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UP TO PAGE 53 S&S [Neurologic presentation in haemolytic-uraemic syndrome] [Article in Spanish] Roche-Martínez A, Póo P, Maristany-Cucurella M, Jiménez-Llort A, Camacho JA, Campistol J. Servicio de Neurología, Hospital Sant Joan de Deu, 08950 Esplugues de Llobregat, España. [email protected] INTRODUCTION: Haemolytic-uraemic syndrome (HUS) is characterized by microangiopathic hemolytic anaemia, thrombopenia and multiorganic aggression, specially renal, gastrointestinal and central nervous system disturbances. Sporadic in Spain (2/1,500,000 inhabitants), its clinical onset includes acute renal failure, hypertension and central nervous system symptoms (irritability, drowsiness, convulsions, cortical blindness, hemiparesia or coma), due to metabolic distress, hypertension or central nervous system microangiopathy. Few long-term outcome studies have been published. PATIENTS AND METHODS: A retrospective analysis of a series of 58 patients with HUS between 1981 and 2006, is reported. Clinical onset, laboratory, electrophysiology, neuroimaging tests, and prognosis factors are reviewed, together with long-term clinical outcome. RESULTS:

22 children presented neurologic symptoms, seven had some neurological test; one patient died; in five some neurological sequelae persisted (hemiparesia, cognitive deficit, visual-perception deficit), the other 16 remaining asymptomatic. CONCLUSIONS: Neurological morbility is high in HUS (27% of the children with neurological symptoms), with a 1.7% mortality. Seizure at onset was not a poor prognosis factor in our group. No positive correlation can be established between neuroimaging and long-term outcome.

. Kathmandu Univ Med J (KUMJ). 2007 Oct-Dec;5(4):468-74.

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Clinico-laboratory profile of haemolytic uremic syndrome. Jha DK, Singh R, Raja S, Kumari N, Das BK.

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Department of Pediatrics and Adolescent Medicine, B P Koirala Institute of Health Sciences, Dharan, Nepal. Objective: To study the clinical profile, the spectrum of functional abnormalities, prognostic factors and outcome of children with haemolytic uremic syndrome (HUS). Materials and methods: This is a prospective, descriptive, single centre, cohort study, conducted on 42 children during the period of January 2004 to January 2005. Results: The maximum numbers of cases were below 24 months of age with mean age of 26.6 months and male: female ratio of 2.8:1. Most of the

The common clinical presentations were bloody diarrhoea, pallor, oliguria & anuria, fever, vomiting, abdominal distension and pain, involvement of central nervous system, chest and cardiovascular system and bleeding manifestations. The common haematological abnormalities were leucocytosis, thrombocytopenia, anaemia and features of haemolysis in the peripheral blood. Electrolyte abnormalities observed were in the form of hyponatremia, hypokalemia and hyperkalemia. Arterial blood gas analysis showed metabolic acidosis in 64% cases, where the estimations were done. The mean blood urea and serum creatinine levels were 113.7 mg/dL and 2.5 mg/dL, respectively. Stool examination showed blood in all cases. Urine examination showed microscopic haematuria and significant proteinuria in 74% and 38% cases, respectively. E. coli and Shigella were isolated in stool in three cases each and one case showed mixed growth of E. coli and Salmonella. The mortality rate was 21%. Significantly higher mortality was observed in females, patients presenting with complete anuria, leucocytosis, hyperkalemia and systemic involvement like central nervous system, cardio vascular system and chest. Conclusions: Female sex, complete anuria, leucocytosis, extra renal involvement and hyperkalemia were associated with poor outcome. Key words: Haemolytic Uremic Syndrome, Clinical Features, Outcome, Prognostic Factors. cases (79%) occurred in the warmer months (April-September).

77: Kathmandu Univ Med J (KUMJ). 2004 Oct-Dec;2(4):291-6.

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Prognostic indicators in haemolytic uraemic syndrome. Malla K, Malla T, Hanif M. Kathmandu Medical College, Sinamangal. [email protected] OBJECTIVE: This study aims to review the clinical presentations of Haemolytic Uraemic Syndrome (HUS) and to compare the poor prognostic indicators with mortality. METHODS AND MATERIALS: Prospective study carried out in Renal Dialysis ward of Dhaka Shishu Hospital, Bangladesh from September 2001 November 2003 for a period of 26 months. All children admitted to renal dialysis ward with oliguria or anuria with pallor was included in this study. HUS was confirmed after laboratory investigations showing features of hemolytic anaemia, thrombocytopenia and renal insufficiency. Various clinical presentations were reviewed. Then bad prognostic factors were compared with mortality. RESULTS: There were total 25 cases of HUS in 26 months.17 (68%) were males and 8(32%) females.21 (84%) children were < 5 years. Only 4(16%) were > 5 years. Before onset of HUS 40% children had bloody diarrhoea, 36% had acute watery diarrhoea and 24% had others symptoms. The other presentations noted were fever 88%, respiratory distress and convulsion 52% and oliguria 40%, anuria 60%, reluctant to feed 40% and cough 28%. The main physical findings noted were irritability 40%, pallor 100%, dehydration 28%, puffy face with oedema 32%, high blood pressure 16%, hepatomegaly 28%, jaundice, sclerema and petechial rashes 8%, lethargic 16%, acidotic breathing 48% and rectal prolapse 12%. 44% children died after HUS and 56% recovered from the illness. Mortality was 66% when duration of illness before

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onset of HUS was > 14 days. With duration of anuria < 3 days there was no mortality but it was 91% and 100% with anuria > 3 to 8 days and >8 days respectively. Mortality was 78% when age was < 18 months and it was 75% when age was > 5 years. Diarrhoea associated HUS had 27% and non diarrhoea associated HUS had 85% mortality. Mortality was 77% and 100% respectively when HUS was associated with convulsion and hypertension. WBC > 30,000 had mortality 100% and decreased platelet count < 30,000 had mortality 80%. With creatinine level > 700 micromol/L mortality was 80% and with

CONCLUSION: HUS comprised of varieties of presentations. Diarrhoea was the commonest preceding illness before onset of HUS. The bad prognostic indicators carrying high mortality was duration of illness before onset of HUS >14 days, anuria > 3 days, age < 18 months and >5 years, Non diarrhoea associated HUS, HUS associated with convulsion and hypertension, WBC > 30,000/cumm, platelets < 30,000/cumm, creatinine level > 700 micromol/L and serum potassium level 5.6 to 7.5 mmol/L. Since bad prognostic factors may progress rapidly to mortality, consultation with paediatrician and transfer to a tertiary care centre should be done when HUS is diagnosed so that it can be managed appropriately in time. Serum potassium level 5.6 to 7.5 mmol/L mortality was 67%.

AETIOLOGY Pathogenesis 28: Annu Rev Pathol. 2008;3:249-77.

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Pathogenesis of thrombotic microangiopathies. Zheng XL, Sadler JE. Department of Pathology and Laboratory Medicine, The Children's Hospital of Philadelphia and The University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. [email protected]

Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangiopathy, which includes two major disorders: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). TTP has at least three types: congenital or familial, idiopathic, and nonidiopathic. The congenital and idiopathic TTP syndromes are caused primarily by deficiency of ADAMTS13, owing to mutations in the ADAMTS13 gene or autoantibodies that inhibit ADAMTS13 activity. HUS is similar to TTP, but is associated with acute renal failure. Diarrhea-associated HUS accounts for more than 90% of cases and is usually caused by infection with Shiga-toxin-producing Escherichia coli (O157:H7). Diarrhea-negative HUS is associated with complement dysregulation in up to 50% of cases, caused by mutations in complement factor H, membrane cofactor protein, factor I or factor B, or by autoantibodies against factor H. The incomplete penetrance of mutations in either ADAMTS13 or complement regulatory

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genes suggests that precipitating events or triggers may be required to cause thrombotic microangiopathy in many patients.

93: Thromb Haemost. 2005 Aug;94(2):312-8.

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Consequences of enterohaemorrhagic Escherichia coli infection for the vascular endothelium. Bielaszewska M, Karch H. Institute for Hygiene and National Consulting Laboratory on Haemolytic Uraemic Syndrome, University Hospital Münster, Germany. Microvascular endothelial damage underlies the pathological changes in haemorrhagic colitis and the haemolytic uraemic syndrome (HUS) caused by enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) are presently the best characterised EHEC virulence factors that can cause the microvascular endothelium injury. Stxs are released by EHEC in the intestine, absorbed across the gut epithelium into the circulation, and transported to small vessel endothelial cells. Then, they presumably injure the host cell by inhibiting protein synthesis, stimulating prothrombotic messages, or inducing apoptosis. The net result is a multi-organ thrombotic process. Moreover, Stxs stimulate a variety of nonendothelial cells to produce and secrete inflammatory mediators (cytokines, chemokines, adhesion molecules) which could potentiate the effects of Stxs on endothelial cells. The association of HUS with Stx-negative E. coli strains stimulated intensive research on putative non-Stx virulence factors, which might also contribute to the pathogenesis of HUS and haemorrhagic colitis. Based on current data, cytolethal distending toxin, EHEC haemolysin, and subtilase cytotoxin might be such candidates. 111: J Thromb Haemost. 2005 Apr;3(4):752-62.

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Shiga toxin enhances functional tissue factor on human glomerular endothelial cells: implications for the pathophysiology of hemolytic uremic syndrome. Nestoridi E, Tsukurov O, Kushak RI, Ingelfinger JR, Grabowski EF.

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Cardiovascular Thrombosis Laboratory, MassGeneral Hospital for Children, Harvard Medical School, Boston, MA, USA. BACKGROUND: The pathogenesis of Shiga toxin (Stx)-mediated childhood hemolytic uremic syndrome (HUS) is not fully delineated, although current evidence implicates a prothrombotic state. We hypothesized that the tissue factor (TF) pathway plays a major role in the pathophysiology of HUS. MATERIALS AND METHODS: We measured cell surface TF activity in response to tumor necrosis factor-alpha (TNFalpha) (20 ng mL(-1), 2-144 h), Stx-1 (10(-11) mol L(-1), 4-144 h), or their combination (TNF-alpha 22 h and Stx-1 for the last 0.5-4 h of TNF-alpha incubation) on human glomerular (microvascular) endothelial cells (HGECs) and human

RESULTS AND CONCLUSIONS: We observed that while TNF-alpha caused an increase in cell surface TF activity on both cell types, the combination of TNF-alpha and Stx-1 differentially affected HGECs. On these cells, TF activity was increased further by 2.67 +/0.38-fold (n = 38, P < 0.001), consistent with our parallel observation that Stx1 binds to HGECs but not to HUVECs. Anti-TF antibody abolished functional TF while anti-tissue factor pathway inhibitor antibody enhanced TF activity. Stx-1 alone did not induce TF activity on either cell type. Measurement of TF antigen levels and quantitative real-time polymerase chain reaction demonstrated that exposure to TNF-alpha markedly increased TF protein and TF mRNA for HGECs, but the exposure to the combination of TNF-alpha and Stx-1 did not increase further the amount of either TF protein or TF mRNA. We conclude that cytokine-activated HGECs, but not HUVECs, undergo a significant augmentation of cell surface TF activity following exposure to Stx, suggesting an important role for TF in the coagulopathy observed in HUS. umbilical vein (macrovascular) endothelial cells (HUVECs).

213: Semin Thromb Hemost. 2002 Apr;28(2):167-72.

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Assays of von Willebrand factor-cleaving protease: a test for diagnosis of familial and acquired thrombotic thrombocytopenic purpura. Furlan M, Lämmle B. Central Hematology Laboratory, University Hospital, Inselspital, Bern, Switzerland. Endothelial cells secrete von Willebrand factor (vWF) multimers that are larger than those found in the circulating plasma. These very large multimeric forms of vWF, capable of spontaneously binding to and agglutinating the blood platelets under conditions of high fluid shear rate, are degraded by a specific metalloprotease cleaving the peptide bond 842Tyr843Met of the vWF subunit. The vWF-cleaving protease was found to be 5

deficient in patients with familial thrombotic thrombocytopenic purpura (TTP). The acute events in these patients can be successfully treated and prophylactically prevented by repletion of the missing protease using fresh frozen plasma (FFP). In another, apparently more common, form of TTP, the protease deficiency is due to inhibiting circulating antibodies directed against the vWF-cleaving protease. Therapy of these patients

Normal activity of vWF-cleaving protease was established in patients with a clinically similar disorder: hemolytic-uremic syndrome (HUS). should include immunosuppressive treatment in addition to plasma exchange and replacement with FFP.

The level of vWF-cleaving protease activity is thus a laboratory parameter that provides important information for the differential diagnosis and treatment of patients with TTP/HUS. Several assays of vWF-cleaving protease have been described and are summarized here.

GOOOOOD 7: Eur J Pediatr. 2008 Sep;167(9):965-71. Epub 2008 Jun 25. Links

What's new in haemolytic uraemic syndrome? Johnson S, Taylor CM. Department of Paediatric Nephrology, Birmingham Children's Hospital, Birmingham, B4 6NH, UK, [email protected]. Recent advances in understanding the aetiology of the disorders that make up the haemolytic uraemic syndrome (HUS) permit a revised classification of the syndrome. With appropriate laboratory support, an aetiologically-based subgroup diagnosis can be made in all but a few cases. HUS caused by enterohaemorrhagic Escherichia coli remains by far the most prevalent subgroup, and new insights into this zoonosis are discussed. The most rapidly expanding area of interest is the subgroup of inherited and acquired abnormalities of complement regulation. Details of the pathogenesis are incomplete but it is reasonable to conclude that local activation of the alternative pathway of complement in the glomerulus is a central event. There is no evidence-based treatment for this diagnostic subgroup. However, in circumstances where there is a mutated plasma factor such as complement factor H, strategies to replace the abnormal protein by plasmapheresis or more radically by liver transplantation are logical, and anecdotal successes are reported. In summary, the clinical presentation of HUS gives a strong indication as to the underlying cause. Patients without evidence of EHEC infection should be fully investigated to determine the aetiology. Where complement abnormalities are suspected there is a strong argument for

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empirical and early plasma exchange, although rapid advances in this field may provide more specific treatments in the near future. GOOD 88: Br J Haematol. 2005 Oct;131(2):247-52.

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Haemolytic uraemic syndrome is an immunemediated disease: role of anti-CD36 antibodies. Rock G, Clark W, Sternbach M, Kolajova M, McLaine P. Division of Hematology and Transfusion Medicine, Department of Pathology and Laboratory Medicine, Ottawa Hospital, Ottawa, ON, Canada. [email protected] Haemolytic uraemic syndrome (HUS) is a disorder in which platelet microthrombi are formed that have a particular propensity to deposit in the kidney microvasculature, resulting in impaired renal function and thrombocytopenia. The mechanism of formation of these microthrombi is not known. In this study, we showed that plasma from five adult and six paediatric cases of HUS caused aggregation and release of adenosine triphosphate from normal platelets. The plasma reacted against platelet lysate in a protein blot and all samples showed reactivity against a band at 88 kDa, corresponding to the membrane antigen CD36. This was confirmed by probing with Mo91, a monoclonal antibody to CD36. CD36 was also identified in the immune complex formed by incubation of patient plasmas with normal platelet lysate. In other studies, bands of 32 and 7.7 kDa were obtained when purified verotoxin was protein blotted and probed with either patient plasma or with anti-CD36 antibody Mo91 suggesting structural homologies between CD36 and verotoxin. While a direct cause-effect relationship is not yet established, the data support the concept of an immunological pathogenesis for HUS and suggest that molecular mimicry involving one or both of the homologous domains in membrane-bound CD36 and verotoxin lead to the development of antibodies capable of inducing the pathophysiological events characteristic of HUS.

CAUSES CHILDREN 87: Int J Med Microbiol. 2005 Oct;295(6-7):405-18.

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Enterohaemorrhagic Escherichia coli in human medicine. Karch H, Tarr PI, Bielaszewska M. Institute for Hygiene, and National Consulting Laboratory on Haemolytic Uraemic Syndrome, University Hospital Münster, Robert Koch Str. 41, D-48149 Münster, Germany. [email protected] Enterohaemorrhagic Escherichia coli (EHEC) are the pathogenic subgroup of Shiga toxin (Stx)-producing E. coli. EHEC can cause non-bloody and bloody diarrhoea, and the haemolytic uraemic syndrome (HUS). HUS is a major cause of acute renal failure in children. E. coli O57:H7 is the predominant, but far from being the only, serotype that can cause HUS. The cascade leading from gastrointestinal infection to renal impairment is complex, with the microvascular endothelium being the major histopathological target. EHEC also produce nonStx molecules, such as cytolethal distending toxin, which can contribute to the endothelial or vascular injury. Because there are no specific therapies for EHEC infections, efficient reservoir and human preventive strategies are important areas of ongoing investigations. This review will focus on the microbiology, epidemiology, and pathophysiology of EHEC-associated diseases, and illustrate future challenges and opportunities for their control.

10: Pediatr Infect Dis J. 2008 Jul;27(7):595-601.

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Surveillance of hemolytic uremic syndrome in children less than 15 years of age, a system to monitor O157 and non-O157 Shiga toxinproducing Escherichia coli infections in France, 1996-2006. Espié E, Grimont F, Mariani-Kurkdjian P, Bouvet P, Haeghebaert S, Filliol I, Loirat C, Decludt B, Minh NN, Vaillant V, de Valk H. Institut de Veille Sanitaire, Saint-Maurice, France. [email protected] BACKGROUND: Since the 1980s, Shiga toxin-producing Escherichia coli (STEC), especially E. coli O157:H7, has been an important cause of food borne disease in industrial countries. In France, as there was no routine screening for STEC in clinical laboratories, enhanced surveillance of hemolytic uremic syndrome (HUS) in children less than 15 years of age was established in 1996 to monitor trends in the incidence of STEC infections. METHODS: The surveillance system was based on a voluntary national network of pediatricians of 31 pediatric nephrology units in public hospitals. RESULTS: From 1996 to 2006, the mean annual incidence of HUS was 0.71 cases per 100,000 children less than 15 years of age and 1.87

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STEC infections were confirmed in 66% of patients; STEC O157 was the most common serogroup identified in STECrelated HUS (83%). In this 11-year period, 96% of HUS cases were sporadic and only 2 outbreaks caused by STEC O157 and by a dual infection of STEC O26 and O80 were detected. CONCLUSIONS: An evaluation of the surveillance of pediatric HUS cases per 100,000 children less than 5 years of age.

showed that it is a simple and useful system for monitoring trends in STEC infections in France. It provides the information needed to measure the impact of new and changing vehicles of STEC transmission, and evaluate the effectiveness of prevention measures

12: Pediatr Nephrol. 2008 Sep;23(9):1425-31. Epub 2008 May 21. Links

Enterohaemorrhagic Escherichia coli and Shigella dysenteriae type 1-induced haemolytic uraemic syndrome. Mark Taylor C. Department of Nephrology, Birmingham Children's Hospital, Birmingham, B4 6NH, UK. [email protected] Haemolytic uraemic syndrome (HUS) can be classified according to the aetiology of the different disorders from which it is composed. The most prevalent form is that induced by shigatoxin producing Escherichia coli (STEC) and, in some tropical regions, by Shigella dysenteriae type 1. STEC cause a zoonosis, are widely distributed in nature, enter the food chain in different ways, and show regional differences. Not all STEC are human pathogens. Enterohaemorrhagic E. coli usually cause attachment and effacing lesions in the intestine. This is not essential, but production of a shigatoxin (Stx) is. Because Stx are encoded by a bacteriophage, this property is transferable to naïve strains. Laboratory methods have improved by identifying STEC either via the toxin or its bacteriophage. Shigella dysenteriae type 1 produces shigatoxin, identical to Stx-1, but also has entero-invasive properties that enterohaemorrhagic Escherichia coli (EHEC) do not. Shigella patients risk bacteremia and benefit from early antibiotic treatment, unlike those with EHEC. PMID: 18493800 [PubMed - in process] Euro Surveill. 2008 Feb 14;13(7). pii: 8041. Links Comment in: Euro Surveill. 2008 Feb 28;13(9). pii: 8053; author reply pii/8054.

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Outbreak of verocytotoxin-producing E. coli O145 and O26 infections associated with the consumption of ice cream produced at a farm, Belgium, 2007. De Schrijver K, Buvens G, Possé B, Van den Branden D, Oosterlynck O, De Zutter L, Eilers K, Piérard D, Dierick K, Van Damme-Lombaerts R, Lauwers C, Jacobs R. Department of Control of Infectious Diseases, Antwerp, Belgium. [email protected] In October 2007, an outbreak of verocytotoxin-producing Escherichia coli (VTEC) O145 and E. coli O26 occurred among consumers of ice cream produced and sold in September 2007 at a farm in the province of Antwerp (Belgium). The ice cream was consumed at two birthday parties and also eaten at the farm. Five children, aged between two and 11 years, developed haemolytic uraemic syndrome (HUS), and seven other co-exposed persons contracted severe diarrhoea. In three of the five HUS cases VTEC O145 infections were laboratory confirmed, one in association with VTEC O26. Identical isolates of E. coli O145 and O26 were detected with PCR and PFGE in faecal samples of patients and in ice cream leftovers, in faecal samples taken from calves, and in samples of soiled straw from the farm at which the ice cream was produced. Ice cream was made from pasteurised milk and most likely contaminated by one of food handlers. 78: Epidemiol Infect. 2006 Aug;134(4):724-8. Epub 2005 Dec 22. Links

Haemolytic uraemic syndrome associated with interfamilial spread of E. coli O26:H11. Sayers G, McCarthy T, O'Connell M, O'Leary M, O'Brien D, Cafferkey M, McNamara E. Department of Public Health, Health Service Executive Eastern Region, Dublin. [email protected] In September 2000, haemolytic uraemic syndrome (HUS) was diagnosed in a 10-month-old child with a prodromal history of vomiting and diarrhoea (non-bloody). Investigation revealed that a self-limiting gastrointestinal illness (mean duration 48 h) had occurred among immediate and extended family in the 2 weeks prior to the child's admission.

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epidemiology of the illness suggested person-to-person spread.

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79: Epidemiol Infect. 2006 Aug;134(4):719-23. Epub 2005 Dec 22. Links

Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157: indications for an animal reservoir. Orth D, Grif K, Dierich MP, Würzner R. Department of Hygiene, Microbiology and Social Medicine, Innsbruck Medical University and Austrian Reference Laboratory for EHEC/STEC/VTEC, Innsbruck, Austria. This study investigates a sorbitol-fermenting enterohaemorrhagic Escherichia coli (SF EHEC) O157 infection in a farmer's family in the Austrian province of Salzburg. The investigation commenced after a 10-month-old boy was admitted to hospital with the clinical diagnosis of a haemolytic-uraemic syndrome (HUS) and his stool specimen grew SF EHEC O157:H-. In a subsequent environmental survey, a stool specimen of the 2-year-old brother and faecal samples of two cattle from the family's farm were also found to be positive for SF EHEC O157:H-. All four isolates had indistinguishable phenotypic and molecular characteristics and were identical to the first strain detected in Bavaria in 1988. Despite identical isolates being demonstrated in Bavaria after 1988, and until this report, increased surveillance in neighbouring Austria had not found this organism. We propose that the strain may have recently spread

Although SF EHEC O157:H- strains are still rare, they may represent a considerable health threat as they can spread from farm animals to humans and between humans. from Bavaria to Austria.

87: Int J Med Microbiol. 2005 Oct;295(6-7):405-18.

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Enterohaemorrhagic Escherichia coli in human medicine. Karch H, Tarr PI, Bielaszewska M. Institute for Hygiene, and National Consulting Laboratory on Haemolytic Uraemic Syndrome, University Hospital Münster, Robert Koch Str. 41, D-48149 Münster, Germany. [email protected] Enterohaemorrhagic Escherichia coli (EHEC) are the pathogenic subgroup of Shiga toxin (Stx)-producing E. coli. EHEC can cause non-bloody and bloody diarrhoea, and the haemolytic uraemic syndrome (HUS). HUS is a major cause of acute renal failure in children. E. coli O57:H7 is the predominant, but far from 11

being the only, serotype that can cause HUS. The cascade leading from gastrointestinal infection to renal impairment is complex, with the microvascular endothelium being the major histopathological target. EHEC also produce nonStx molecules, such as cytolethal distending toxin, which can contribute to the endothelial or vascular injury. Because there are no specific therapies for EHEC infections, efficient reservoir and human preventive strategies are important areas of ongoing investigations. This review will focus on the microbiology, epidemiology, and pathophysiology of EHEC-associated diseases, and illustrate future challenges and opportunities for their control.

30: J Appl Microbiol. 2008 Jan;104(1):14-25.

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Escherichia coli serogroup O26--a new look at an old adversary. Jenkins C, Evans J, Chart H, Willshaw GA, Frankel G. Department of Medical Microbiology, Royal Free Hospital, NW3 2QG, London, UK. [email protected] Escherichia coli serogroup O26 played an important part in the early work on Verocytotoxin and is an established diarrhoeal pathogen. Recently, Verocytotoxigenic E. coli (VTEC) O26 has been increasingly associated with diarrhoeal disease and frequently linked to outbreaks and cases of haemolytic uraemic syndrome (HUS). This review investigates the pathogenicity, geographical distribution, changing epidemiology, routes of transmission and improved detection of VTEC O26. Laboratory data on VTEC O26 isolates and clinical data on HUS suggest a true difference in the incidence of VTEC O26 in different geographic locations. However, few diagnostic laboratories use molecular methods to detect VTEC and so it is difficult to assess the role of VTEC O26 in causing diarrhoeal disease.

VTEC O26 is frequently found in the cattle population but rarely in food. However, the small number of outbreaks analysed to date are thought to be food-borne rather than associated with direct or indirect contact with livestock or their faeces. The increase in awareness of VTEC O26 in the clinical and veterinary setting has coincided with the development of novel techniques that have improved our ability to detect and characterize this pathogen.

153: Mol Biotechnol. 2004 Feb;26(2):117-22.

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Infection by Shiga toxin-producing Escherichia coli: an overview. Karmali MA.

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Laboratory for Foodborne Zoonoses, Health Canada, 110 Stone Road West, Guelph, Ontario, Canada, N1G 3W4. [email protected] Shiga toxin-producing Escherichia coli (STEC), especially of serotype O157:H7, cause a zoonotic food or waterborne enteric illness that is often associated with large epidemic outbreaks as well as the hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. After ingestion, STEC colonize enterocytes of the large bowel with a characteristic attaching and effacing pathology, which is mediated by components of a type III secretion apparatus encoded by the LEE pathogenicity island. Shiga toxins are translocated from the bowel to the circularoty system and transported by leukocytes to capillary endothelial cells in renal glomeruli and other organs. After binding to the receptor globotriaosylceramide on target cells, the toxin is internalized by receptormediated endocytosis and interacts with the subcellular machinery to inhibit protein synthesis. This leads to pathophysiological changes that result in HUS. Specific therapeutic or preventive strategies are presently not available. The recent sequencing of genomes of two epidemic E. coli O157 strains has revealed novel pathogenicity islands which will likely provide new insights into the virulence of these bacteria. PMID: 14764937 [PubMed - indexed for MEDLINE]

32: Pediatr Nephrol. 2008 Feb;23(2):221-31. Epub 2007 Nov 30. Links

Role of the renin angiotensin system in TNF-alpha and Shiga-toxin-induced tissue factor expression. Nestoridi E, Kushak RI, Tsukurov O, Grabowski EF, Ingelfinger JR. Pediatric Nephrology Laboratory, MassGeneral Hospital for Children at Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. Current evidence implicates a prothrombotic state in the development of Shiga-toxin (Stx)-mediated hemolytic uremic syndrome (HUS). We recently reported that Stx modulates procoagulant activity by enhancing functional tissue factor (TF) activity on cytokine-activated human glomerular endothelial cells (HGECs). Since angiotensin II (Ang II), the key effector of the renin angiotensin system (RAS), has been shown to increase TF expression in vascular tissue, we examined the possible involvement of Ang II in TF expression in HGECs. HGECs were exposed to tumor necrosis factor (TNF)-alpha +/- Stx-1 +/- Ang II. Exogenous Ang II significantly increased TF activity and TF mRNA in TNF-alpha- +/- Stx-1-activated HGECs. This

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increase was mediated via Ang II type I receptor (AT(1)R), as losartan, an AT(1)R inhibitor, attenuated Ang-II-induced TF activity. To study the effect of endogenous Ang II in TF expression by TNF-alpha +/- Stx-1, HGECs were incubated with losartan or an AT(2)R inhibitor (PD 123319) or an angiotensin-converting enzyme inhibitor (enalapril). Losartan but not PD 123319 decreased TF activity induced by TNF-alpha +/- Stx-1 (P < 0.05). Enalapril, also, dose dependently, downregulated TF expression in HGECs exposed to TNF-alpha +/- Stx-1 (P < 0.05). AT(1)R mRNA was upregulated in

These data indicate that TF expression in TNF-alpha- and Stx-1-activated HGECs is enhanced by exogenous Ang II and that endogenous Ang II production may be upregulated by TNF-alpha +/- Stx-1. Hence, local RAS activation may be important in the development of the thrombotic microangiopathy observed in HUS. TNF-alpha- +/- Stx-1-activated HGECs (P < 0.05).

PMID: 18060435 [PubMed - indexed for MEDLINE]

Shiga toxin-mediated hemolytic uremic syndrome: time to change the diagnostic paradigm? Bielaszewska M, Köck R, Friedrich AW, von Eiff C, Zimmerhackl LB, Karch H, Mellmann A. Institute of Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Münster, Münster, Germany. [email protected] BACKGROUND: Hemolytic uremic syndrome (HUS) is caused by enterohemorrhagic Escherichia coli (EHEC) which possess genes encoding Shiga toxin (stx), the major virulence factor, and adhesin intimin (eae). However, the frequency of stx-negative/eae-positive E. coli in stools of HUS patients and the clinical significance of such strains are unknown. METHODOLOGY/PRINCIPAL FINDINGS: Between 1996 and 2006, we sought stx-negative/eae-positive E. coli in stools of HUS patients using colony blot hybridization with the eae probe and compared the isolates to EHEC causing HUS. stx-negative/eae-positive E. coli were isolated as the only pathogens from stools of 43 (5.5%) of 787 HUS patients; additional 440 (55.9%) patients excreted EHEC. The

majority (90.7%) of the stx-negative/eaepositive isolates belonged to serotypes O26:H11/NM (nonmotile), O103:H2/NM, O145:H28/NM, and O157:H7/NM, which were also the most frequent serotypes identified among EHEC. The stx-negative isolates shared non-stx virulence and fitness genes with EHEC of the corresponding serotypes and clustered with them into the same clonal complexes in multilocus sequence typing, demonstrating their close relatedness to EHEC. CONCLUSIONS/SIGNIFICANCE: At the time of microbiological analysis, approximately 5% of HUS patients shed no longer the causative EHEC, but do excrete stx-negative derivatives of EHEC that lost stx during infection. In such patients, the EHEC etiology of HUS is missed using current methods detecting solely stx or Shiga toxin; this can hamper epidemiological investigations and lead to inappropriate clinical management. While maintaining the paradigm that HUS is triggered by Shiga toxin, our data demonstrate the necessity of considering genetic changes of the pathogen during infection to adapt appropriately diagnostic strategies.

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PMID: 17925872 [PubMed - indexed for MEDLINE] PMCID: PMC1995754 37: Berl Munch Tierarztl Wochenschr. 2007 Jul-Aug;120(7-8):279-87.

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Enterohaemorrhagic Escherichia coli O26:H11/H-: a human pathogen in emergence. Bielaszewska M, Zhang W, Mellmann A, Karch H. Institut für Hygiene, und Nationales Konsiliarlaboratorium für HämolytischUrämisches Syndrom, Universität Münster, Robert Koch Str. 41, 48149 Münster, Germany. [email protected] Enterohaemorrhagic Escherichia coli (EHEC) O26:H11 have emerged as the most important non-O157:H7 EHEC, with respect to their ability to cause diarrhoea and the haemolytic uraemic syndrome (HUS). HUS is a leading cause of acute renal failure in children, and is mainly caused by EHEC expressing Shiga toxins (Stx) 1 and/or 2. Since 1996, EHEC O26, which produce Stx2 only and appear to have enhanced virulence, have been increasingly isolated from HUS patients in Germany. In contrast, EHEC O26 found in cattle predominantly produce Stx1 as the sole Stx. Additional potential virulence factors of EHEC O26 include cytolysins (EHEC hemolysin), serine proteases (EspP), lymphotoxins (Efal) and adhesins (intimin). The genes encoding the virulence factors are located within pathogenicity islands (eae, efa1), bacteriophages (stx) or plasmids (EHEChlyA, espP). In addition, EHEC O26 possess, in contrast to other EHEC, the "high pathogenicity island" (HPI), which is also present in pathogenic Yersiniae.This island contains genes involved in the biosynthesis, regulation and transport of the siderophore yersiniabactin. Comparative genomic analyses between EHEC O26 and non-pathogenic E. coli, as well as investigations of mechanisms involved in the transfer of virulence genes, provide a deeper insight into the evolution of EHEC O26.These studies demonstrate how horizontal transfer of virulence genes, even from distantly related organisms, can lead in brief intervals to the rise of a highly virulent clone within a particular E. coli serotype.The classical bacteriological methods are no longer sufficient to determine the risk posed by EHEC O26. However, knowledge of the complete virulence profiles of these pathogens and understanding their stepwise evolution form a foundation for developing new strategies to prevent human infections and new methods for their laboratory diagnosis. PMID: 17715820 [PubMed - indexed for MEDLINE

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40: Clin Infect Dis. 2007 Jul 1;45(1):39-45. Epub 2007 May 21.

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Prevalence, virulence profiles, and clinical significance of Shiga toxin-negative variants of enterohemorrhagic Escherichia coli O157 infection in humans. Friedrich AW, Zhang W, Bielaszewska M, Mellmann A, Köck R, Fruth A, Tschäpe H, Karch H. National Consulting Laboratory on Hemolytic Uremic Syndrome, Institute for Hygiene, University of Münster, Münster, Germany. BACKGROUND: Escherichia coli O157, of the H7 clone, exists in humans and in the environment as Shiga toxin (Stx)-positive and Stx-negative variants. Stx production by infecting organisms is considered to be a critical requirement for the development of hemolytic uremic syndrome (HUS), which occurs in approximately 15% of E. coli O157-infected patients. It is unknown if loss of the stx gene during the early stage of an enterohemorrhagic E. coli infection prevents HUS, or if absence of the stx gene from E. coli O157 reduces or ablates virulence. METHODS: We determined the frequency of stx-positive and stx-negative E. coli O157 isolates in stool samples obtained from patients who experienced sporadic cases of diarrhea or HUS, as well as the frequency in samples obtained during outbreaks, and investigated the clinical course of the disease. RESULTS: Among E. coli O157 isolates obtained from samples related to sporadic cases of diarrhea, stx-negative strains accounted for 4%. The proportion of stx-negative strains was significantly higher among sorbitol-fermenting, nonmotile E. coli O157 isolates (12.7%) than among non-sorbitol-fermenting E. coli O157:H7 or nonmotile isolates (0.8%; P<.001). stx-Negative sorbitol-fermenting E. coli O157 isolates were also observed in samples related to 3 HUS outbreaks and 1 outbreak of diarrhea caused by sorbitol-fermenting, nonmotile enterohemorrhagic E. coli O157; additionally, they were the only pathogens that were isolated in 2 other outbreaks of diarrhea without HUS.

CONCLUSIONS: Strains of stx-negative E. coli O157 isolated from stool samples of patients are either inherently stx-negative strains that cause mostly uncomplicated diarrhea, or strains that descended from enterohemorrhagic E. coli O157 by the loss of the stx gene during infection; the latter strains may still cause severe disease. PMID: 17554698 [PubMed - indexed for MEDLINE] 41: Microbes Infect. 2007 Jun;9(7):891-7. Epub 2007 Mar 21.

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Presence of virulence and fitness gene modules of enterohemorrhagic Escherichia coli in atypical enteropathogenic Escherichia coli O26. Bielaszewska M, Sonntag AK, Schmidt MA, Karch H.

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Institute for Hygiene, and the National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Münster, Robert Koch Strasse 41, D-48149 Münster, Germany. [email protected] Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O26 cause hemolytic-uremic syndrome (HUS) whereas atypical enteropathogenic E. coli (aEPEC) O26 typically cause uncomplicated diarrhea but have been also isolated from HUS patients. To gain insight into the virulence of aEPEC O26, we compared the presence of O island (OI) 122, which is associated with enhanced virulence in EHEC strains, among aEPEC O26 and EHEC O26 clinical isolates. We also tested these strains for the high pathogenicity island (HPI) which is a fitness island. All 20 aEPEC O26 and 20 EHEC O26 investigated contained virulence genes located within OI-122 (efa1/lifA, nleB, nleE, ent). In

both aEPEC O26 and EHEC O26, OI-122 was linked to the locus for enterocyte effacement, forming a mosaic island which was integrated in pheU. Moreover, strains of these two pathotypes shared a conserved HPI. These data support a close relatedness between aEPEC O26 and EHEC O26 and have evolutionary implications. The presence of OI-122 in aEPEC O26 might contribute to their pathogenic potential.

: Microbes Infect. 2007 Mar;9(3):282-90. Epub 2006 Dec 22.

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Hemolysin from Shiga toxin-negative Escherichia coli O26 strains injures microvascular endothelium. Aldick T, Bielaszewska M, Zhang W, Brockmeyer J, Schmidt H, Friedrich AW, Kim KS, Schmidt MA, Karch H. Institute of Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, University of Münster, Robert Koch Str. 41, 48149 Münster, Germany. We identified Shiga toxin gene (stx)-negative Escherichia coli O26:H11 and O26:NM (nonmotile) strains as the only pathogens in the stools of five patients with hemolytic-uremic syndrome (HUS). Because the absence of stx in E. coli associated with HUS is unusual, we examined the strains for potential virulence factors and interactions with microvascular endothelial cells which are the major targets affected during HUS. All five isolates possessed the enterohemorrhagic E. coli (EHEC)-hlyA gene encoding EHEC hemolysin (EHEC-Hly), expressed the enterohemolytic phenotype, and were cytotoxic, in dose- and time-dependent manners, to human brain microvascular endothelial cells (HBMECs). Significantly reduced cytotoxicity in an EHEC-Hly-negative spontaneous derivative of one of these strains,

the endothelial cytotoxicity of these strains was mediated by EHEC-Hly. The toxicity of EHEC-Hly to microvascular endothelial cells plausibly contributes and a dose- and time-dependent cytotoxicity of recombinant E. coli O26 EHEC-Hly to HBMECs, suggest that

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to the virulence of the stx-negative E. coli O26 strains and to the pathogenesis of HUS. 61: J Infect Dis. 2006 Sep 15;194(6):819-27. Epub 2006 Aug 11. Links

Bacterial genetic determinants of non-O157 STEC outbreaks and hemolytic-uremic syndrome after infection. Wickham ME, Lupp C, Mascarenhas M, Vazquez A, Coombes BK, Brown NF, Coburn BA, Deng W, Puente JL, Karmali MA, Finlay BB. Michael Smith Laboratories, University of British Columbia, Vancouver BC, Canada. Although O157:H7 Shiga toxin-producing Escherichia coli (STEC) are the predominant cause of hemolytic-uremic syndrome (HUS) in the world, nonO157:H7 serotypes are a medically important cause of HUS that are underdetected by current diagnostic approaches. Because Shiga toxin is necessary but not sufficient to cause HUS, identifying the virulence determinants that predict severe disease after non-O157 STEC infection is of paramount importance. Disease caused by O157:H7 STEC has been associated with a 26-gene pathogenicity island known as O island (OI) 122. To assess the public-health significance of this pathogenicity island, we examined the association between OI122 genes and outbreaks and HUS after non-O157 STEC infection. We found that a subset of OI122 genes is independently associated with outbreaks and HUS after infection with non-O157 STEC. The presence of multiple virulence genes in non-O157 serotypes strengthened this association, which suggests that the additive effects of a variable repertoire of virulence genes contribute to disease severity. In vivo, Citrobacter rodentium mutants lacking outbreak- and HUS-associated genes were deficient for virulence in mice; in particular, nleB mutant bacteria were unable to cause mortality in mice. The

present study shows that virulence genes associated epidemiologically with outbreaks and HUS after non-O157 STEC infection are pivotal to the initiation, progression, and outcome of in vivo disease.

90: J Infect Dis. 2005 Oct 15;192(8):1422-9. Epub 2005 Sep 14.

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Non-O157 Shiga toxin-producing Escherichia coli infections in the United States, 1983-2002. Brooks JT, Sowers EG, Wells JG, Greene KD, Griffin PM, Hoekstra RM, Strockbine NA. BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a well-recognized cause of bloody diarrhea and hemolytic-uremic syndrome (HUS). Non-O157 STEC contribute to this burden of illness but have been

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underrecognized as a result of diagnostic limitations and inadequate surveillance. METHODS: Between 1983 and 2002, 43 state public health laboratories submitted 940 human nonO157 STEC isolates from persons with sporadic illnesses to the Centers for Diseases Control and Prevention reference laboratory for confirmation and serotyping. RESULTS: The most common serogroups were O26 (22%), O111 (16%), O103 (12%), O121 (8%), O45 (7%), and O145 (5%). Non-O157 STEC infections were most frequent during the summer and among young persons (median age, 12 years; interquartile range, 3-37 years). Virulence gene profiles were as follows: 61% stx(1) but not stx(2); 22% stx(2) but not stx(1); 17% both stx(1) and stx(2); 84% intimin (eae); and 86% enterohemolysin (E-hly). stx(2) was strongly associated with an increased risk of HUS, and eae was strongly associated with an increased risk of bloody diarrhea. STEC O111 accounted for most cases of HUS and was also the cause of 3 of 7 non-O157 STEC outbreaks reported in the United States. CONCLUSIONS:

Non-O157 STEC can cause severe illness that is comparable to the illness caused by STEC O157. Strains that produce Shiga toxin 2 are much more likely to cause HUS than are those that produce Shiga toxin 1 alone. Improving surveillance will more fully elucidate the incidence and pathological spectrum of these emerging agents. These efforts require increased clinical suspicion, improved clinical laboratory isolation, and continued serotyping of isolates in public health laboratories.

70: Blood. 2006 Jul 1;108(1):167-76. Epub 2006 Mar 2. Links

Lipopolysaccharide from enterohemorrhagic Escherichia coli binds to platelets through TLR4 and CD62 and is detected on circulating platelets in patients with hemolytic uremic syndrome. Ståhl AL, Svensson M, Mörgelin M, Svanborg C, Tarr PI, Mooney JC, Watkins SL, Johnson R, Karpman D. This study presents evidence that human platelets bind lipopolysaccharide (LPS) from enterohemorrhagic Escherichia coli (EHEC) through a complex of toll-like receptor 4 (TLR4) and CD62, leading to their activation. TLR4 colocalized with CD62 on the platelet membrane, and the TLR4 specificity of LPS binding to platelets was confirmed using C57BL/10ScN mice lacking Tlr4. Only platelets from TLR4 wild-type mice bound O157LPS in vitro. After in vivo injection, O157LPS bound to platelets from wild-type mice, which had lower platelet counts than did mice lacking TLR4. Mouse experiments confirmed that O157LPS binding to TLR4 is the primary event leading to platelet activation, as shown by CD40L expression, and that CD62 further contributes to this process. Activation of human platelets by EHECLPS was demonstrated by expression of the activated GPIIb/IIIa receptor, CD40L, and fibrinogen binding. In perfusion experiments, platelet activation on endothelial cells was TLR4 and CD62 dependent. O157LPS was detected 19

on platelets from 12 of 14 children with EHEC-associated hemolytic uremic syndrome (HUS) and on platelets from 2 children before the development of HUS but not on platelets of EHEC-infected children in whom HUS did not develop (n = 3). These data suggest that O157LPS on platelets might contribute to platelet consumption in HUS 168: Eur J Clin Microbiol Infect Dis. 2003 Dec;22(12):726-30. Epub 2003 Nov 12. Links

Strong association between shiga toxin-producing Escherichia coli O157 and virulence genes stx2 and eae as possible explanation for predominance of serogroup O157 in patients with haemolytic uraemic syndrome. Werber D, Fruth A, Buchholz U, Prager R, Kramer MH, Ammon A, Tschäpe H. Department for Infectious Disease Epidemiology, Robert Koch-Institut, Seestrasse 10, 13353 Berlin, Germany. [email protected] The aim of this study was to investigate the association between Shiga toxinproducing Escherichia coli (STEC) O157 and the simultaneous presence of the virulence genes stx2 and eae in STEC from patients with gastroenteritis. In Germany, the proportion of serogroup O157 is substantially higher among STEC isolates from patients with haemolytic uraemic syndrome (HUS) than among STEC isolates from patients with gastroenteritis. The reason for this is unknown. Independent of serogroup, the virulence genes stx2 and eae have been associated with severe disease. Data collected in 2000-2001 from a Germany-wide laboratory-based surveillance system for STEC-associated gastroenteritis in patients <15 years were analysed. Overall, 18% of the STEC isolates belonged to serogroup O157. Compared with non-O157 strains, 0157 isolates were strongly associated with the simultaneous presence of both an stx2 gene and the eae gene (OR, 76; 95%CI, 27-230). Within the subset of STEC isolates that carried both virulence genes, 60% belonged to serogroup O157, a proportion similar to that found in STEC isolates from pediatric patients with HUS in Germany and Austria (67%, P=0.35).

These data suggest that the more frequent carriage of both virulence genes, i.e. stx2 and eae, forms the basis of why STEC O157 predominates in patients with HUS. PMID: 14614596 [PubMed - indexed for MEDLINE] •

169: J Clin Microbiol. 2003 Nov;41(11):4930-40. Links

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Association of genomic O island 122 of Escherichia coli EDL 933 with verocytotoxin-producing Escherichia coli seropathotypes that are linked to epidemic and/or serious disease. Karmali MA, Mascarenhas M, Shen S, Ziebell K, Johnson S, Reid-Smith R, Isaac-Renton J, Clark C, Rahn K, Kaper JB. Laboratory for Foodborne Zoonoses, Population and Public Health Branch, Health Canada, Guelph, Ontario, Canada. [email protected] The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five "seropathotypes" (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolyticuremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an "incomplete" OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant (P < 0.0001). PMID: 14605120 [PubMed - indexed for MEDLINE] 172: J Med Microbiol. 2003 Nov;52(Pt 11):941-7.

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Subtyping of virulence genes in verocytotoxinproducing Escherichia coli (VTEC) other than serogroup O157 associated with disease in the United Kingdom. Jenkins C, Willshaw GA, Evans J, Cheasty T, Chart H, Shaw DJ, Dougan G, Frankel G, Smith HR. Verocytotoxin-producing Escherichia coli (VTEC) causes a wide spectrum of disease in humans, from mild diarrhoea to haemolytic uraemic syndrome (HUS). The verocytotoxin (vtx) and intimin (eae) genes of VTEC strains, other than those of serogroup O157, were subtyped to identify common properties that may be associated with increased pathogenicity. Strains were isolated from patients with HUS, those with diarrhoea or from asymptomatic individuals. Strains of VTEC that carried vtx(2) gene subtypes vtx(2) and vtx(2c) were most commonly associated with HUS, whereas strains from patients with less severe disease and from the healthy control group were more likely to have vtx(1c) or vtx(2d) genes. The eae gene was detected more frequently in strains isolated from HUS patients than in those associated with cases of diarrhoea; beta-intimin was the most common intimin subtype in strains isolated from both groups of patients. None of the strains from the healthy control group carried the eae gene.

57: Mol Immunol. 2007 Mar;44(8):1835-44. Epub 2006 Nov 7. Links

A mutation in factor I that is associated with atypical hemolytic uremic syndrome does not affect the function of factor I in complement regulation. Nilsson SC, Karpman D, Vaziri-Sani F, Kristoffersson AC, Salomon R, Provot F, Fremeaux-Bacchi V, Trouw LA, Blom AM. Lund University, Department of Laboratory Medicine, University Hospital Malmö, S-205 02 Malmö, Sweden. Factor I (FI) is the major complement inhibitor that degrades C3b and C4b in the presence of cofactors such as factor H (FH) and membrane cofactor protein (MCP). Recently, mutations and polymorphisms in complement regulator molecules FH and MCP but also in FI have been associated with atypical hemolytic uremic syndrome (aHUS). HUS is a disorder characterized by

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hemolytic anemia, thrombocytopenia and acute renal failure. In this study, we report three unrelated patients with an identical heterozygous mutation, G261D, in the FI heavy chain who developed severe aHUS at different time points in their lives. Two of the patients also have polymorphisms in FH previously associated with risk of developing aHUS. Testing in particular one patient and control serum samples we did not observe major differences in complement hemolytic activity, FI plasma levels or the capability to degrade C4b or C3b. A recombinant protein was produced in order to analyze the functional consequences of the mutation. Mutant FI had a slightly different migration pattern during electrophoresis under reducing conditions. An alteration due to alternative splicing or glycosylation was ruled out, thus the altered migration may be due to proximity of the mutation to a cysteine residue. The recombinant mutant FI degraded C3b and C4b in a manner comparable to wild-type protein.

In conclusion, despite the association between the heterozygous mutation in FI and aHUS we did not observe any abnormalities in the function of FI regarding complement regulation. 89: Am J Kidney Dis. 2005 Oct;46(4):e59-63.

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An adult with acute poststreptococcal glomerulonephritis complicated by hemolytic uremic syndrome and nephrotic syndrome. Izumi T, Hyodo T, Kikuchi Y, Imakiire T, Ikenoue T, Suzuki S, Yoshizawa N, Miura S. Second Department of Internal Medicine, National Defense Medical College, Tokorozawa, Saitama, Japan. We report the case of a 47-year-old man with the simultaneous occurrence of clinical and laboratory features consistent with acute poststreptococcal glomerulonephritis (APSGN), hemolytic uremic syndrome (HUS), and nephrotic syndrome. Acute nephritic syndrome occurred 3 weeks after having pharyngeal pain and diarrhea. He presented with edema and hypertension on admission. Laboratory evaluation showed hemolytic anemia with fragmentation, thrombocytopenia, elevated lactic dehydrogenase level, low haptoglobin level, low complement C3 level, and elevated antistreptolysin-O titer. Serum creatinine level was 1.22 mg/dL (108 micromol/L), and urinalysis showed marked proteinuria, with protein of 8.7 g/d, and hematuria. The renal biopsy specimen was characteristic of APSGN, but not HUS. Moderate expansion of the mesangial matrix, moderate proliferation of epithelial and endothelial cells, and marked infiltration of neutrophils was seen by means of light microscopy, and many subepithelial humps were seen by means of electron microscopy. Neither fibrin deposition nor evidence of thrombotic microangiopathy was found. Complement C3 deposition along the capillary wall and tubules was seen in an immunofluorescence study. The patient was administered plasma infusion at 320 mL/d and antihypertensive drugs. Serum complement C3 and haptoglobin levels returned to normal within 3 weeks. This is a rare case of the simultaneous occurrence of APSGN, HUS, and nephrotic syndrome.

Renal complications associated with human parvovirus B19 infection in early childhood. Härtel C, Herz A, Vieth S, Lensing C, Schultz C. Department of Pediatrics of Medical Microbiology at University of Lübeck. [email protected] 23

A previously healthy two-year-old girl presented with proteinuria and macroscopic haematuria. Laboratory findings included haemolytic anaemia with thrombocytopenia. Interestingly, continuing reticulocytopenia was noted. Therefore an acute parvovirus B19 infection was suspected, which could be confirmed by serological and molecularbiological evidence.

This case report underlines renal complications of parvovirus B19 infection in early childhood including haemolytic-uraemic syndrome (HUS)-like episodes, and potential pathogenetic mechanisms are discussed. 65: Pediatr Infect Dis J. 2006 Jul;25(7):608-10.

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T-antigen activation for prediction of pneumococcus-induced hemolytic uremic syndrome and hemolytic anemia. Huang DT, Chi H, Lee HC, Chiu NC, Huang FY. Department of Pediatrics, Mackay Memorial Hospital, Taipei, Taiwan. BACKGROUND: Among the most severe complications of invasive pneumococcal infection are hemolytic uremic syndrome (P-HUS) and hemolytic anemia (P-HA), which occur when the Thomsen-Freidenreich antigen (TA) is exposed on erythrocytes, platelets and glomeruli. METHODS: To determine the positive predictive value, sensitivity, and specificity of early TA activation testing for P-HUS or P-HA and to compare the microbiologic features of pneumococcus isolates associated or not associated with TA activation. The case records for 36 patients with invasive pneumococcal infection who had been tested for TA activation were retrospectively

TA activation. RESULTS: Positive TA activation was 86% sensitive and 57% specific for P-HUS or P-HA. The positive predictive value was 76%. There were no between-group differences in antibiotic susceptibility reviewed. Clinical and laboratory data were compared between patients with and without

of the pneumococcal isolates. Pneumococcal serotype 14 was the most frequent (5/10 isolates tested) serotype causing PHUS. Of the 36 patients, 13 required packed red blood cell transfusion, 3 died, and 2 required extracorporeal membrane

CONCLUSIONS: TA activation is a reasonable predictor of P-HUS or P-HA and could be useful if tested soon after invasive pneumococcal disease is first diagnosed. oxygenation. No patient had long-term renal sequelae.

144: Am J Kidney Dis. 2004 Jun;43(6):976-82.

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Non-enteropathic hemolytic uremic syndrome: causes and short-term course. Constantinescu AR, Bitzan M, Weiss LS, Christen E, Kaplan BS, Cnaan A, Trachtman H. Division of Pediatric Nephrology, University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, New Brunswick, NJ, USA.

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BACKGROUND: Nondiarrheal or Streptococcus pneumoniae-related hemolytic uremic syndrome (HUS) represents a heterogeneous group of disorders. This study was performed to: (1) describe the current incidence, causes, demographic features, hospital courses, and short-term outcomes of nonenteropathic HUS; (2) compare findings in patients with non-enteropathic HUS with those obtained from a contemporaneous cohort of children with enteropathic or diarrhea-associated HUS (D+ HUS) diagnosed and treated at the same clinical sites; and (3) identify clinical or laboratory features that differentiate these 2 groups and predict disease severity and the short-term outcome in patients with non-enteropathic HUS. METHODS: Data were collected from patients screened between 1997 and 2001 for enrollment in a multicenter trial of SYNSORB Pk (SYNSORB Biotech Inc, Calgary, Alberta, Canada) in D+ HUS, but who were ineligible because of lack of a diarrhea prodrome. The following features were recorded: age; sex; ethnicity; prodromal symptoms; cause; nadir values for hemoglobin, hematocrit, and platelet count; use of dialysis; and length of hospitalization. RESULTS: Twenty-seven of 247 children with HUS had non-enteropathic HUS (11%). Twenty-four patients (15 boys, 9 girls), whose medical records were complete and available for review, comprise the study cohort. Mean age at onset was 4.2 +/- 0.9 (SE) years. Infection caused by S pneumoniae was diagnosed in 9 patients (38%). Dialysis was performed in 17 patients (71%) for 40 +/- 27 days. Median length of hospitalization was 22 days (range, 2 to 71 days). Children with S pneumoniae-related HUS had a longer hospital stay than those with other causes of non-enteropathic HUS, but all patients with S pneumoniae-related HUS recovered kidney function. Dialysis therapy was required more often (17 of 24 versus 59 of 145 children; P = 0.025) and hospital stays were longer (median, 22 versus 9 days; P = 0.002) in children with non-enteropathic HUS compared with patients with D+ HUS who were enrolled in the SYNSORB Pk clinical trial. CONCLUSION:

(1) The incidence of non-enteropathic HUS is approximately one tenth that of D+ HUS; (2) patients with nonenteropathic HUS require dialysis therapy more often and are hospitalized more than twice as long during the acute episode compared with those with D+ HUS; (3) infection caused by S pneumoniae accounts for nearly 40% of cases of non-enteropathic HUS; and (4) although S pneumoniae-related HUS is associated with a less favorable short-term course than other types of nonenteropathic HUS or D+ HUS, the long-term prognosis for recovery of renal function appears to be good in these patients. 148: Lett Appl Microbiol.

ADULTS 140: Ther Apher Dial. 2004 Apr;8(2):102-11.

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Drug-induced thrombotic thrombocytopenic purpura/hemolytic uremic syndrome: a concise review. Dlott JS, Danielson CF, Blue-Hnidy DE, McCarthy LJ. Department of Pathology and Laboratory Medicine (Transfusion Medicine), Indiana University School of Medicine, Indianapolis, IN, USA. An extensive variety of drugs have been associated with thrombotic thrombocytopenic purpura and hemolytic uremic syndrome (TTP/HUS). Although a direct causal effect has usually not been proven, the cumulative evidence linking several drugs with TTP/HUS is strong. This paper reviews 25

several categories of drugs including antineoplastics, immunotherapeutics and anti-platelet agents that have been reported to induce TTP/HUS. The pathogenesis of drug-induced TTP/HUS and the effectiveness of treatment regimens are also reviewed. A consensus on diagnostic criteria to accurately and consistently diagnose drug-induced TTP is needed.

162: Am J Med Sci. 2003 Dec;326(6):378-80.

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Quinine induced HUS-TTP: an unusual presentation. Baliga RS, Wingo CS. Department of Internal Medicine, University of Florida & The Malcolm Randall VA Medical Center, Gainsville, Florida 32608, USA. [email protected] A 67-year-old white woman developed severe nausea, vomiting, diffuse abdominal cramping pain, and blurred vision followed by a syncopal episode after taking 1 tablet of quinine for leg cramps. Examination was significant for fever, elevated blood pressure, and confusion without any focal neurological deficits.

Laboratory

studies confirmed HUS, showed markedly elevated liver enzymes, elevated lactate dehydrogenase, anemia, thrombocytopenia, and acute renal failure. Peripheral smear showed many schistocytes and burr cells. She later recalled taking quinine more than 40 years before while on a trip to the Philippines. The patient was treated with 7 sessions of plasmapheresis with a rapid normalization of her hematological parameters. Three weeks of dialysis support were required

Re-exposure to quinine can cause a rapid onset of hemolytic uremic syndrome-like syndrome. We are not aware of any cases of hemolytic uremic syndrome-thrombotic thrombocytopenic purpura in response to re-exposure to a single tablet of the drug 40 years after first use. before return of renal function to baseline.

: Transplant Proc. 2008 May;40(4):1114-6.

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Postrenal transplant hemolytic uremic syndrome/thrombotic microangiopathy: Ahmedabad experience. Goplani KR, Vanikar AV, Shah PR, Gumber M, Feroz A, Patel HV, Kasat P, Falodia J, Saboo D, Kaswaan K, Geerish MS, Pandya T, Trivedi HL. Department of Nephrology and Transplantation Medicine, G.R. Doshi and K.M. Mehta Institute of Kidney Diseases and Research Centre, Dr H.L. Trivedi Institute of Transplantation Sciences, Gujarat, India. [email protected]

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BACKGROUND: Hemolytic uremic syndrome (HUS)/thrombotic microangiopathy (TMA) (tissue-limited HUS) is a well-recognized serious complication of renal transplantation, affecting 3% to 14% patients who are administered calcineurin inhibitor-based immunosuppression. We performed a retrospective study to examine the incidence, etiology, course, and outcome of HUS/TMA in our experience. PATIENTS AND METHODS: This retrospective study of 1540 renal allograft biopsies performed between January 2000 and October 2007 was performed to assess the incidence of HUS/TMA. Institute Transplant Registry records were reviewed for clinical history, laboratory findings, medications, and outcome. The offending drug was substituted in all subjects and plasmapheresis was added as an adjuvant until recovery of allograft function. RESULTS: TMA was observed in 17 (1.1%) biopsies. Two of 17 patients experienced recurrent HUS; 15 were drug-induced (12 with cyclosporine, three with Sirolimus); 10 were TMA; and five HUS. Nine patients developed HUS/TMA within 3 months of transplantation with eight developing it within 1 year posttransplantation. Graft function recovered in 12, while five did not recover. The HUS group showed 60% recovery compared with 80% among the TMA group. Two patients were lost; both displayed HCV seropositivity and one also showed anti-cardiolipin antibody. CONCLUSION:

Early allograft biopsy with prompt diagnosis & management OF DRUG-NDUCED HUS by drug substitution +/- plasmapheresis in posttransplant HUS/TMA plays an important role in allograft outcome. TMA showed better recovery than HUS. 19: Ren Fail. 2008;30(3):331-4.

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Unusual manifestation of diarrhea-associated haemolytic uraemic syndrome in an adult. Anagnostis P, Stangou M, Leontsini M, Athyros VG, Karagiannis A. Second Propedeutic Department of Internal Medicine, Aristotle University, Hippokration Hospital, Thessaloniki, Greece. [email protected] Hemolytic-Uremic Syndrome (HUS) is an uncommon disease characterized by microangiopathic hemolytica anaemia, thrombocytopenia, and acute renal failure. There are two forms of HUS: diarrhoea (D+)- and non-diarrhoea (D-)-associated HUS. We report the case of a 21-year-old woman presented to our department with jaundice, anaemia, thrombocytopenia, and anuria, preceded by a diarrheal prodrome, secondary to infection with Escherichia coli O157:H7. The whole clinical and laboratory investigation led to the diagnosis of HUS. Her condition was complicated with cholestasis, liver dysfunction, bleeding from the vagina, and myocardial involvement. She was treated only with fresh frozen plasma transfusions and hemodialysis, and despite the long duration of anuria (22 days), the patient showed subsequent improvement over days until full recovery one month later. 134: Intern Med. 2004 Jul;43(7):620-3.

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Hemolytic uremic syndrome associated with Shiga toxin producing Escherichia coli infection in a healthy adult woman. Kanno Y, Kobayashi H, Rai T, Kanno K, Watanabe K, Obara K, Sato Y. Second Department of Internal Medicine, Fukushima Medical University School of Medicine, Hikarigaoka, Fukushima. A 49-year-old healthy Japanese woman presented with hemorrhagic diarrhea because of Shiga toxin producing Escherichia coli infection, and then hemolytic uremic syndrome (HUS) developed in the patient. She was successfully treated with continuous hemodiafiltration, plasma exchange, and endotoxin adsorption therapy. An analysis of previous case reports suggests that females aged between 16 and 65 years are at an increased risk of HUS resulting from hemorrhagic colitis. We propose that adult female patients with hemorrhagic colitis should be carefully monitored regardless of their medical history, physical presentation, or laboratory data.

115: J Pak Med Assoc. 2005 Feb;55(2):84-7.

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Systemic lupus erythematosus presenting as hemolytic uremic syndrome: a case report. Rabbani MA, Ahmad B, Mekan SF, Muzaffar S, Ali SS. Department of Medicine, Aga Khan University Hospital, Karachi. Associating systemic lupus erythematosus (SLE), with an initial presentation of hemolytic uremic syndrome (HUS) is rare. We report a case of 21-year old Afghani female admitted to our hospital with an initial complaint of high grade fever and diffuse maculopapular rash and swelling of lower limbs. Diagnosis of atypical HUS was established according to the clinical triad of HUS without a veriotoxin-producing organism in her stool and the pathological finding compatible to thrombotic microangiopathy. In addition, her symptoms fulfilled the 1982 revised criteria for the classification of SLE. After pulse methylprednisolone, cyclophosphamide and plasmapheresis therapies, her laboratory findings and general condition improved. Unfortunately she was lost to follow up as she decided to return back to Afghanistan.

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16: Mod Rheumatol. 2008;18(4):403-6. Epub 2008 Apr 22.

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Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome and adult onset Still's disease: case report and review of the literature. Sayarlioglu M, Sayarlioglu H, Ozkaya M, Balakan O, Ucar MA. Department of Internal Medicine, Division of Rheumatology, Sutcu Imam University Medical Faculty, Kahramanmaras, Turkey, [email protected]. Thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP/HUS) is a multisystem disorder characterized by consumptive thrombocytopenia, microangiopathic hemolytic anemia, neurologic symptoms, renal function abnormalities, and fever. Coexistence of TTP/HUS and adult onset Still's disease (ASD) is extremely rare. We report the case of a 46year-old woman who presented with fever, arthritis, myalgias, petechia on skin and confusion five years after the onset of ASD. Thrombocytopenia, renal failure, marked elevation lactate dehydrogenase, and red cell fragmentation on peripheral blood smear were observed. We made a diagnosis of TTP/HUS associated with ASD, according to physical examination and characteristic laboratory data. She recovered from the TTP/HUS following daily sessions of therapeutic plasma exchange with fresh frozen plasma replacement and glucocorticoid therapy.

Awareness of the possible development of TTP/HUS in ASD is important for early diagnosis and treatment.

Thrombotic microangiopathy after kidney transplantation. Ponticelli C, Banfi G. Department of Immunology, IRCCS, Istituto Auxologico Italiano, Milan, Italy. [email protected] Two forms of post-transplant thrombotic microangiopathy (TMA) may be recognized: recurrent TMA and de novo TMA. Recurrent TMA may occur in patients who developed a nondiarrhoeal form of haemolytic uraemic syndrome (HUS) being particularly frequent in patients with autosomal recessive or dominant HUS. The recurrence is almost the rule in patients with mutation in complement factor H gene. Most patients eventually lose the graft. Treatment with plasma infusions or plasmapheresis is often disappointing, but few cases may be rescued. Intravenous immunoglobulins and rituximab have also been successful in anedoctic cases. De novo TMA is rarer. A number of factors including viral infection may be responsible of de novo TMA, but in most cases TMA is triggered by calcineurin inhibitors or mTOR inhibitors. The clinical presentation of de novo TMA may be variable with some patients showing clinical and laboratory features of HUS while others showing only a progressive renal failure. The prognosis is less severe than with recurrent TMA. Complete withdrawal of the offending drug may lead to improvement in many cases. The addition of plasma exchange may result in graft salvage in about 80% of cases.

2: Egypt J Immunol. 2006;13(2):95-9.

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Tacrolimus (FK506) associated thrombotic thrombocytopenic purpura/hemolytic uremic syndrome in lung transplant salvage with a plasmapheresis and cyclosporin. Boctor FN. Department of Laboratory Medicine, Geisinger Medical Center, USA. Thrombotic microangiopathy (TMA), a microvascular hemolytic disorder is a rare, but well described complication in organ transplant patients receiving immunosuppressant drugs. We report a 56-year-old female with a history of left lung transplant that presented to the hospital with microangiopathic hemolytic anemia and thrombocytopenia while receiving tacrolimus (FK 506) for 36 months. The patient was diagnosed with tacrolimus-induced TTP/HUS 76: Semin Thromb Hemost. 2005 Dec;31(6):681-90.

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Drug-induced thrombotic microangiopathy. Zakarija A, Bennett C. Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA. Drug-associated thrombotic thrombocytopenic purpura and hemolytic uremic syndrome (TTP/HUS) has been recognized for several years. The most commonly implicated drugs are mitomycin-C, cyclosporine, quinine, and ticlopidine. As with idiopathic cases of TTP/HUS, basic science discoveries in the late 1990s now suggest that the likely mechanisms by which these agents lead to a thrombotic microangiopathy (TMA) include either an immune-mediated phenomenon involving the ADAMTS13 metalloprotease or direct endothelial toxicity. This article reviews the current understanding of the pathogenesis, the clinical and laboratory features, and the recommended treatments, prognosis, and outcomes of drug-associated TMA.

2: Egypt J Immunol. 2006;13(2):95-9.

Tacrolimus (FK506) associated thrombotic thrombocytopenic purpura/hemolytic uremic 30

syndrome in lung transplant salvage with a plasmapheresis and cyclosporin. Boctor FN. . The ADAMTS-13 activity was subnormal and no inhibitory antibody was detected.

PATHOLOGY INVESTIGATIONS laboratory investigations showing features of hemolytic anaemia, thrombocytopenia and renal insufficiency. 160: J Nephrol. 2003 Jul-Aug;16(4):586-90.

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Successful treatment of hemolytic uremic syndrome after liver-kidney transplantation. Gatti S, Arru M, Reggiani P, Como G, Rossi F, Fassati LR, Ponticelli C. Liver Transplant Unit, IRCCS Maggiore Hospital, Milan, Italy. [email protected] INTRODUCTION: Hemolytic-uremic syndrome (HUS) is a rare complication in organ transplantation, characterized by hemolytic microangiopathic anemia, thrombocytopenia, and severe renal failure. The syndrome is a well-recognized complication in bone marrow transplantation, and has been likewise described in several cases of solid organs transplantation, but never in patients receiving combined liver and kidney transplantation. CASE REPORT: We describe a case of HUS in a 59-year-old woman who underwent combined liver-kidney transplantation for hepatorenal polycystic disease. Clinical and laboratory manifestations of the syndrome were severe and included renal failure, hemolytic anemia, severe thrombocytopenia, hypertension, and neurological damage. The initial treatment consisted of withdrawal of cyclosporine, introduction of low-dose tacrolimus, and administration of fresh frozen plasma (FFP) transfusion and heparin. Since there was no improvement in clinical or biochemical features, plasmapheresis with FFP replacement (2000 mL/day) followed by intravenous immunoglobulin (0.4 mg/Kg/day) was started. A rapid improvement in renal function, platelet count, and hemolytic anemia was 31

observed. CONCLUSIONS: Based on the good response observed in our patient, we feel that an aggressive treatment with plasmapheresis and intravenous immunoglobulin should be offered to organ transplant recipients with severe HUS.

31: Clin Lab Sci. 2007 Fall;20(4):216-20.

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Thrombotic microangiopathy (TTP and HUS): advances in differentiation and diagnosis. Schneider M. William Beaumont Hospital, 35212 Banbury Court, Livonia, MI 48152, USA. [email protected]

Detection of the proteolytic enzyme ADAMTS-13 may be used to differentiate between the forms of thrombotic microangiopathy (TMA), thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS).

3: Emerg Infect Dis. 2008 Aug;14(8):1287-90.

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Analysis of collection of hemolytic uremic syndrome-associated enterohemorrhagic Escherichia coli. Mellmann A, Bielaszewska M, Köck R, Friedrich AW, Fruth A, Middendorf B, Harmsen D, Schmidt MA, Karch H. Institute for Hygiene and the National Consulting Laboratory on Hemolytic Uremic Syndrome, Münster, Germany. [email protected] Multilocus sequence typing of 169 non-O157 enterohemorrhagic Escherichia coli (EHEC) isolated from patients with hemolytic uremic syndrome (HUS) demonstrated 29 different sequence types (STs); 78.1% of these strains clustered

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in 5 STs. From all STs and serotypes identified, we established a reference panel of EHEC associated with HUS (HUSEC collection). 101: Rev Argent Microbiol. 2005 Jan-Mar;37(1):1-10.

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[Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli] [Article in Spanish] Leotta GA, Chinen I, Epszteyn S, Miliwebsky E, Melamed IC, Motter M, Ferrer M, Marey E, Rivas M. Servicio Fisiopatogenia, INEI-ANLIS Dr. Carlos G. Malbrán Av. Vélez Sarsfield 563 (1281) Ciudad Autónoma de Buenos Aires, Argentina. Shiga toxin-producing Escherichia coli (STEC) cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 10(7) and 1.0 x 10(4) CFU/50 microl. The detection limit was 1.0 x 10(4) CFU/50 microl and the cut limit 1.0 x 10(5) CFU/50 ml. A good robustness was observed when different variables were introduced. Inclusivity, exclusivity, positive predictivity, negative predictivity and analytical accuracy were of 100%. Interference was not shown when different concentrations of STEC strains, carrying different genes, were used. The validated technique is an appropriate alternative for detection and confirmation of STEC O157 and non-O157 strains from bacterial cultures. PMID: 15991473 [PubMed - indexed for MEDLINE]

71: Clin Chim Acta. 2006 Apr;366(1-2):281-6. Epub 2006 Feb 14. Links

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Detection of Escherichia coli O157 using equallength double-stranded fluorescence probe in a real-time polymerase chain reaction assay. Yu G, Niu J, Shen M, Shao H, Chen L. The Key Laboratory of Cell Biology and Tumor Cell Engineering of the Ministry of Education, School of Life Sciences, Xiamen University, 422 Siming Nan Road, Xiamen 361005, Fujian, People's Republic of China. BACKGROUND: Enterohemorrhagic Escherichia coli (E. coli) O157 is a dangerous pathogen, which causes bloody diarrhea and severe hemolytic uremic syndrome (HUS). Although several assay systems based on real-time polymerase chain reaction (PCR) have been integrated to detect this pathogen, most of them are not specific. We report a real-time quantitative PCR method targeting rfbE, a gene specifically expressed in E. coli O157. This method can therefore be used to diagnose enterohemorrhagic Escherichia coli (E. coli) O157. METHODS: A nucleic acid based diagnostic assay system, combining equal-length double-stranded fluorescence probe technique and real-time PCR, was developed to detect E. coli O157. This assay system take advantage of the highly conserved rfbE O-antigen synthesis gene, and a pair of fluorescence-quenching probes complementary to rfbE gene were used in a real-time PCR to quantify the presence of the pathogen. RESULTS: The specificity of the diagnostic method was assessed by comparing test results on 14 different related pathogens including common E. coli, enteroinvasive Escherichia coli (EIEC), Salmonella, Shigella and E. coli O157. The detection limit of the method was determined using 10-fold serial dilutions of an E. coli O157 standard sample, and as few as 1.49 x 10(3) CFU/ml could be detected. All E. coli with serotype O157, which expresses rfbE gene, were positive in this assay, while all other species without rfbE gene expression were negative.

CONCLUSIONS: By combining equal-length double-stranded fluorescence probe technique and real-time PCR, we have developed a simple, rapid, specific and sensitive method to detect E. coli O157. 148: Lett Appl Microbiol. 2004;38(5):351-4.

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The serological response to Verocytotoxigenic Escherichia coli in patients with haemolytic uraemic syndrome. Chart H, Perry NT. Laboratory of Enteric Pathogens, Specialist and Reference Microbiology Division, Health Protection Agency, London, UK. [email protected] AIMS: To screen sera from 80 patients with clinical haemolytic uraemic syndrome (HUS) and serum antibodies to the lipopolysaccharide (LPS) of Escherichia coli O157, for antibodies to Verocytotoxin-producing Escherichia coli (VTEC) belonging to serogroups O5, O26, O104, O111, O128, O145, O153 and O165. METHODS AND RESULTS: Sera were screened by an LPS-based ELISA and SDS-PAGE/immunoblotting. 34

None of the 80 sera contained antibodies binding to long-chain LPS of any of the LPS types employed; however, nine sera

CONCLUSIONS: The presence of patients' serum antibodies to the LPS of E. coli O157, in the absence of antibodies to the LPS of a range of other VTEC, demonstrated that cases of HUS may be caused by strains of O157 VTEC alone and that concurrent infection with multiple strains of VTEC is not a prerequisite for cases of HUS. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibodies to long-chain LPS of VTEC other than O157 were not detected, and so there was no evidence of infection with VTEC belonging to more than one serogroup. The results of immunoassays such as ELISAs and micro-agglutinations must take into consideration antibodies binding to R3 epitopes located on LPS-core. contained antibodies binding to R3 LPS-core epitopes.

213: Semin Thromb Hemost. 2002 Apr;28(2):167-72.

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Assays of von Willebrand factor-cleaving protease: a test for diagnosis of familial and acquired thrombotic thrombocytopenic purpura. Furlan M, Lämmle B. Central Hematology Laboratory, University Hospital, Inselspital, Bern, Switzerland. The vWF-cleaving protease was found to be deficient in patients with familial thrombotic thrombocytopenic purpura (TTPNormal activity of vWFcleaving protease was established in patients with a clinically similar disorder: hemolytic-uremic syndrome (HUS). The level of vWF-cleaving protease activity is thus a laboratory parameter that provides important information for the differential diagnosis and treatment of patients with TTP/HUS. Several assays of vWF-cleaving protease have been described and are summarized here. 72: Thromb Res. 2006;118(6):723-31. Epub 2006 Jan 23.

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Novel, semi-automated, 60-min-assay to determine von Willebrand factor cleaving activity of ADAMTS-13. Kostousov V, Fehr J, Bombeli T.

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Coagulation Laboratory, Division of Haematology, University Hospital of Zürich, Raemistrasse 100, 8091 Zürich, Switzerland. [email protected] INTRODUCTION: The clinical entity of idiopathic thrombotic thrombocytopenic purpura (TTP) has been closely associated with a severe deficiency of the von Willebrand factor (vWF)-cleaving protease (ADAMTS-13). Levels below 5% are highly specific for TTP basically excluding hemolytic uremic syndrome (HUS). . In such cases rapid knowledge of the ADAMTS-13 activity is very useful in helping to establish a correct diagnosis We have therefore designed a rapid, sensitive and simple assay for the determination of ADAMTS-13 activity and ADAMTS-13 inhibitors. The test is based on the measurement of the ristocetin-cofactor activity of purified, urea-denaturated vWF concentrate after incubation with diluted patient plasma that has been preactivated with barium chloride. 8: J Immunoassay Immunochem. 2008;29(3):234-43.

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Age-dependent decrease in serum soluble interferon-gamma receptor (sIFN-gammaR) in healthy Japanese individuals; population study of serum sIFN-gammaR level in Japanese. Okamoto Y, Seta K, Nakajo I, Gotoh Y, Nagai T, Fujita N, Fukui T, Masuzawa T. Laboratory of Immunology and Microbiology, Faculty of Pharmacy, Chiba Institute of Science, Choshi, Chiba, Japan. [email protected] We planned to investigate the clinical significance of serum soluble interferongamma receptor (sIFN-gammaR) level in pediatric patients. The diagnostic application of the measurement of serum sIFN-gammaR level depends critically on the control value. However, there is no information of the control value of serum sIFN-gammaR for children. In the present study, we determined the serum sIFN-gammaR level of healthy Japanese children using an ELISA. The serum sIFN-gammaR level of children (0-14 years old) was significantly higher than that of adults (over 15 years old) (p < 0.01, n = 104). Thus, it is recommended that, when the serum sIFN-gammaR level of patients is evaluated, it should be compared against age-matched controls. We also preliminarily applied this assay as a diagnostic parameter for the patients with diarrhea positive (D+) hemolytic uremic syndrome (HUS).

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