Dorothea Orth-Höller

Enterohemorrhagic Escherichia coli O26:H11/H−: A New Virulent Clone Emerges in Europe

BACKGROUND Enterohemorrhagic Escherichia coli (EHEC) O26 causes diarrhea and hemolytic uremic syndrome (HUS). Strains harboring the stx1a gene prevail, but strains with stx2a as the sole Shiga toxin-encoding gene are now emerging. The traits and virulence of the latter set of strains are unknown. We correlated stx genotypes of 272 EHEC O26 strains isolated in 7 European countries between 1996 and 2012 with disease phenotypes. We determined phylogeny, clonal structure, and plasmid gene profiles of the isolates and portray geographic and temporal distribution of the different subgroups. METHODS The stx genotypes and plasmid genes were identified using polymerase chain reaction, phylogeny was assigned using multilocus sequence typing, and clonal relatedness was established using pulsed-field gel electrophoresis. RESULTS Of the 272 EHEC O26 isolates, 107 (39.3%), 139 (51.1%), and 26 (9.6%) possessed stx1a, stx2a, or both genes, respectively. Strains harboring stx2a only were significantly associated with HUS (odds ratio, 14.2; 95% confidence interval, 7.9-25.6; P < .001) compared to other stx genotypes. The stx2a-harboring strains consist of 2 phylogenetically distinct groups defined by sequence type (ST) 21 and ST29. The ST29 strains are highly conserved and correspond by plasmid genes to the new virulent clone of EHEC O26 that emerged in Germany in the 1990s. This new clone occurred in 6 of the 7 countries and represented approximately 50% of all stx2a-harboring EHEC O26 strains isolated between 1996 and 2012. CONCLUSIONS A new highly virulent clone of EHEC O26 has emerged in Europe. Its reservoirs and sources warrant identification.

Role of complement in enterohemorrhagic Escherichia coli-Induced hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in childhood, is mainly caused by infection with Shiga toxin (Stx)-producing enterohemorrhagic Escherichia coli (EHEC). Besides its cytotoxic activity, Stx has been shown to interact with the complement system. Complement breakdown products have been found in serum of HUS patients suggesting complement activation and in vitro studies have demonstrated that Stx2 directly activates complement leading to formation of terminal complement complex. Furthermore, Stx2 has been found to bind to factor H (FH) resulting in a reduced cofactor activity on the cell surface. Binding of Stx2 has also been shown for other members of the FH family, namely FH-like protein 1 and FH-related protein 1. Both proteins also compete with FH for Stx binding, so that in the presence of FHR-1 less FH is bound to Stx and therefore more is available for endothelial cell protection. In addition, Stx2 has been demonstrated to downregulate the membrane-bound regulator CD59 on the surface of glomerular endothelial and tubulus epithelial cells on protein and at the mRNA level. In conclusion, Stx modulates complement regulator proteins leading to an impaired control and thus to enhanced complement activation. Its implication in the pathogenesis of EHEC-induced HUS in vivo and whether complement blockage might be a therapeutic option still has to be elucidated.

Treatment of enterohemorrhagic Escherichia coli-induced hemolytic uremic syndrome (eHUS).

Treatment of enterohemorrhagic Escherichia coli-induced hemolytic uremic syndrome (eHUS) still mostly relies on supportive intensive care regimens. Antibiotic treatment, as administered to eHUS patients during the 2011 O104:H4 outbreak, may reduce the shedding period, but this may apply only to this particular strain. In any case, there is no evidence for a beneficial use in the diarrheal phase and earlier warnings that antibiotic therapy at this stage may actually increase the likelihood of HUS remain unrefuted. Plasma exchange, a frequently chosen therapy in acute atypical HUS, was not beneficial for the outbreak patients and a prospective study of 274 pediatric eHUS patients even indicates a poorer long-term outcome. As eHUS is a disease where complement plays a pathophysiological role and individual beneficial treatments had been published, eculizumab was broadly administered during the outbreak, in particular to severely ill patients. The equally good outcome of treated versus untreated patients obviously does not allow a clear-cut statement, but rather points toward an advantageous use, at least for the severe cases. Although the role of complement should not be overestimated, the use of a complement blocker--not necessarily being a therapeutic option for uncomplicated eHUS--in severe disease may actually make the difference between favorable or detrimental outcome.

Interaction of Shiga toxin 2 with complement regulators of the factor H protein family.

Shiga toxin 2 (Stx2) is believed to be a major virulence factor of enterohemorrhagic Escherichia coli (EHEC) contributing to hemolytic uremic syndrome (HUS). The complement system has recently been found to be involved in the pathogenesis of EHEC-associated HUS. Stx2 was shown to activate complement via the alternative pathway, to bind factor H (FH) at short consensus repeats (SCRs) 6-8 and 18-20 and to delay and reduce FH cofactor activity on the cell surface. We now show that complement factor H-related protein 1 (FHR-1) and factor H-like protein 1 (FHL-1), proteins of the FH protein family that show amino acid sequence and regulatory function similarities with FH, also bind to Stx2. The FHR-1 binding site for Stx2 was located at SCRs 3-5 and the binding capacity of FHR-1*A allotype was higher than that of FHR-1*B. FHR-1 and FHL-1 competed with FH for Stx2 binding, and in the case of FHR-1 this competition resulted in a reduction of FH cofactor activity. FHL-1 retained its cofactor activity in the fluid phase when bound to Stx2. In conclusion, multiple interactions of key complement inhibitors FH, FHR-1 and FHL-1 with Stx2 corroborate our hypothesis of a direct role of complement in EHEC-associated HUS.

Primary antifungal prophylaxis with micafungin in patients with haematological malignancies: real-life data from a retrospective single-centre observational study.

Mould‐active antifungal prophylaxis is increasingly used in patients at risk for invasive fungal disease. Between June 2011 and June 2012, one hundred patients with various haematological malignancies at risk for invasive fungal disease received primary antifungal prophylaxis with intravenous micafungin at a daily dosage of 50 mg during neutropenia. The median number of days on micafungin prophylaxis was 14 (range, 6–48 d). The incidence of proven and probable breakthrough invasive fungal diseases (bIFDs) was 6% and 3%, respectively. There were two bloodstream infections caused by yeasts or yeast‐like fungi (Candida krusei, Trichosporon asahii) in two patients during the neutropenic phase after allogeneic haematopoietic stem cell transplantation. Four proven bIFDs caused by non‐Aspergillus moulds and three cases of probable pulmonary bIFDs were documented during the neutropenic phase after induction/consolidation chemotherapy for acute leukaemia. Colonisation with Candida spp. was documented in 51% of the patients with none of the isolates being in vitro micafungin resistant. Compared to a historical control, receiving primary prophylaxis with posaconazole micafungin is at least as effective in preventing IFD. In both cohorts, bIFDs were exclusively caused by emerging pathogens with a highly preserved in vitro sensitivity to amphotericin B.

Shiga Toxin 2 Reduces Complement Inhibitor CD59 Expression on Human Renal Tubular Epithelial and Glomerular Endothelial Cells

ABSTRACT Infections with enterohemorrhagic Escherichia coli (EHEC) are a primary cause of hemolytic-uremic syndrome (HUS). Recently, Shiga toxin 2 (Stx2), the major virulence factor of EHEC, was reported to interact with complement, implying that the latter is involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate the effect of Stx2 on the expression of membrane-bound complement regulators CD46, CD55, and CD59 on proximal tubular epithelial (HK-2) and glomerular endothelial (GEnC) cells derived from human kidney cells that are involved in HUS. Incubation with Stx2 did not influence the amount of CD46 or CD55 on the surface of HK-2 and GEnC cells, as determined by fluorescence-activated cell sorter analysis. In contrast, CD59 was significantly reduced by half on GEnC cells, but the reduction on HK-2 cells was less pronounced. With increasing amounts of Stx2, reduction of CD59 also reached significance in HK-2 cells. Enzyme-linked immunosorbent assay analyses showed that CD59 was not present in the supernatant of Stx2-treated cells, implying that CD59 reduction was not caused by cleavage from the cell surface. In fact, reverse transcription-quantitative PCR analyses showed downregulation of CD59 mRNA as the likely reason for CD59 cell surface reduction. In addition, a significant increase in terminal complement complex deposition on HK-2 cells was observed after treatment with Stx2, as a possible consequence of CD59 downregulation. In summary, Stx2 downregulates CD59 mRNA and protein levels on tubular epithelial and glomerular endothelial cells, and this downregulation likely contributes to complement activation and kidney destruction in EHEC-associated HUS.

Inhibition of terminal complement activation in severe Shiga toxin-associated HUS - perfect example for a fast track from bench to bedside

Typical haemolytic uraemic syndrome (HUS) is a thrombotic microangiopathy, leading to severe renal disease as well as life‐threatening extrarenal complications. HUS is primarily caused by infections with enterohaemorrhagic Escherichia coli (EHEC) and 5–20% of EHEC infections result in HUS. EHEC bacteria produce several virulence factors, of which Shiga toxins are believed to play a central role. About 10% of all HUS cases are not caused by E. coli and are thus termed atypical HUS. These are observed as familial or sporadic forms and are most commonly caused by a dysregulation of the alternative pathway of the complement system due to inherited mutations of, or acquired auto‐antibodies against, complement regulator proteins. The, thereby, impaired control of complement, which plays a prominent role in the humoral immune system and in immune homeostasis, leads to a hyperactive state, including activation of endothelial cells and platelets, inflammation of small vessel and eventually to the destruction of the kidney and other organs (summarized in Orth et al, 2009). Lapeyraque et al have published the successful use of the licensed terminal complement C5 inhibitor eculizumab for the treatment of severe Shiga toxin‐associated HUS in three 3‐year‐old children with devastating prognoses (Lapeyraque et al, 2011). The three patients continued to show progression of the HUS despite having received multiple plasma exchanges. Treatment with eculizumab markedly improved their clinical status, in particular their neurological and renal functions, after two to four administrations of this complement inhibitor. Dialysis was reported to be discontinued after 3–16 days in all three patients. Recovery was attributed to eculizumab and absence of mutations of complement proteins …

Protection of human podocytes from Shiga toxin 2-induced phosphorylation of mitogen-activated protein kinases and apoptosis by human serum amyloid P component

ABSTRACT Hemolytic uremic syndrome (HUS) is mainly induced by Shiga toxin 2 (Stx2)-producing Escherichia coli. Proteinuria can occur in the early phase of the disease, and its persistence determines the renal prognosis. Stx2 may injure podocytes and induce proteinuria. Human serum amyloid P component (SAP), a member of the pentraxin family, has been shown to protect against Stx2-induced lethality in mice in vivo, presumably by specific binding to the toxin. We therefore tested the hypothesis that SAP can protect against Stx2-induced injury of human podocytes. To elucidate the mechanisms underlying podocyte injury in HUS-associated proteinuria, we assessed Stx2-induced activation of mitogen-activated protein kinases (MAPKs) and apoptosis in immortalized human podocytes and evaluated the impact of SAP on Stx2-induced damage. Human podocytes express Stx2-binding globotriaosylceramide 3. Stx2 applied to cultured podocytes was internalized and then activated p38α MAPK and c-Jun N-terminal kinase (JNK), important signaling steps in cell differentiation and apoptosis. Stx2 also activated caspase 3, resulting in an increased level of apoptosis. Coincubation of podocytes with SAP and Stx2 mitigated the effects of Stx2 and induced upregulation of antiapoptotic Bcl2. These data suggest that podocytes are a target of Stx2 and that SAP protects podocytes against Stx2-induced injury. SAP may therefore be a useful therapeutic option.

An Update on the Thrombotic Microangiopathies Hemolytic Uremic Syndrome (HUS) and Thrombotic Thrombocytopenic Purpura (TTP)

Welcome to this special issue of Seminars in Thrombosis & Hemostasis, which summarizes up-to-date knowledge on pathogenesis, diagnosis, and treatment of thrombotic microangiopathies (TMAs) such as (enterohemorrhagic) E. coli (EHEC)–associated hemolytic uremic syndrome (HUS) (eHUS), atypical HUS (aHUS), (aHUS) and thrombotic thrombocytopenic purpura (TTP). This issue also reviews C3 glomerulopathies (C3G), emphasizing the expanding spectrum of TMAs. The introduction to this issue is provided by Sethi and Fervenza who revise, from a morphological and histological view, the current classification of C3G based on renal pathology and explaining the typical features of the different complement-mediated diseases on biopsy. They introduce the role of uncontrolled complement activation of the alternative pathway in the pathogenesis of C3G, using new methods such as mass spectrometry of microdissected glomeruli.1 de Cordoba et al then review up-to-date knowledge on the inherited form of aHUS and thus on genetics, including recent findings such as complement factor H (CFH) and/or factor H– related (CFHR) genomic rearrangements and mutations in the coagulation cascade. It is commonly understood that multiple hits, involving genetic/autoimmune defects of complement regulators and proteins, are likely required to impair protection of endothelial cells. The genetic susceptibility is increased if an individual carries a combination of inherited defects.2 For the acquired form of aHUS, Hofer et al describe in their review on CFH antibody–associated HUS (CFH-Ab HUS) the current knowledge on CFH-Ab detection, the associationwith CFHR3-CFHR1 deletion, and the different treatment options. The significance of a rapid CFH-Ab HUS diagnosis is crucial, as the treatment algorithm differs from other aHUS patients.3 Insights into endothelial cell activation and inflammation involved in TMA pathogenesis are reviewed in detail by Riedl et al. Increasing knowledge on aHUS and an expanding TMA spectrum have evolved over the last years (►Table 1). Complement emerges as a crucial regulator, as it plays a central role in maintaining this homeostasis within the microvasculature. Defects in the complement system, raising the individual susceptibility to TMA evolvement, were discovered in affected patients. The successful use of eculizumab in patients with aHUS or the extended TMA spectrum corroborates the role of complement in TMA pathogenesis.4 Xiao et al focus on the functional aspects of the complement system and, in particular, on the genetic complement disorders in C3G patients, such as CFH-related protein dimerization and complex formation. Abnormal CFHR proteins detected in patientswith C3Gmay promote formation of unusual dimers and/or multimers impacting complement control.5 Eculizumab, a monoclonal antibody binding C5, has been used in 11 C3G patients so far, as reported by Vivarelli and Emma. A significant response was seen in eight patients with evidence for terminal complement activation. As patients with C3G show a great deal of interindividual variety, a patient-tailored complement-targeting treatment might be the ideal approach in the future.6 Safouh and coworkers (Hofer et al) share the experience of a pediatric nephrologist in Cairo, Egypt, in the review on HUS in the developing world. This is an important contribution, as patients outnumber every cohort in the developed world and

Diagnosing filamentous fungal infections in immunocompromised patients applying computed tomography-guided percutaneous lung biopsies: a 12-year experience

BackgroundInvasive fungal diseases (IFD) are an important cause of morbidity and mortality in immunocompromised patients, and early diagnosis and management are a challenge. We evaluated the clinical utility of computed tomography (CT)-guided percutaneous lung biopsies in diagnosing IFD.MethodsBetween 2003 and 2014, we analyzed 2671 CT-guided lung biopsies, from which 157 were IFD associated; we aimed to determine microbiological-based diagnostic accuracy of calcofluor white staining (CFWS), culture, Aspergillus antigen detection (GM), broad-range fungal PCR, and Aspergillus PCR per sample.Results127 (81%) specimens were microscopically positive for any fungal elements, 30 (19%) negative. Aspergillus and non-Aspergillus like hyphae were obtained in 85 (67%) and 42 (33%) specimens, respectively. CFWS positivity was defined as proof of infection. Sensitivity, specificity, and positive (PPV) and negative predictive (NPV) values for CT scan were 100, 44, 80, and 100%, for Aspergillus PCR 89, 58, 88, and 58%, for broad-range fungal PCR 90, 83, 95, and 90%, and for GM 94, 83, 95, and 90%. The most common CT features were patchy opacifications with central necrosis (78%) or cavern defects (50%), less common were air bronchograms (39%) or ground glass halos (39%), and all other features were rare. The overall pneumothorax rate subsequent to biopsy was 19%, but in only 2% of all cases the placement of a chest tube was indicated. One case of fatal air embolism occurred.ConclusionsCT-guided lung biopsies have high diagnostic accuracy in terms of microscopic examination, and complication rates are low. Molecular-based and antigen tests applied on fungal hyphae-positive specimens showed comparable results.