in the parC gene, resulting in an amino acid change of Ser83 -> Ile, explaining both azithromycin and moxifloxacin treatment failure. The SNPs associated with resistance were probably generated de novo, as a link with high-prevalence areas was not established. It is, thus, predictable that there is going to be an increase of multidrug-resistant M. genitalium strains in Europe. As treatment options for multidrug-resistant M. genitalium are limited, the treatment of M. genitalium infections needs to be carefully considered in order to limit the rapid increase of resistance to macrolides and fluoroquinolones.

Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg MacrolideR qPCR). Specificity of the qPCR was tested using urogenital samples that were tested positive for a range of other micro-organisms. M. genitalium was detected in 196/236 (83.1%) samples by the MgPa qPCR, versus 172/236 (72.9%) by the combined Mg23S qPCR/Sequencing, and 202/236 (85.6%) by the Mg MacrolideR qPCR. The Mg MacrolideR qPCR showed high concordance to the Mg23S qPCR/Sequencing assay (201 vs 202 could be genotyped, respectively) for the detection of the macrolide resistant mutations. None of the other urogenital pathogens were tested positive in the Mg MacrolideR qPCR, indicating specificity. The Mg MacrolideR qPCR is fast, sensitive, specific, and can easily be implemented in the routine diagnostics.

Dientamoeba fragilis(D. fragilis) is an intestinal parasite frequently detected in humans with abdominal pain and diarrhoea, but it is also commonly found in asymptomatic subjects. Hence its clinical relevance is often disputed. The introduction of polymerase chain reaction (PCR) is a versatile and sensitive diagnostic technique for the detection of intestinal parasites, and in some Western world countries PCR has almost completely replaced microscopic diagnostics. PCR has however resulted in an increase in the number ofD. fragilis-positive patients. The disputed pathogenic nature of this intestinal parasite and an apparent increase in the incidence of patients with positive PCR results have renewed the discussions between clinicians and microbiologists on how to deal with an infected patient. Moreover, treatment guidelines differ throughout the world which makes it difficult for clinicians to choose an optimal therapeutic regimen.AimTo summarize and discuss the current knowledge on the pathogenicity, best diagnostic approach, treatment and follow-up of children and adults infected withD. fragilis.

Patients infected by Mycoplasma genitalium are often treated empirically with the macrolide azithromycin. Macrolide resistance is becoming quite common; empirical treatment is compromised. Sequencing was initially used to detected azithromycin resistance-associated mutations. As this was laborious, qPCRs have been developed for their detection. In the present study, we describe a fast, sensitive, and specific qPCR assay that enables routine testing of M. genitalium and macrolide resistance-associated mutations in a single assay. M. genitalium positive clinical samples were used to compare (i) the commonly used MgPa assay for the detection of M. genitalium infections (MgPa qPCR), (ii) a combined 23S rRNA gene PCR/sequencing assay (Mg23S qPCR/Sequencing) to identify macrolide resistance-associated mutations, and (iii) our newly developed probe-based melt curve qPCR for simultaneous detection of M. genitalium and macrolide resistance-associated mutations (Macrolide-R/MG ELITe MGB Kit, Elitech Bothel USA in short Mg Macrolide

Objectives: Mycoplasma genitalium is a sexually transmitted pathogen, and infection with it is usually treated with macrolides. Unfortunately, emerging resistance to the macrolides has been associated with mutations in region V of the 23S rRNA gene. The aim of this retrospective study was to describe the incidence of macrolide resistance-associated mutations in M. genitalium from patients in the Netherlands. Methods: All urogenital samples obtained from patients visiting a general practitioner or hospital in the east of the Netherlands that tested positive using the routine M. genitalium real-time PCR (February 2012-November 2014) were included. Following a PCR targeting the 23S rRNA gene, sequencing of the PCR fragments was performed to identify possible macrolide resistance-associated mutations. Results: Forty-eight of the 153 samples (31.4%) included in this study contained a mutation in the 23S rRNA gene. This was reduced to 44/146 (30.1%) if only samples from unique patients were included. The predominant mutations identified were A2058G (16/44; 36.3%), A2059G (14/44; 31.8%) and a unique high proportion of A2058T (12/44; 27.3%). Treatment failure was observed in four patients initially infected with M. genitalium containing macrolide resistance-associated mutations, while in one of these patients subsequent treatment with moxifloxacin resulted in a microbiological cure. Conclusions: This study shows that macrolide resistance-associated mutations in M. genitalium occur with a high frequency. In contrast to studies from other regions, Dutch M. genitalium isolates carry the A2058T mutation at high frequency. Our data confirm the need for prospective detection of macrolide resistance-associated mutations prior to treating patients.

Metaplasia in Barrett’s esophagus (BE) is characterized by the transition of squamous epithelium into intestinal-type columnar epithelium. The immune response in BE shares many similarities with the response found in the gut, which is different from the response found in a normal-looking esophagus. Here, we investigated the role of the genes associated with the retinoic acid (RA) pathway in BE, as RA is important not only in shaping the gut’s immune response but also in the induction of metaplasia in vitro. mRNA was isolated from esophageal and duodenal biopsies from BE (n = 14), reflux esophagitis patients (n = 9), and controls (n = 12). cDNA was made and qPCR was performed. The expression of RALDH1, CYP26A1, MAdCAM1 were similar for both the BE and duodenum, but different when compared to squamous esophageal epithelium. BE was characterized by a higher expression of RALDH2 and FOXP3, compared to the duodenum. In BE, RALDH2 correlated with expression of the myeloid dendritic cell-specific genes: CD11c and CD1c. Also, RALDH2 expression correlated with RAR-β and FOXP3. Hierarchical clustering on the expression of multiple relevant genes demonstrated that BE, duodenum, and SQ tissues are clustered as three different groups. The differential expression of RA-specific genes and dendritic cell (DC)-subsets indicates that BE resembles duodenal tissue. The higher expression of RALDH2 and FOXP3 in BE points at a mechanism associated with a possible anti-inflammatory microenvironment. This aberrant immune regulation might contribute to the altered tissue and immune responses found in BE.

SUMMARY Whipple's disease is a rare infectious disease that can be fatal if left untreated. The disease is caused by infection with Tropheryma whipplei , a bacterium that may be more common than was initially assumed. Most patients present with nonspecific symptoms, and as routine cultivation of the bacterium is not feasible, it is difficult to diagnose this infection. On the other hand, due to the generic symptoms, infection with this bacterium is actually quite often in the differential diagnosis. The gold standard for diagnosis used to be periodic acid-Schiff (PAS) staining of duodenal biopsy specimens, but PAS staining has a poor specificity and sensitivity. The development of molecular techniques has resulted in more convenient methods for detecting T. whipplei infections, and this has greatly improved the diagnosis of this often missed infection. In addition, the molecular detection of T. whipplei has resulted in an increase in knowledge about its pathogenicity, and this review gives an overview of the new insights in epidemiology, pathogenesis, clinical manifestations, diagnosis, and treatment of Tropheryma whipplei infections.

Borrelia burgdorferi non-sensu lato (s.l.) strains occurred in the Netherlands. A multiplex OspA, FlaB, IGS real time PCR was compared to 16S rRNA/rDNA RT-qPCR with lower average Cycle threshold (Ct) and LOD on strain dilutions. Multiplexing increased sensitivity on CSF samples (n=74), distinguishing B. burgdorferi s.l. from non-s.l. strains.

Seven weeks after being kicked in the face by a cow, a 34-year-old male patient developed a posttraumatic mycobacterial lymphadenitis. A rapidly growing mycobacterial isolate cultured from a surgically drained lymphadenitis pus specimen was identified as Mycobacterium smegmatis by matrix-assisted laser desorption/ionization mass spectrometry and a combination of ITS-, hsp65-, and 16S rRNA-DNA sequence analysis, but as Mycobacterium fortuitum complex using the commercial INNO-LiPA Mycobacteria v2 line probe assay. As it is unclear if the misidentification of this strain is an exception, more research is required.

Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) identification (ID) of Candida yeast isolates is fast and reliable, but the optimal workflow is debated ([1][1], [2][2]). Two different steel target plates are available for the Microflex system (Bruker

Introduction The aim of this study was to determine the prevalence of Mycoplasma genitalium infection and the resistance to macrolides within a general population in Madrid in 2015. Methods We collected 359 urine samples from a general population with symptoms of sexually transmitted infections (STIs). All samples underwent a real-time PCR. For the detection of macrolide resistance, a 283 bp fragment of region V of the 23S rRNA gene of M. genitalium was amplified and sequenced. Results We found a prevalence of 3.34% of M. genitalium and a macrolide resistance rate of 20%. In males, the prevalence was 6.62% and in women 0.96%, being significantly higher in males. Conclusions The prevalence obtained shows that it is a pathogen to consider in our environment. These findings stress the need for routine testing of M. genitalium infections and would seem to suggest the advisability of resistance testing. Introducción El objetivo de este estudio fue determinar en 2015 en una población general de Madrid la prevalencia de infección por Mycoplasma genitalium (M. genitalium) y la resistencia a macrólidos. Mèc)todos Se recogieron 359 muestras de orina procedentes de una población general con síntomas de infección de transmisión sexual. A todas las muestras se les realizó una PCR a tiempo real. Para la detección de resistencias a macrólidos, se amplificó y secuenció un fragmento de 283 pb de la región v del gen 23S rRNA de M. genitalium. Resultados Se encontró una prevalencia de un 3,34% de M. genitalium y un 20% de resistencia a macrólidos. En varones la prevalencia fue del 6,62% y en mujeres del 0,96%, siendo significativamente superior en varones. Conclusión La prevalencia obtenida muestra que es un patógeno a considerar en nuestro entorno. Estos hallazgos hacen hincapièc) en la necesidad de realizar pruebas de rutina de infección por M. genitalium y argumentan que es recomendable realizar pruebas de resistencia. Sin financiación 1.685 JCR (2018) Q4, 72/89 Infectious Diseases, 106/133 Microbiology 0.348 SJR (2018) Q3, 82/127 Microbiology (medical) No data IDR 2018 UEM

BACKGROUND: Infection with Helicobacter pylori is associated with severe digestive diseases including chronic gastritis, peptic ulcer disease, and gastric cancer. Successful eradication of this common gastric pathogen in individual patients is known to prevent the occurrence of peptic ulcer disease and gastric cancer. DISCUSSION: With half of the world's population being infected with H, pylori and only few antibiotics result in an effective eradication, a successful antibiotic driven worldwide eradication program seems unlikely. In addition, H. pylori eradication is not always beneficial as it has been described that eradication can be associated with an increased frequency of other disorders such as pediatric asthma, inflammatory bowel diseases and Barrett's Esophagus. We have to accept that eradication of this infection is a two-edged sword that is both useful and harmful and we should therefore focus our H. pylori eradication policy toward selectively identify and destroy only the virulent strains. CONCLUSION: In order to still be able to effectively treat H. pylori infections in the future we need an alternative diagnostic/treatment algorithm. This would involve a shift towards more precise and enhanced disease predicting diagnosis that tries to identify patients with chance of developing severe diseases such as gastric cancer, rather than the current regime that is geared towards find and destroy all H. pylori.

Identification of Neisseria gonorrhoeae by the Bruker matrix-assisted laser desorption ionization−time of flight mass spectrometry (MALDI-TOF MS) system may be affected by “B consistency categorization.” A supplementary database of 17 N. gonorrhoeae main spectra was constructed. Twelve of 64 N. gonorrhoeae identifications were categorized with B consistency, which disappeared using the supplementary database. Database extension did not result in misidentification of Neisseria meningitidis .

In our hospital, mupirocin has increasingly been used for peri-operative decolonization of Staphylococcus aureus . The target for mupirocin is isoleucyl tRNA synthetase ( ileS ). High-level resistance to mupirocin is conferred by acquisition of plasmids expressing a distinct ileS gene ( ileS2 ). Here we evaluated the longitudinal trends in high-level mupirocin resistance in coagulase-negative staphylococci (CoNS) and linked this to the presence of ileS2 genes and mupirocin use. We assessed mupirocin resistance in CoNS bloodstream isolates from 2006 to 2011 tested by Phoenix automated testing (PAT). We evaluated the reliability of PAT results using Etest. PAT species determination was confirmed by MALDI-TOF (matrix-assisted laser desorption ionization–time of flight) mass spectrometry. We investigated the presence of ileS2 in the first 100 consecutive CoNS bloodstream isolates of each year using RT-PCR. Mupirocin use increased from 3.6 kg/year in 2006 to 13.3 kg/year in 2010 and correlated with the increase in the percentage of CoNS isolates carrying ileS2 (8% in 2006 to 22% in 2011; Spearman's rho, 0.137; P = 0.01). The sensitivity and specificity of PAT for detecting high-level mupirocin resistance were 0.97 and 0.97, respectively. ileS2 was detected in 81 of 82 phenotypically highly mupirocin-resistant strains and associated with resistance to ciprofloxacin, erythromycin, and clindamycin. In conclusion, we found a rapid increase in high-level resistance to mupirocin and resistance to other antibiotics in CoNS associated with an increase in mupirocin use. The associated resistance to other antibiotics may result in a reduction of oral antibiotic options for prolonged treatment of prosthetic infections with CoNS.

Objective. Esophageal cancer development is a sequence that starts with reflux esophagitis (RE), followed by Barrett's esophagitis (BE), dysplasia, and finally esophageal adenocarcinoma (EAC). Tumor necrosis factor (TNF) is a potent antineoplastic agent, hence DNA polymorphisms that reduce TNF levels potentially enhance the development of BE and EAC. The aim of the study was to determine the impact of TNF gene variation on the RE-BE-EAC cascade. Methods. DNA from 887 Caucasian participants (197 controls, 305 RE, 257 BE, 128 EAC) was tested for the gene polymorphism TNF-beta NcoI, and TNF production was determined by TNF-alpha specific immunohistochemistry on esophageal biopsies from these BE (n = 31) and EAC (n = 4) patients. Results. As compared with healthy controls, the TNF-beta NcoI A/A genotype was significantly more prevalent in BE (p = 0.04) and EAC patients (p = 0.02), but not in RE patients (p = 0.1). While TNF-alpha protein levels were invariably high in esophageal biopsies from EAC patients, most esophageal BE samples showed low to moderate TNF levels. Conclusions. Chronic inflammation, like in BE, markedly increase the risk of malignant transformation. In this study, the significantly higher frequency of the TNF-beta NcoI A/A genotype and the local TNF expression indicate that the pro-inflammatory cytokine TNF plays a role in the development of BE and EAC.

Reflux esophagitis (RO) and Barrett's esophagus (BO) can cause esophageal adenocarcinoma (OAC). The esophageal mucosa in the RO-BO-OAC cascade is chronically exposed to gastro-esophageal reflux. Epidermal growth factor (EGF) has an important role in the protection and repair of mucosal damage, and non-physiologic levels are associated with gastrointestinal tumors. The aim is to determine the functional effect of EGF gene polymorphisms on RO, BO and OAC development. A cohort of 871 unrelated Dutch Caucasians consisted of 198 healthy controls, 298 RO patients, 246 BO patients and 129 OAC patients. The frequency of the EGF-production-associated 5'UTR A+61G polymorphism was determined in these four groups. EGF immunohistochemistry was performed on BO biopsies. EGF expression was significantly lower in the G/G genotype compared with the A/G (P=0.008) and A/A (P=0.002) group. The G/G genotype was significantly more prevalent in RO (odds ratios (OR)=2.6; 95% confidence intervals (95% Cl): 1.3-5.2), BO (OR=3.0; 95% Cl: 1.5-6.2) and OAC (OR=4.1; 95% Cl: 1.8-9.7) than in controls. The G allele is associated with reduced EGF expression and increased risk for RO, BO and OAC development. This indicates that reduced mucosal protection resulting from genetically decreased EGF expression enhances esophageal tumor development. Journal of Human Genetics (2012) 57, 26-32; doi: 10.1038/jhg.2011.124; published online 1 December 2011

Background Regular colonoscopic surveillance for detection of dysplasia is recommended in longstanding inflammatory bowel disease (IBD), however, its sensitivity is disputed. Screening accuracy may increase by using a biomarker-based surveillance strategy. Methods A case-control study was performed to determine the prognostic value of DNA ploidy and p53 in IBD-related neoplasia. Cases with IBD-related colorectal cancer (CRC), detected in our surveillance program between 1985-2008, were selected and matched with two controls, for age, gender, disease characteristics, interval of follow-up, PSC, and previous surgery. Biopsies were assessed for DNA ploidy, p53, grade of inflammation and neoplasia. Progression to neoplasia was analyzed with Cox regression analysis, adjusting for potentially confounding variables. Results Adjusting for age, we found statistically significant Hazard ratios (HR) between development of CRC, and low grade dysplasia (HR5.5; 95% CI 2.6-11.5), abnormal DNA ploidy (DNA index (DI) 1.06-1.34, HR4.7; 95% CI 2.9-7.8 and DI>1.34, HR6.6; 95% CI 3.7-11.7) and p53 immunopositivity (HR3.0; 95% CI 1.9-4.7) over time. When adjusting for all confounders, abnormal DNA ploidy (DI 1.06-1.34, HR4.7; 95% CI 2.7-7.9 and DI>1.34, HR5.0; 95% CI 2.5-10.0) and p53 immunopositivity (HR1.7; 95% CI 1.0-3.1) remained statistically significant predictive of neoplasia. Conclusion In longstanding IBD, abnormal DNA ploidy and p53 immunopositivity are important risk factors of developing CRC. The yield of surveillance may potentially increase by adding these biomarkers to the routine assessment of biopsies.

Helicobacter pylori infections are thought to eventually lead to symptoms as a result of the long-lasting interactions between the bacterium and its host. Mechanisms that allow this bacterium to cause a life-long infection involve modulation of both the immune response and host cellular processes. Last year many novel findings that improve our knowledge on how H. pylori virulence factors interact with the host were reported, but because of space limitations we can only discuss a limited number of these studies. Among those are studies on the genetic variation of genes encoding outer membrane proteins and the mimicry of host antigens, factors that alter host-cell metabolism and factors that modulate the host’s immune response.

SUMMARYBarrett’s esophagus (BE) affects approximately 2% of the Western population and progresses to esophageal adenocarcinoma (EAC) in 0.5% of these patients each year. In BE, the stratified epithelium is replaced by an intestinal-type epithelium owing to chronic gastroduodenal reflux. Since self-renewal of intestinal crypts is driven by Notch signaling, we investigated whether this pathway was active in the proliferative crypts of BE. Immunohistochemistry confirmed the presence of an intact and activated Notch signaling pathway in metaplastic BE epithelium, but not in the normal human esophagus. Similar observations were made in two well-known human Barrett’s-derived EAC cell lines, OE33 and SKGT-5. We then sought to investigate the effects of Notch inhibition by systemic treatment with a γ-secretase inhibitor in a well-validated rodent model for BE. As we have shown previously in normal intestinal epithelium, Notch inhibition converted the proliferative Barrett’s epithelial cells into terminally differentiated goblet cells, whereas the squamous epithelium remained intact. These data imply that local application of γ-secretase inhibitors may present a simple therapeutic strategy for this increasingly common pre-malignant condition.

OBJECTIVES: Surveillance of patients with Barrett's esophagus (BE) aims at early detection and treatment of neoplastic changes, particularly esophageal adenocarcinoma (EAC). The histological evaluation of biopsy samples has its limitations, and biomarkers may improve early identification of BE patients at risk for progression to EAC. The aim of this study was to determine the predictive value of p53, Ki67, and aneuploidy as markers of neoplastic progression in BE. METHODS: A total of 27 BE patients with histologically proven progression to high-grade dysplasia (HGD) or EAC (cases) and 27 BE patients without progression (controls) were selected and matched for age, gender, and duration of follow-up. Dysplasia grade was determined in 212 biopsy samples obtained during surveillance endoscopies from cases and in 231 biopsy samples collected from controls. DNA ploidy status was determined by flow cytometry, whereas Ki67 and p53 expression was determined by immunohistochemistry. Hazard ratios (HRs) were calculated by Cox regression adjusted for potentially confounding variables. RESULTS: A univariate analysis showed that low-grade dysplasia (LGD) increased the risk of developing HGD/EAC compared with no dysplasia (HR 3.6; 95 % confidence interval (CI): 1.6-8.1). Aneuploidy (HR 3.5; 95 % CI: 1.3-9.4), strong Ki67 overexpression (HR 5.2; 95 % CI: 1.5-17.6), and moderate p53 overexpression (HR 6.5; 95 % CI: 2.5-17.1) were also associated with an increased risk of developing HGD/EAC, independent of the histological result. A multivariable analysis showed that in the presence of LGD, p53 overexpression, and to a lesser extent, Ki67 overexpression remained important risk factors for neoplastic progression, whereas aneuploidy was no longer predictive. CONCLUSIONS: p53 overexpression and, to a lesser extent, Ki67 overexpression could predict neoplastic progression in BE irrespective of the histological result. These markers may be useful for identifying patients at an increased risk of developing EAC, either alone or used as a panel.

The acidic gastric environment of mammals can be chronically colonized by pathogenic Helicobacter species, which use the nickel-dependent urea-degrading enzyme urease to confer acid resistance. Nickel availability in the mammal host is low, being mostly restricted to vegetarian dietary sources, and thus Helicobacter species colonizing carnivores may be subjected to episodes of nickel deficiency and associated acid sensitivity. The aim of this study was to investigate how these Helicobacter species have adapted to the nickel-restricted diet of their carnivorous host. Three carnivore-colonizing Helicobacter species express a second functional urea-degrading urease enzyme (UreA2B2), which functions as adaptation to nickel deficiency. UreA2B2 was not detected in seven other Helicobacter species, and is in Helicobacter mustelae only expressed in nickel-restricted conditions, and its expression was higher in iron-rich conditions. In contrast to the standard urease UreAB, UreA2B2 does not require activation by urease or hydrogenase accessory proteins, which mediate nickel incorporation into these enzymes. Activity of either UreAB or UreA2B2 urease allowed survival of a severe acid shock in the presence of urea, demonstrating a functional role for UreA2B2 in acid resistance. Pathogens often express colonization factors which are adapted to their host. The UreA2B2 urease could represent an example of pathogen adaptation to the specifics of the diet of their carnivorous host, rather than to the host itself.

Summary Background Chronic oesophageal inflammation and related oxidative stress are important in the pathogenesis of erosive oesophagitis (EO) and its malignant progression. Aim To study the effect of proton pump inhibitors (PPIs) on oesophageal cellular immune response and oxidative damage in EO patients. Methods Forty gastro-oesophageal reflux disease (GERD) patients [non-erosive reflux disease (NERD): 15, EO: 25] were included, after 7 days off antisuppressive drugs. EO patients were randomized to 20-mg rabeprazole once daily for either 4 or 8 weeks with baseline and follow-up endoscopy with distal oesophageal biopsies. T lymphocytes, macrophages and mast cells were quantified by immunohistochemistry. DNA adducts were measured by analysis of 8-oxo-deoxyguanosine levels. Results Erosive oesophagitis patients had more T lymphocytes and CD8+ T lymphocytes in squamous epithelium than NERD patients (P = 0.001, P = 0.002, respectively). Levels of DNA adducts between both groups were, however, not different (P = 0.99). Four- and eight-week rabeprazole treatment in EO patients resulted in a significant decrease in number of T lymphocytes and CD8+ T lymphocytes (all P

Unless detected at an early stage, esophageal adenocarcinoma (EAC) has a poor prognosis. Changes in cellular DNA content and expression levels of p53 and Ki67 in Barrett esophagus (BE) are associated with the development EAC and might serve as markers to identify EAC at an early stage. The aim of this study was to examine the presence of these three markers in various steps of neoplastic progression in BE towards EAC. Dysplasia was graded in 212 biopsy sets taken during follow-up upper endoscopy in 27 patients in whom ultimately high-grade dysplasia (HGD) or EAC was detected. Ploidy status was determined by flow cytometry, whereas Ki67 and p53 expression were determined by immunohistochemistry. Smoothing splines were used to analyze trends in time. We found an increasing fraction of Ki67 overexpression and, to a lesser extent, abnormal DNA content in biopsies closer to the time-point of detecting HGD/EAC in BE, suggesting the potential value of these biomarkers in identifying patients at increased risk of progression towards HGD/EAC. Accumulation of p53 was seen several years before development of HGD/EAC, and may therefore be an early marker in BE at a stage when dysplasia is not yet detected. A prospective follow-up study is needed to confirm these findings.

OBJECTIVES: Barrett's esophagus (BE) is a premalignant condition of the esophagus. It is a consequence of mucosal injury from chronic gastroesophageal reflux in which bile acids are an important toxic component. The farnesoid X receptor (FXR) is a nuclear receptor involved in the regulation of bile acid synthesis, transport, and absorption. FXR activation is also involved in the induction of the innate immune response. This suggests that FXR is involved in the pathogenesis and the inflammation seen in BE. METHODS: mRNA levels of FXR and the FXR-regulated genes, ileal bile acid-binding protein (IBABP), small heterodimer partner (SHP), and chemokines interleukin (IL)-8 and macrophage inflammatory protein 3 alpha (MIP3 alpha), were determined by real time-polymerase chain reaction (RT-PCR). Protein expression was determined by immunohistochemistry. RESULTS: FXR was not expressed in squamous epithelium of healthy subjects (N = 7), but was present in both squamous and columnar epithelium of BE patients. Compared to the squamous epithelium of BE patients, their columnar epithelium displayed a 2.3-fold (P = 0.02) increase in FXR mRNA. Also, IBABP (2.2-fold; P = 0.0029), SHP (2.7-fold; P = 0.007), IL-8 (1.5-fold; P = 0.04), and MIP3 alpha (1.7-fold; P = 0.019) transcription levels were increased. Exposure of esophageal cell line TE7 to deoxycholic acid (DCA) resulted in a similar induction. The induction was abolished by the FXR antagonist guggulsterone. CONCLUSIONS: Expression levels of the bile acid receptor FXR, the bile acid metabolism genes IBABP and SHP, and the chemokines IL-8 and MIP3 alpha are increased in Barrett's epithelium. The in vitro induction of FXR by DCA suggests that bile acids can actively induce the inflammatory response in BE by recruiting immune cells.

Only in a minority of patients with chronic hepatitis B (CHB) will treatment with interferon (IFN)-alpha or nucleoside analogues lead to sustained virological response. In vivo immunization (IVI) following virus suppression aims to optimize conditions for an effective immune response: following rapid and profound virus suppression by interferon-lamivudine combination therapy, lamivudine is withdrawn intermittently during continued interferon therapy. It is thought that withdrawal of lamivudine will lead to increased viral replication and increased antigen expression with subsequent immune stimulation. The aim of this prospective pilot study was to evaluate IVI as a therapeutic approach for CHB. Fourteen HBeAg-positive CHB patients were treated for 42 weeks with a combination of pegylated interferon-alpha 2b and lamivudine. After 12 weeks of combination therapy lamivudine was withdrawn intermittently for three consecutive periods of 4 weeks until it was permanently stopped on week 36. At the end of follow-up (week 52) all patients had remained HBeAg positive and the median viral load was similar to baseline. During the initial 12 weeks of treatment, there was a reduction of both the hepatitis B virus (HBV)-specific proliferation capacity of Th-cells and the frequencies of IFNgamma-producing cells. During the lamivudine interruption-cycle there was an inverse relation between the increase of HBV-DNA, and the decrease in proliferation capacity and frequency of IFN-gamma-producing cells. The intrahepatic fraction of CD8(+) T-cells increased during lamivudine withdrawal. In conclusion, IVI was able to transiently stimulate the HBV-specific immune responsiveness of T-cells, but the magnitude of the response was insufficient to cause a beneficial virological effect.

Background Treatment with interferon-α (IFN-α) leads to a response in only a minority of patients with chronic hepatitis B virus (HBV) infection, but the reasons for this are poorly understood. It was recently shown that in patients with chronic HBV infection, CD4+CD25+ regulatory T-cells (Treg) can suppress the HBV-specific immune response. We aimed to investigate whether in non-responders to IFN-α therapy Treg contribute to treatment failure by downregulating the HBV-specific T-cell responses. Patients and methods Fourteen patients positive for hepatitis B e antigen received pegylated IFN-α monotherapy for 52 weeks and were followed for 26 weeks. Results Compared with non-responders, responders displayed an increased HBV-specific T-helper cell proliferation. At the start of treatment there was no difference in the frequencies of CD4+CD25+ Treg between responders and non-responders. During therapy, the frequency of CD4+CD25+ Treg increased in non-responders, but not in responders. In contrast to the responders, the non-responders showed a significant increase in the frequency of interleukin-10-producing cells. Treg depletion resulted in increased proliferation capacity, but did not affect the frequency of interleukin-10-producing cells measured during the course of the treatment. Conclusion This study indicates that Treg might have an important role in HBV persistence during and after pegylated IFN-α therapy.

Background and study aims In patients with presumed Barrett esophagus we evaluated clinical risk factors that could predict the presence of intestinal metaplasia and dysplasia in biopsies of columnar-lined esophagus (CLE), independently of histological results. Patients and methods In 908 patients with CLE of length > or = 2 cm, data on age, sex, reflux symptoms, tobacco and alcohol use, medication use, and upper gastrointestinal endoscopy findings were prospectively collected. Multivariate logistic regression analysis was performed, and a model for predicting the histological results was developed. Results In 127/908 patients, biopsies of CLE did not contain intestinal metaplasia. Of the 781 patients with intestinal metaplasia, 663 patients (85%) had no dysplasia, and 118 (15%) had low grade dysplasia (LGD). The most important predictors for the presence of intestinal metaplasia were length of CLE, size of hiatal hernia, and male sex, while among those with intestinal metaplasia, age and male sex were most important for the presence of LGD. Multivariate combinations of these predictors yielded reliable models, which were able to discriminate intestinal metaplasia well from no intestinal metaplasia (area under receiver operating characteristic [ROC] curve 0.82), but only reasonably discriminated LGD from no dysplasia (area under ROC 0.65). Conclusions A simple model based on clinical findings can be used to predict the presence of intestinal metaplasia in biopsies from CLE. In contrast, predicting the presence of LGD versus no dysplasia in intestinal metaplasia is more difficult. Predictions from these models may aid decision making on whether a patient with CLE should have surveillance, in view of the known sampling error at endoscopy and interobserver variability at histology.

Helicobacter mustelae is a gastric pathogen of ferrets, where it causes disorders similar to those caused by Helicobacter pylori in humans. The H. mustelae ferret model therefore has potential for the in vivo study of Helicobacter pathogenesis in general. In this study a library of 500 individual H. mustelae mutants was generated using an in vitro random insertion mutagenesis technique. Mutants were subsequently tested for motility and adherence, and 43 of the 500 mutants tested were found to be nonmotile in a soft agar assay. Of these 43 mutants, seven were subsequently identified as deficient in their ability to adhere to AGS cells. Insertion had taken place in different positions in the H. mustelae genome, and included mutants in or near to genes involved in motility and urease activity (e.g. the chemotaxis gene cheV and the urease accessory gene ureH). The development of a mutant library for a natural animal model of Helicobacter infection provides the opportunity to study in vivo the role of candidate Helicobacter virulence genes.

Summary Background Risk factors for adenocarcinoma of the oesophagus (OAC) and gastric cardia (GCA) are not yet established. Aim To compare environmental risk factors between patients with OAC and GCA. Methods One-hundred and twenty-six patients with OAC, 43 with GCA and 57 with squamous cell carcinoma filled out a questionnaire with information on demographic and lifestyle characteristics, physical activity levels, family history, gastro-oesophageal reflux disease symptoms and medication use. Results OAC and GCA patients were similar with regard to male predominance and age, alcohol intake and smoking, use of fruits and vegetables, body posture and occupational activities (P > 0.05). GCA patients less often had heartburn compared with OAC patients [odds ratio (OR) 0.5, 95% confidence interval (CI) 0.2–0.96] and had these symptoms less frequently and for a shorter period (OR 0.3, CI 0.1–1.0 and OR 0.1, CI 0.03–0.6, respectively). Former and current aspirin use was lower among GCA patients than OAC patients (OR 0.2, CI 0.05–0.7 and OR 0.4, CI 0.1–0.9, respectively), whereas no difference in non-steroidal anti-inflammatory drug use was detected. Conclusion Although OAC and GCA share several environmental risk factors, OAC is more frequently associated with a history of gastro-oesophageal reflux disease, suggesting a more important role for gastro-oesophageal reflux in OAC compared with GCA.

Regulatory T cells (Treg) play a key role in the impaired immune response that is typical for a chronic Hepatitis B virus (HBV) infection. To gain more insight in the mechanism that is responsible for this impaired immune response, the effect of viral load reduction resulting from treatment with the nucleotide analogue adefovir dipivoxil on the percentages of Treg and HBV-specific T-cell responses was analyzed. Peripheral blood mononuclear cells (PBMC) of 12 patients were collected at baseline and during treatment. In parallel to the decline in viral load, we found a decline in circulating Treg, combined with an increase in HBV core antigen-specific IFN-γ production and proliferation. The production of IL10 did not decrease during therapy. In conclusion, adefovir induced viral load reduction results in a decline of circulating Treg together with a partial recovery of the immune response.

Urease activity is vital for gastric colonization by Helicobacter species, such as the animal pathogen Helicobacter felis. Here it is demonstrated that H. felis expresses two independent, and distinct urease systems. H. felis isolate CS1 expressed two proteins of 67 and 70 kDa reacting with antibodies to H. pylori urease. The 67-kDa protein was identified as the UreB urease subunit, whereas the N-terminal amino acid sequence of the 70-kDa protein displayed 58% identity with the UreB protein and was tentatively named UreB2. The gene encoding the UreB2 protein was identified and located in a gene cluster named ureA2B2. Inactivation of ureB led to complete absence of urease activity, whereas inactivation of ureB2 resulted in decreased urease activity. Although the exact function of the UreA2B2 system is still unknown, it is conceivable that UreA2B2 may contribute to pathogenesis of H. felis infection through a yet unknown mechanism.

BACKGROUND: Neoplastic progression of BE towards EAC is associated with increased expression of COX-2. Increased COX-2 expression and enzyme activity is linked to the COX-2 CA haplotype, which consists of two gene polymorphisms in the COX-2 promoter. AIM: To study the impact of COX-2 haplotypes on the risk of developing EAC in patients with different forms of gastroesophageal reflux disease including BE. METHODS: DNA was obtained from a total of 635 Dutch white patients comprised of 140 patients with EAC, 255 with BE, and 240 with reflux esophagitis. COX-2 haplotypes were based on the gene polymorphisms at -765C/G and -1195A/G, as determined by PCR-RFLP. RESULTS: The tested population contained 170 (14%) CA- (-765C and -1195A) haplotypes, 829 (65%) GA and 271 (21%) GG-haplotypes, and no GC-haplotypes. The haplotype distribution in patients with reflux esophagitis and BE was similar (CA 12%, GA 68%, GG 21%), but differed significantly from that in patients with EAC (CA 21%, GA 58%, GG 20%). Particularly, the CA-haplotype was more common (P < 0.001) in EAC patients. CA-carriership was associated with EAC (OR 2.8, 95% CI 1.3-6.2, P = 0.008), with homozygosity for the CA-allele being statistically most significantly associated (OR 6.1, 95% CI 1.6-24.2, P = 0.01). CONCLUSION: The COX-2 CA-haplotype is more frequently observed in patients with EAC than in patients with BE and reflux esophagitis. These data suggest a direct link between COX-2 activity and neoplastic progression in patients with BE and reflux esophagitis.

Background: Patients who have undergone esophagectomy with gastric tube reconstruction often have complaints of gastro-esophageal reflux. A subset of these patients will develop columnar epithelium in the remnant esophagus, which can be of the gastric or intestinal type (Barrett esophagus). Goals: To determine whether gastric-type mucosa (GM) in the esophagus is a precursor stage of intestinal metaplasia (IM). Study: The medical records of 613 patients having undergone esophagectomy were reviewed for the endoscopic presence of segments with columnar mucosa in the remnant esophagus. Of them, 45 patients underwent endoscopic follow-up at least 6 months after resection. The presence of IM in the remnant esophagus was determined histologically in archival biopsy samples. Intestinal characteristics were identified by immuno-histochemistry for CDX2, MUC2, and cytokeratins 7 and 20. CDX2 transcription was assessed by reverse transcription polymerase chain reaction. Results: In 18 of 45 patients (40%) GM was identified, and 7 of these patients also had foci of IM. CDX2 and MUC2 expression was observed in IM, and in 2 patients, CDX2 expression was also observed in gastric-type glands at a distance from intestinal glands. CDX2 transcription was identified in 2 patients without IM. Conclusions: In the majority of patients after esophageal resection, expression of CDX2 and MUC2 in the remnant esophagus was only detectable in IM, but CDX2 was also observed in 4 cases with only GM. This could indicate that induction of formation of GM and IM may share a common pathway, eventually leading to the development of specialized intestinal epithelium.

UNLABELLED Chronicity of hepatitis B virus (HBV) infection is characterized by a weak immune response to the virus. CD4+CD25+ regulatory T cells (Treg) are present in increased numbers in the peripheral blood of chronic HBV patients, and these Treg are capable of suppressing the HBV-specific immune response. The aim of this study was to abrogate Treg-mediated suppression of the HBV-specific immune response. Therefore, Treg and a Treg-depleted cell fraction were isolated from peripheral blood of chronic HBV patients. Subsequently, the suppressive effect of Treg on the response to HBV core antigen (HBcAg) and tetanus toxin was compared, and the effect of exogenous tumor necrosis factor alpha (TNF-alpha), interleukin-1-beta (IL-1beta), or neutralizing antibodies against interleukin-10 (IL-10) or transforming growth factor beta (TGF-beta) on Treg-mediated suppression was determined. The results show that Treg of chronic HBV patients had a more potent suppressive effect on the response to HBcAg compared with the response to tetanus toxin. Neutralization of IL-10 and TGF-beta or exogenous IL-1beta had no effect on Treg-mediated suppression of the anti-HBcAg response, whereas exogenous TNF-alpha partially abrogated Treg-mediated suppression. Preincubation of Treg with TNF-alpha demonstrated that TNF-alpha had a direct effect on the Treg. No difference was observed in the type II TNF receptor expression by Treg from chronic HBV patients and healthy controls. CONCLUSION Treg-mediated suppression of the anti-HBV response can be reduced by exogenous TNF-alpha. Because chronic HBV patients are known to produce less TNF-alpha, these data implicate an important role for TNF-alpha in the impaired antiviral response in chronic HBV.

Patients with Barrett's esophagus (BE) are at risk of developing esophageal adenocarcinoma, which is usually preceded by dysplastic changes of the metaplastic mucosa. The aim of this study was to increase the understanding of the development of dysplastic lesions in BE through the identification of genes that are differentially transcribed in these tissue types.Paired biopsy samples from non-dysplastic BE, and high-grade dysplasia from a single patient were used for histological evaluation and gene expression profile analysis. In addition, relative mRNA levels of differentially expressed genes were tested to validate the association with the presence or absence of dysplasia by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) (58 biopsy samples containing squamous epithelium, non-dysplastic BE, high-grade dysplasia, or adenocarcinoma from 23 unrelated patients) and immunohistochemistry (9 sets of paired non-dysplastic/high-grade dysplasiac samples from 9 unrelated patients).Microarray results from high-grade dysplasia showed 866 genes with a2-fold difference in mRNA levels compared with non-dysplastic BE. Subsequent comparison of mRNA levels of the 22 genes with a10-fold difference in 76 unrelated biopsies showed that only two of these genes, i.e. calgranulin A (S100A8; p=0.017) and calgranulin B (S100A9; p=0.022), were consistently up-regulated in high-grade dysplasia, as were protein levels for calgranulin A and B.This is the first report of an association between the calprotectin complex, which is involved in chemotaxis of neutrophils, and the progression towards high-grade dysplasia in BE. It remains to be established whether differentially expressed proteins in biopsies form BE can be used to facilitate the diagnosis of advanced dysplasia in BE.

Because the molecular mechanism of amoxicillin resistance in Helicobacter pylori seems to be partially explained by several mutational changes in the pbp1A gene, the aim of the present study was to evaluate the gene expression pattern in response to amoxicillin in the Amx(R) Hardenberg strain using RNA arbitrarily primed PCR (RAP-PCR). In the experiments, c. 100 differentially expressed RAP-PCR products were identified using five arbitrary primers. The cDNAs that presented the highest levels of induction or repression were cloned and sequenced, and the sequences were compared with those present in databases using the blast search algorithm. The differential expression of the isolated cDNAs was confirmed by real-time PCR. The preliminary results showed that amoxicillin alters the expression of five cDNAs involved in biosynthesis, two involved with pathogenesis, four related to cell envelope formation, two involved in cellular processes, three related with transport and binding proteins, one involved with protein degradation, one involved with energy metabolism and seven hypothetical proteins. Further analysis of these cDNAs will allow a better comprehension of both the molecular mechanism(s) of amoxicillin resistance and the adaptative mechanism(s) used by H. pylori in the presence of this antibiotic.