Valid Presumption of Shiga Toxin-Mediated Damage of Developing Erythrocytes in EHEC-Associated Hemolytic Uremic Syndrome.

The global emergence of clinical diseases caused by enterohemorrhagic Escherichia coli (EHEC) is an issue of great concern. EHEC release Shiga toxins (Stxs) as their key virulence factors, and investigations on the cell-damaging mechanisms toward target cells are inevitable for the development of novel mitigation strategies. Stx-mediated hemolytic uremic syndrome (HUS), characterized by the triad of microangiopathic hemolytic anemia, thrombocytopenia, and acute renal injury, is the most severe outcome of an EHEC infection. Hemolytic anemia during HUS is defined as the loss of erythrocytes by mechanical disruption when passing through narrowed microvessels. The formation of thrombi in the microvasculature is considered an indirect effect of Stx-mediated injury mainly of the renal microvascular endothelial cells, resulting in obstructions of vessels. In this review, we summarize and discuss recent data providing evidence that HUS-associated hemolytic anemia may arise not only from intravascular rupture of erythrocytes, but also from the extravascular impairment of erythropoiesis, the development of red blood cells in the bone marrow, via direct Stx-mediated damage of maturing erythrocytes, leading to "non-hemolytic" anemia.

A rapid and sensitive method to measure the functional activity of Shiga toxins in human serum.

Shiga toxins (Stx) have a definite role in the development of hemolytic uremic syndrome in children with hemorrhagic colitis caused by pathogenic Stx-producing Escherichia coli (STEC) strains. The dramatic effects of these toxins on the microvasculature of different organs, particularly of the kidney, are well known, whereas there is no consensus on the mechanism by which Stx reach the endothelia of target organs and/or indirectly injure these body sites. We hereby describe a quick (4 h), radioactive, Raji cell-based method designed for the detection of Stx in human sera. The assay monitors the translation impairment induced by these powerful inhibitors of protein synthesis, which are identified properly by neutralizing their activity with specific monoclonal antibodies. By this method, we detected for the first time the functional activity of Stx in sera of STEC-infected patients during hemorrhagic colitis. Recent research has pointed to a dynamic process of Stx-induced renal intoxication in which concurrent and interactive steps are involved. Our rapid and specific method could be useful for studying the kinetics of Stx during the natural course of STEC infection and the interplay between Stx activity in serum and Stx presence in different blood fractions (neutrophils, monocytes, platelets, leukocyte-platelet aggregates, microvesicles, lipoproteins).

Patient and sample-related factors that effect the success of in vitro isolation of Orientia tsutsugamushi.

Orientia tsutsugamushi is the causative agent of scrub typhus infection, a major cause of human disease in rural areas of Southeast Asia. Twenty-six blood samples collected from patients with serologically proven scrub typhus during a six month period were sent to Bangkok (535 km from the clinical site) by road at ambient temperature (average daily temperature range: 27.1-29.1 degrees C) for attempted in vitro isolation in Vero cells. O. tsutsugamushi was isolated from 12 samples (sensitivity 46.7%) with the time to isolation ranging from 16 to 37 days [median 27 days, inter-quartile range (IQR) 22.5-33.5 days]. Patient factors such as days of fever and O. tsutsugamushi IgM antibody titer, transport factors such as transit time, and isolate genotype (Karp and Gilliam/Kawasaki) were assessed to determine their influence on the outcome of in vitro isolation. None of the factors significantly influenced the isolation outcome. This study demonstrates that O. tsutsugamushi can often be isolated in vitro from the blood of scrub typhus patients when transported at ambient tropical temperatures for many days.

Plasma exchange therapy for the treatment of Escherichia coli O-157 associated hemolytic uremic syndrome.

Plasma exchange (PE) therapy was administered to three patients with Escherichia coli O-157 associated hemolytic uremic syndrome (HUS) in early phase. Following several PE treatments, all cases completely recovered without any apparent complications. The usefulness of PE therapy in removing microbial fragments and inflammatory cytokines was evaluated. The peptidoglycan (PG) level, interleukin-1 receptor antagonist (IL-1Ra) were higher in HUS patients starting PE therapy than in patients who had received several sessions of PE therapy. PE therapy was an effective early phase treatment for Escherichia coli O-157 associated HUS.

Detection of verocytotoxin bound to circulating polymorphonuclear leukocytes of patients with hemolytic uremic syndrome.

The epidemic form of hemolytic uremic syndrome (HUS) is the most common cause of acute renal failure in children and is characterized by a prodromal phase of sometimes bloody diarrhea. The role of verocytotoxin (VT)-producing Escherichia coli has been strongly implicated. Although antibodies against VT have been detected in the serum of patients with HUS, VT itself has never been detected in circulating blood. In this study, VT-2 was detected in the systemic circulation in 9 of 10 patients with the epidemic form of HUS. In those cases, VT-2 was bound exclusively to polymorphonuclear leukocytes (PMN). The detection of VT-2 bound to PMN was associated with the presence of diarrhea at the time the blood samples were obtained. The one patient for whom VT was not detected presented with atypical HUS. For 5 of the 10 patients with HUS who were studied, the time course of VT binding was analyzed; binding decreased in four patients. The finding of VT bound to PMN in the systemic circulation of patients with HUS is important for a clearer understanding of the pathogenesis of HUS and suggests new approaches for treatment in the future.