Antimicrobial resistance of Helicobacter pylori in an eastern German region.

BACKGROUND: Antimicrobial therapy is recommended to eradicate Helicobacter (H.) pylori in infected individuals. As first-line treatments are empiric, knowledge of antimicrobial resistance is key to successful eradication. AIMS: We investigated primary resistance in an eastern German region to derive recommendations for eradication treatment. METHODS: We used molecular genetic methods to examine Helicobacter rapid urease test (RUT) positive gastric specimens of 533 patients from Berlin and the federal state of Brandenburg with allegedly no prior eradication treatment. Tissue samples were removed from RUT and screened by real-time PCR for mutations conferring resistance to clarithromycin. In addition, 182 samples out of 533 were tested for resistance to levofloxacin and tetracycline. RESULTS: Primary resistances were 10.9% (58 out of 533) to clarithromycin; 13.7% (25/182) to levofloxacin; and 2.2% to tetracycline (4/182). Combined resistance to clarithromycin/levofloxacin was low (2.2%, 4/182). Female sex was significantly associated with clarithromycin resistance. CONCLUSION: Clarithromycin may be a suitable first-line antibiotic for about 90% of outpatients. A simple molecular test may help physicians avoid prescription of an ineffective first-line regimen.

Hematopoietic Stem Cell Transplantation From Unrelated Donors in 2 Cases of Interleukin-10 Receptor Deficiency: Is Surgery Not a Requirement?

Mutations in interleukin-10 and its receptors cause infantile inflammatory bowel disease (IBD), a hyperinflammatory disorder characterized by severe, treatment-refractory colitis, multiple abscesses, and enterocutaneous fistulas. Patients with infantile IBD often require several surgical interventions, including complete colectomy, and hematopoietic stem cell transplantation is currently the only known medical therapy. Traditionally, operative management has been preferred before stem cell transplantation because of the latter's increased susceptibility to procedural complications; however, surgical intervention could be delayed, and possibly reconsidered, because our 2 patients with infantile IBD demonstrated a rapid response to treatment via engraftment.

Retrospective study on outcome of salvage Helicobacter pylori eradication therapies based on molecular genetic susceptibility testing.

BACKGROUND: Antimicrobial susceptibility of Helicobacter (H.) pylori is usually determined by phenotypic methods. When H. pylori cannot be grown owing to contaminations or delay in transport of gastric tissue samples to the microbiological laboratory, molecular genetic testing is a reasonable alternative. The aim of this retrospective study was to assess the outcome of salvage eradication treatments based on molecular genetic susceptibility testing. METHODS: Data on 144 H. pylori PCR-positive gastric tissue samples of patients primarily with prior unsuccessful eradication treatments were retrospectively analyzed. Eradication treatments were recommended based on genotypic clarithromycin and/or levofloxacin susceptibility as tested by real-time PCR or reverse hybridization. Treatment success was assessed by attending physicians using urea breath test; stool-antigen ELISA; and microbiology/histopathology. RESULTS: Overall success rate of molecular genetic testing-guided salvage treatments was low (68%); none of the regimens chosen was significantly better than another. Multivariable logistic regression analysis did not reveal any factors that may predict treatment failure. CONCLUSIONS: Eradication success was poor despite susceptibility testing. Gastroenterologists are advised to prescribe recommended salvage treatments, considering recommended dosages and prolonged treatment duration.

Antimicrobial susceptibility testing of Arcobacter butzleri: development and application of a new protocol for broth microdilution.

Objectives: To develop a standard reference broth microdilution method for antimicrobial susceptibility testing (AST) of Arcobacter butzleri. The protocol was subsequently applied to a collection of A. butzleri isolates from different sources. Methods: Broth microdilution susceptibility testing was performed on eight A. butzleri isolates in three media: non-supplemented CAMHB, CAMHB + 2% FBS and CAMHB + 5% FBS. The MIC values were read after 24 and 48 h of incubation at 35 ±âŸ2 °C in ambient air. A logistic regression model was used to determine the combination of medium and incubation time yielding the most homogeneous results. Subsequently, the protocol was applied to 65 A. butzleri isolates to determine their MICs of 31 antimicrobial agents. Results: The statistical analysis revealed that the most homogeneous MIC values were obtained with CAMHB + 5% FBS and reading of MIC values after 24 h of incubation. The standardized method was successful for AST of all 65 A. butzleri isolates. MIC values were distributed unimodally for most antimicrobial agents. However, one field isolate showed elevated MIC values of gentamicin, streptomycin, tetracycline and trimethoprim/sulfamethoxazole. Conclusions: This study presents a new protocol for AST of A. butzleri by broth microdilution and shows the distribution of MIC values of 31 antimicrobial agents for a collection of A. butzleri isolates from different origins.

Targeted Gene Panel Sequencing for Early-onset Inflammatory Bowel Disease and Chronic Diarrhea.

BACKGROUND: In contrast to adult-onset inflammatory bowel disease (IBD), where many genetic loci have been shown to be involved in complex disease etiology, early-onset IBD (eoIBD) and associated syndromes can sometimes present as monogenic conditions. As a result, the clinical phenotype and ideal disease management in these patients often differ from those in adult-onset IBD. However, due to high costs and the complexity of data analysis, high-throughput screening for genetic causes has not yet become a standard part of the diagnostic work-up of eoIBD patients. METHODS: We selected 28 genes of interest associated with monogenic IBD and performed targeted panel sequencing in 71 patients diagnosed with eoIBD or early-onset chronic diarrhea to detect causative variants. We compared these results to whole-exome sequencing (WES) data available for 25 of these patients. RESULTS: Target coverage was significantly higher in the targeted gene panel approach compared with WES, whereas the cost of the panel was considerably lower (approximately 25% of WES). Disease-causing variants affecting protein function were identified in 5 patients (7%), located in genes of the IL10 signaling pathway (3), WAS (1), and DKC1 (1). The functional effects of 8 candidate variants in 5 additional patients (7%) are under further investigation. WES did not identify additional causative mutations in 25 patients. CONCLUSIONS: Targeted gene panel sequencing is a fast and effective screening method for monogenic causes of eoIBD that should be routinely established in national referral centers.

Autoantibodies against BAFF, APRIL or IL21 - an alternative pathogenesis for antibody-deficiencies?

BACKGROUND: The ability of anti-cytokine antibodies to play a disease-causing role in the pathogenesis of immunodeficiencies is widely accepted. The aim of this study was to investigate whether autoantibodies against BAFF (important B cell survival signal), APRIL (important plasma cell survival signal), or Interleukin-21 (important cytokine for immunoglobulin class switch) present an alternative mechanism for the development of the following primary antibody deficiencies (PADs): common variable immune deficiency (CVID) or selective IgA deficiency (sIgAD). RESULTS: Two hundred thirty-two sera from patients with PADs were screened for autoantibodies against cytokines by ELISA. Statistical data analysis yielded a significant difference (p < 0.01) between the healthy donor sera and both PAD cohorts. The analysis was deepened by subdividing the patient collective into groups with distinct B cell phenotypes but no significant differences were found. For selected sera with notable high ELISA-read outs functional analysis ensued. Anti-BAFF and anti-APRIL antibodies were further examined by a B cell survival assay, whilst the functional relevance of putative anti-IL-21 autoantibodies was investigated by means of a STAT3 phosphorylation assay. However, the results of these experiments revealed no discernible functional effect. CONCLUSION: Whilst statistical analysis of ELISA results showed significant differences between patients and healthy controls, in our set of patients functional tests yielded no evidence for an involvement of autoantibodies against BAFF, APRIL, or IL-21 in the pathogenesis of CVID or sIgAD.

Outcome of second- and third-line Helicobacter pylori eradication therapies based on antimicrobial susceptibility testing.

OBJECTIVES: The objective of this study was to assess the outcome of antimicrobial susceptibility-guided therapies in Helicobacter pylori-infected individuals who had undergone unsuccessful prior eradication treatments. METHODS: From October 2004 to December 2013, 481 H. pylori-positive patients with prior unsuccessful eradication treatments were administered susceptibility-guided salvage eradication treatments. Six months on, treatment outcome was assessed by urea breath test, stool antigen ELISA, Helicobacter urease test or microbiology and/or histopathology. RESULTS: Resistance to metronidazole and clarithromycin was high in patients with prior unsuccessful eradication treatments and was dependent on the number of treatment failures. Susceptibility-guided salvage eradication treatments achieved eradication rates of nearly 70% in these patients. No particular regimen was significantly better than another. CONCLUSIONS: Antimicrobial susceptibility testing prevents prescription of inefficient antimicrobials and enables individualized and promising salvage treatments in patients with prior unsuccessful eradication treatments.

Prospective multicentre study on antimicrobial resistance of Helicobacter pylori in Germany.

OBJECTIVES: Antimicrobial resistance of Helicobacter pylori endangers the successful eradication of the bacteria. The aim of this prospective surveillance study (ResiNet) is to continuously keep antimicrobial resistance of H. pylori in Germany under surveillance and to identify risk factors for its development. METHODS: From July 2001 until December 2012, we tested the antimicrobial susceptibility of H. pylori strains isolated from 1651 prospectively enrolled patients. We analysed clinical and epidemiological data and identified risk factors for the development of resistance. RESULTS: Average primary resistances were 29.4% for metronidazole, 6.7% for clarithromycin and 3.1% for both antimicrobials. Prior unsuccessful eradication treatments, female sex and country or continent of origin were identified as independent risk factors for development of resistance. CONCLUSIONS: H. pylori-positive patients without prior eradication therapy can be treated empirically; antimicrobial susceptibility testing is recommended in previously unsuccessfully treated patients and in patients who have received antimicrobial chemotherapies due to unrelated bacterial infections.

Evidence for non-neutralizing autoantibodies against IL-10 signalling components in patients with inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease constitutes a heterogeneous group of conditions, whose aetiology is only partly understood. The prevailing hypothesis on its pathogenesis is that IBD is the result of an inadequate immune response to the resident bacterial flora of the intestine. An autoimmune background, however, has been discussed since the 1950s. Lately, it has been shown that failures in interleukin-10 (IL-10) signalling due to IL-10- and IL-10 receptor (IL-10R) mutations result in IBD. Our study aimed at investigating the existence of inhibitory autoantibodies against IL-10 and IL-10R in IBD patients capable of down-modulating IL-10 signalling thereby mimicking IL-10 or IL-10R deficiency. RESULTS: Thirteen IBD patients had IgG autoantibodies against IL-10, IL-10RA and/or IL-10RB, and three patients had IgA autoantibodies against IL-10. However, the absolute OD values of the serum antibodies measured by ELISA were low, there was overall no significant difference between patients and controls, and positive sera had no neutralizing activity. CONCLUSION: No evidence for an involvement of autoantibodies against IL-10 or IL-10R in the pathogenesis of inflammatory bowel disease could be established.

High secondary resistance to quinolones in German Helicobacter pylori clinical isolates.

OBJECTIVES: The aim of this study was to update data on levofloxacin/ciprofloxacin and triple resistance (resistance to metronidazole, clarithromycin and levofloxacin/ciprofloxacin) in Helicobacter pylori clinical isolates and to identify the impact of prior eradication therapies on their development. METHODS: We tested the antimicrobial susceptibility to amoxicillin, metronidazole, clarithromycin, levofloxacin/ciprofloxacin, tetracycline and rifampicin of 5296 clinical H. pylori strains isolated between 2006 and 2011. Information on prior eradication therapies was gathered and their impact on the development of antimicrobial resistance, in particular to levofloxacin/ciprofloxacin and triple resistance, was analysed. RESULTS: From 2006 onwards, both levofloxacin/ciprofloxacin and triple resistance have steadily increased and peaked in 2011 with 29.1% and 18.6%, respectively. Unsuccessful prior eradication attempts proved a major risk factor for resistance development. Patients who had undergone unsuccessful eradication attempts harboured levofloxacin/ciprofloxacin- and triple-resistant isolates significantly more often than untreated individuals (26.7% and 18.1% versus 10.6% and 1.6%). Levofloxacin/ciprofloxacin and triple resistance occurred significantly more often in patients who had received quinolones when compared with patients who had not (44.5% versus 23.1% and 28.7% versus 15.6%). We did not observe any significant differences in resistance rates in the different German federal states. CONCLUSIONS: Resistance to levofloxacin/ciprofloxacin and triple resistance have continuously risen and reached worrying numbers. Hence we strongly advise against the use of quinolones in empirical second-line therapies for H. pylori without prior susceptibility testing and/or a carefully taken patient medical history.

Clinical outcome in IL-10- and IL-10 receptor-deficient patients with or without hematopoietic stem cell transplantation.

BACKGROUND: Inherited deficiencies of IL-10 or IL-10 receptor (IL-10R) lead to immune dysregulation with life-threatening early-onset enterocolitis. OBJECTIVES: We sought to gather clinical data of IL-10/IL-10R-deficient patients and devise guidelines for diagnosis and management, including hematopoietic stem cell transplantation (HSCT). METHODS: We enrolled 40 patients with early-onset enterocolitis and screened for mutations in IL10/IL10R using genetic studies, functional studies, or both of the IL-10 signaling pathway. Medical records of IL-10/IL-10R-deficient patients were reviewed and compiled. RESULTS: Of 40 patients, we identified 7 with novel mutations, predominantly in consanguineous families with more than 1 affected member. IL-10/IL-10R-deficient patients had intractable enterocolitis, perianal disease, and fistula formation. HSCT was carried out in 2 patients with IL-10 deficiency and 1 patient with IL-10R α chain deficiency and proved to be an effective therapy, leading to rapid improvement of clinical symptoms and quality of life. CONCLUSION: Because the defect in patients with IL-10/IL-10R deficiency resides in hematopoietic lineage cells and their colitis is resistant to standard immunosuppressive therapy, HSCT should be considered early as a potentially curative therapeutic option.

Interleukin-10 and interleukin-10-receptor defects in inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by abdominal pain, bloody diarrhoea, and malabsorption leading to weight loss. It is considered the result of inadequate control of an excessive reaction of the immune system to the resident flora of the gut. Like other primary immunodeficiencies, IL-10 and IL-10 receptor (IL10R) deficiency present with IBD and demonstrate the sensitivity of the intestine to any changes of the immune system. Both IL-10 and IL10R deficiency cause severe early-onset enterocolitis and can be successfully treated by hematopoietic stem cell transplantation (HSCT).

IL-10 and IL-10 receptor defects in humans.

Inflammatory bowel disease (IBD), which includes Crohn's disease (CD) and ulcerative colitis (UC), is chronic in nature and is characterized by abdominal pain, diarrhea, bleeding, and malabsorption. It is considered a complex multigenic and multifactorial disorder that results from disturbed interactions between the immune system and commensal bacteria of the gut. Recent work has demonstrated that IBD with an early-onset within the first months of life can be monogenic: mutations in IL-10 or its receptor lead to a loss of IL-10 function and cause severe intractable enterocolitis in infants and small children. Both IL-10 and IL-10 receptor deficiency can be successfully treated by hematopoietic stem cell transplantation.

Mutations in STAT3 and diagnostic guidelines for hyper-IgE syndrome.

BACKGROUND: The hyper-IgE syndrome (HIES) is a primary immunodeficiency characterized by infections of the lung and skin, elevated serum IgE, and involvement of the soft and bony tissues. Recently, HIES has been associated with heterozygous dominant-negative mutations in the signal transducer and activator of transcription 3 (STAT3) and severe reductions of T(H)17 cells. OBJECTIVE: To determine whether there is a correlation between the genotype and the phenotype of patients with HIES and to establish diagnostic criteria to distinguish between STAT3 mutated and STAT3 wild-type patients. METHODS: We collected clinical data, determined T(H)17 cell numbers, and sequenced STAT3 in 100 patients with a strong clinical suspicion of HIES and serum IgE >1000 IU/mL. We explored diagnostic criteria by using a machine-learning approach to identify which features best predict a STAT3 mutation. RESULTS: In 64 patients, we identified 31 different STAT3 mutations, 18 of which were novel. These included mutations at splice sites and outside the previously implicated DNA-binding and Src homology 2 domains. A combination of 5 clinical features predicted STAT3 mutations with 85% accuracy. T(H)17 cells were profoundly reduced in patients harboring STAT3 mutations, whereas 10 of 13 patients without mutations had low (<1%) T(H)17 cells but were distinct by markedly reduced IFN-gamma-producing CD4(+)T cells. CONCLUSION: We propose the following diagnostic guidelines for STAT3-deficient HIES. Possible: IgE >1000IU/mL plus a weighted score of clinical features >30 based on recurrent pneumonia, newborn rash, pathologic bone fractures, characteristic face, and high palate. Probable: These characteristics plus lack of T(H)17 cells or a family history for definitive HIES. Definitive: These characteristics plus a dominant-negative heterozygous mutation in STAT3.

Inflammatory bowel disease and mutations affecting the interleukin-10 receptor.

BACKGROUND: The molecular cause of inflammatory bowel disease is largely unknown. METHODS: We performed genetic-linkage analysis and candidate-gene sequencing on samples from two unrelated consanguineous families with children who were affected by early-onset inflammatory bowel disease. We screened six additional patients with early-onset colitis for mutations in two candidate genes and carried out functional assays in patients' peripheral-blood mononuclear cells. We performed an allogeneic hematopoietic stem-cell transplantation in one patient. RESULTS: In four of nine patients with early-onset colitis, we identified three distinct homozygous mutations in genes IL10RA and IL10RB, encoding the IL10R1 and IL10R2 proteins, respectively, which form a heterotetramer to make up the interleukin-10 receptor. The mutations abrogate interleukin-10-induced signaling, as shown by deficient STAT3 (signal transducer and activator of transcription 3) phosphorylation on stimulation with interleukin-10. Consistent with this observation was the increased secretion of tumor necrosis factor alpha and other proinflammatory cytokines from peripheral-blood mononuclear cells from patients who were deficient in IL10R subunit proteins, suggesting that interleukin-10-dependent "negative feedback" regulation is disrupted in these cells. The allogeneic stem-cell transplantation performed in one patient was successful. CONCLUSIONS: Mutations in genes encoding the IL10R subunit proteins were found in patients with early-onset enterocolitis, involving hyperinflammatory immune responses in the intestine. Allogeneic stem-cell transplantation resulted in disease remission in one patient.

A homozygous CARD9 mutation in a family with susceptibility to fungal infections.

BACKGROUND: Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described. METHODS: We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect. RESULTS: We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1. CONCLUSIONS: An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9.