The impact of Rotavirus mass vaccination on hospitalization rates, nosocomial Rotavirus gastroenteritis and secondary blood stream infections.

BACKGROUND: The aim of the study was to evaluate the effects of universal mass vaccination (UMV) against rotavirus (RV) on the hospitalization rates, nosocomial RV infections and RV-gastroenteritis (GE)-associated secondary blood stream infections (BSI). METHODS: The retrospective evaluation (2002-2009) by chart analysis included all clinically diagnosed and microbiologically confirmed RV-GE cases in a large tertiary care hospital in Austria. The pre-vaccination period (2002-2005) was compared with the recommended and early funded (2006-2007) and the funded (2008-2009) vaccination periods. Primary outcomes were RV-GE-associated hospitalizations, secondary outcomes nosocomial RV disease, secondary BSI and direct hospitalization costs for children and their accompanying persons. RESULTS: In 1,532 children with RV-GE, a significant reduction by 73.9% of hospitalized RV-GE cases per year could be observed between the pre-vaccination and the funded vaccination period, which was most pronounced in the age groups 0-11 months (by 87.8%), 6-10 years (by 84.2%) and 11-18 years (88.9%). In the funded vaccination period, a reduction by 71.9% of nosocomial RV-GE cases per year was found compared to the pre-vaccination period. Fatalities due to nosocomial RV-GE were only observed in the pre-vaccination period (3 cases). Direct costs of hospitalized, community-acquired RV-GE cases per year were reduced by 72.7% in the funded vaccination period. The reduction of direct costs for patients (by 86.9%) and accompanying persons (86.2%) was most pronounced in the age group 0-11 months. CONCLUSIONS: UMV may have contributed to the significant decrease of RV-GE-associated hospitalizations, to a reduction in nosocomial RV infections and RV-associated morbidity due to secondary BSI and reduced direct hospitalization costs. The reduction in nosocomial cases is an important aspect considering severe disease courses in hospitalized patients with co-morbidities and death due to nosocomial RV-GE.

[ESBL-producing E. coli and EHEC in dogs and cats in the Tyrol as possible source of human infection]. / ESBL-produzierende E. coli und EHEC bei Hunden und Katzen in Tirol als mögliche Quelle für humane Infektionen.

In contrast to infections with enterohaemorrhagic E. coli (EHEC), which are thought to be classical zoonosis, the zoonotic potential of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae is still widely unknown. The aim of our study was to determine the frequency of EHEC and ESBL-producing Enterobacteriaceae in domestic animals (dogs and cats) in the Tyrol. Among 228 fecal samples of dogs (n = 92) and cats (n = 136) three samples (1.3%) were positive in the EHEC-ELISA. In two of the three cases isolation of the organism was not possible, the third sample of a two-year-old crossbreed bitch yielded EHEC O103:H2. In twelve of 228 (5.3%) fecal samples 13 ESBL-producing Enterobacteriaceae (in ten cats and two dogs) were found.These animals mainly derived from homes for animals (ten animals, 83%). 75% of the isolates belonged to the CTX-M-1-group, 8% to the CTX-M-2-group and 17% to the CTX-M-9-group. One isolate was positive for CTX-M-1 and CTX-M-9. Typing of the 13 ESBL-producing isolates by multilocus sequence typing (MLST) showed ten different sequence types, which points out the importance of the horizontal transfer of mainly plasmid-coded ESBL genes. Transmission of EHEC and ESBL-producing Enterobacteriaceae from domestic animals to humans is possible, corroborated by the fact that the EHEC serotype found in one dog and the sequence types detected by MLST in several dogs and cats were previously reported to occur in severe human infection.

N-chlorotaurine, a long-lived oxidant produced by human leukocytes, inactivates Shiga toxin of enterohemorrhagic Escherichia coli.

N-chlorotaurine (NCT), the main representative of long-lived oxidants produced by granulocytes and monocytes, is known to exert broad-spectrum microbicidal activity. Here we show that NCT directly inactivates Shiga toxin 2 (Stx2), used as a model toxin secreted by enterohemorrhagic Escherichia coli (EHEC). Bacterial growth and Stx2 production were both inhibited by 2 mM NCT. The cytotoxic effect of Stx2 on Vero cells was removed by ≥5.5 mM NCT. Confocal microscopy and FACS analyses showed that the binding of Stx2 to human kidney glomerular endothelial cells was inhibited, and no NCT-treated Stx2 entered the cytosol. Mass spectrometry displayed oxidation of thio groups and aromatic amino acids of Stx2 by NCT. Therefore, long-lived oxidants may act as powerful tools of innate immunity against soluble virulence factors of pathogens. Moreover, inactivation of virulence factors may contribute to therapeutic success of NCT and novel analogs, which are in development as topical antiinfectives.

Rapid detection of bloodstream pathogens by real-time PCR in patients with sepsis.

Rapid detection of bloodstream infections is an important issue for a better patient outcome. The aim of our study was thus to evaluate the LightCycler SeptiFast assay for diagnosis of bloodstream pathogens in a tertiary hospital in Western Austria. The 71 blood samples of 61 patients with presumed sepsis were investigated and compared with conventional blood culture system results. In both assays, 51 samples (71.8 %) were negative. In 20 positive samples (28.2 %), 10 different pathogens were detected by either blood culture system or SeptiFast assay or by both methods. Five samples were positive in both assays. The agreement rate of blood culture system and SeptiFast assay was 78.9 %, the negative predictive value of SeptiFast assay versus blood culture system was 0.94, sensitivity was 0.63, and specificity 0.81. In 12 samples where a positive SeptiFast assay and a negative blood culture system result were obtained, the same pathogens as identified by SeptiFast assay were detected in samples from other body sites suggesting a correct positive detection. In 11.3 % of cases, the SeptiFast assay resulted in an adjustment of the patients' therapy. In 3 samples, the blood culture assay was positive whereas the SeptiFast assay yielded negative results. In two of these cases, the pathogens involved were not included in the SeptiFast detection list, in the third case SeptiFast assay failed to detect Candida glabrata.Thus we recommend the SeptiFast assay as a valuable tool for rapid diagnosis of bloodstream infections in addition to, but not as replacement for, the blood culture test.

Emergence of VIM-1-carbapenemase-producing Enterobacter cloacae in Tyrol, Austria.

The rapid emergence and dissemination of carbapenemase-producing Enterobacter species and other members of the Enterobacteriaceae poses a considerable threat to the care of hospitalized patients and to public health. In this study, Enterobacter isolates demonstrating decreased susceptibility to carbapenems detected at the Division of Hygiene and Medical Microbiology, Innsbruck Medical University, between January 2006 and December 2010 were tested for bla(VIM-1), bla(NDM-1), bla(IMP), bla(KPC) and bla(OXA-48) using a multiplex PCR with published primers. PFGE was performed to determine the genetic relatedness. In total, 33 isolates (28 Enterobacter cloacae and 5 Enterobacter aerogenes) were collected during the study period. From 2006 to 2009, between two and seven isolates were found per year. In 2010, a significant increase of carbapenem-resistant strains was observed (n = 12). The bla(VIM-1) gene was detected in all 28 isolates of E. cloacae. Typing of E. cloacae by PFGE revealed three distinct clusters, the biggest of which contained 18 isolates. These findings demonstrate the emergence of VIM-1-producing Enterobacter in Tyrol, western Austria. The clonal relationship confirms the risk of spread of these organisms and their possible persistence over time.

Complement in typical hemolytic uremic syndrome.

Hemolytic uremic syndrome (HUS) is a severe disease characterized by the clinical triad of hemolytic anemia, thrombocytopenia, and acute renal failure. HUS exists in two forms: the atypical diarrhea-negative HUS, which is often associated with complement disorders, and the more frequent diarrheal-associated typical HUS, which is caused by infections with enterohemorrhagic ESCHERICHIA COLI. The virulence factors of the latter have been studied well, and Shiga toxin (Stx)2 is reported to represent the most important one. In contrast, risk factors on the host side have not been intensively studied until recently: Complement activation products have been detected in the serum and plasma of HUS patients, and an in vitro study could show that Stx2 not only damages the kidney directly but also indirectly via complement, in two ways. First, it activates complement, and second, it delays the functions of its control protein factor H on the cell surface, both known to damage the kidney.

EspP, a serine protease of enterohemorrhagic Escherichia coli, impairs complement activation by cleaving complement factors C3/C3b and C5.

Hemolytic-uremic syndrome (HUS) is a life-threatening disorder characterized by hemolytic anemia, thrombocytopenia, and renal insufficiency. It is caused mainly by infections with enterohemorrhagic Escherichia coli (EHEC). Recently, Shiga toxin 2, the best-studied virulence factor of EHEC, was reported to interact with complement, implying that complement may be involved in the pathogenesis of EHEC-induced HUS. The aim of the present study was to investigate whether or not the serine protease EspP, an important virulence factor of EHEC, interacts with complement proteins. EspP did not have any effect on the integrity of factor H or factor I. However, EspP was shown to cleave purified C3/C3b and C5. Cleavage of the respective complement proteins also occurred in normal human serum (NHS) as a source of C3/C3b or C5 or when purified complement proteins were added to the supernatant of an EspP-producing wild-type strain. Edman degradation allowed unequivocal mapping of all three main C3b fragments but not of the three main C5 fragments. Complement activation was significantly downregulated in all three pathways for C5-depleted serum to which C5, preincubated with EspP, was added (whereas C5 preincubated with an EspP mutant was able to fully reconstitute complement activation). This indicates that EspP markedly destroyed the functional activity, as measured by a commercial total complement enzyme-linked immunosorbent assay (Wieslab). Downregulation of complement by EspP in vivo may influence the colonization of EHEC bacteria in the gut or the disease severity of HUS.

Shiga toxin activates complement and binds factor H: evidence for an active role of complement in hemolytic uremic syndrome.

Infections with enterohemorrhagic Escherichia coli (EHEC) are a major cause of hemolytic uremic syndrome (HUS). Shiga toxins (Stxs), especially Stx2, are believed to represent major virulence factors of EHEC, contributing to HUS pathogenesis. Beside EHEC-associated HUS, there are hereditary atypical forms of HUS, which are mostly caused by mutations of complement regulators. The aim of the present study was to investigate whether or not complement is also involved in the pathogenesis of EHEC-induced typical HUS, by being activated either directly or indirectly by involvement of its inhibitors. Purified Stx2 markedly activated complement via the alternative pathway and was found to bind to factor H (FH), however, only when it was active. No apparent cleavage or destruction of FH was visible, and cofactor activity in fluid phase was unaffected, but clearly delayed for surface-attached FH, where it is essential for host cell protection. Binding studies using FH constructs revealed that Stx2 binds to short consensus repeats (SCRs) 6-8 and SCRs18-20, but not to SCRs16-17, i.e., to regions involved in the surface recognition function of FH. In conclusion, complement, and in particular FH, not only plays an important role in atypical HUS, but most probably also in EHEC-induced HUS.

Sorbitol-fermenting Shiga toxin-producing Escherichia coli O157 in Austria.

Infections with enterohemorrhagic Escherichia coli (EHEC) are the major cause of hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in childhood. Shiga toxins are considered to be the most important virulence factor of EHEC strains. Non-sorbitol-fermenting EHEC O157:H7 is still the most prevalent serotype isolated worldwide; however, sorbitol-fermenting (SF) EHEC O157:H- (H- indicates nonmotility) strains are increasingly reported. Thirteen SF EHEC O157:H- strains (11 of human origin, two from animals) were detected in Austria between 2002 and 2008. Among the 11 human cases, seven suffered from HUS, two from diarrhea and the remaining two were asymptomatic. Seven of the cases were identified in patients living in or visiting (in one case) the province Salzburg, four were in patients from the province Vorarlberg. Three outbreaks with no more than three persons involved were detected, the other four cases occurred sporadically. The pulsed-field gel-electrophoresis banding patterns of the 13 SF EHEC O157:H- isolates were grouped into three distinct clusters (groups 1, 2 and 3). Strains of the three outbreaks were identical (except for one outbreak strain with one band difference) within each outbreak. In comparison, the Bavarian epidemic strain showed a pattern different from all SF O157:H- strains isolated in Austria. For effective detection of SF EHEC O157:H-, screening for Shiga toxins by ELISA and/or Shiga toxin genes by PCR is absolutely necessary; screening on the basis of phenotypic characteristics such as sorbitol-non-fermentation is not sufficient. Typing methods relying solely on investigation of O157 will detect these strains but should nevertheless also be avoided, so that the prevalent non-O157 strains causing HUS are not missed.

Tetracycline-resistant Escherichia coli strains are inherited from parents and persist in the infant's intestines in the absence of selective pressure.

The study investigated tetracycline (TC), ampicillin (AMP), cefazolin (CEF), and trimethoprim (TMP) resistance in Escherichia coli (E. coli) in the feces of 21 infants up to 6 months of age and in their parents in the absence of selective antimicrobial pressure. Clonality of strains was assessed by pulsed-field gel electrophoresis. Three infants had resistant E. coli strains in their feces identical to the mothers' from week 1 on, which persisted over weeks. From week 2 on, in another four infants, persisting resistant E. coli were found, two of them identical to the mothers'. All of these persisting E. coli strains (except one family) showed at least resistance to TC. In infants, resistant E. coli strains inherited from their mothers tended to persist over months. Therefore, the persistence of resistant E. coli and their possible capacity to cause symptomatic infection or transfer its resistance genes to other bacteria deserves more attention.

Serine protease espP subtype alpha, but not beta or gamma, of Shiga toxin-producing Escherichia coli is associated with highly pathogenic serogroups.

Besides Shiga toxins (Stx), Stx-producing Escherichia coli (STEC) harbour several other putative virulence factors, including the serine protease EspP. We have investigated 214 STEC strains from Austria belonging to 61 different serotypes from humans, animals, and food for the presence of this serine protease gene and have determined the espP subtypes and their association with clinical outcome. espP was detected in 121 (57%) out of 214 strains. Sixty-five of 68 strains (96%) of non-sorbitol-fermenting (NSF) O157:H7/NM (NM, non-motile) were positive for espP, while none of 8 SF E. coli O157:NM isolates contained this gene. All 9 strains of serotype O145:NM and 17 of 21 strains (81%) of serotype O26:H11/NM were positive for espP. Nineteen STEC serogroups including O103 and O111 serogroups--considered to be highly pathogenic--were completely negative for espP. Only 5 of 12 strains isolated from patients suffering from haemolytic uraemic syndrome (HUS) were espP-positive (all serogroup NSF O157) as well as 28 of 39 strains from patients with bloody diarrhoea, 40 of 63 strains from patients with non-bloody diarrhoea, and 15 of 19 strains from asymptomatic patients. In O157:H7/NM, O26:H11/NM, and O145:NM only espP subtype alpha was found, whereas in most of the other non-O157 serogroups, subtypes beta and gamma were found. Subtype delta was not detected in our strain collection. Regarding the espP subtypes, only subtype alpha, but not beta and gamma, were found in HUS patients. Moreover, we could demonstrate that espP, and in particular subtype alpha, is associated with highly pathogenic serogroups.

Prevention and treatment of enterohemorrhagic Escherichia coli infections in humans.

Infections with enterohemorrhagic Escherichia coli (EHEC) result in various clinical symptoms and outcomes ranging from watery or bloody diarrhea to the life-threatening hemolytic-uremic syndrome (HUS). Shiga toxins (Stxs) are supposed to play a major role in the pathogenesis of EHEC infections; however, the role of other putative virulence factors is not fully elucidated. So far, there is only supportive therapy available for the treatment of both EHEC-associated diarrhea and HUS. Antibiotic therapy for the treatment of EHEC-associated diarrhea is discussed. In recent years other therapeutic strategies have been developed, including Gb3 receptor analogues, that bind Stx in the gut or in the circulation, passive immunization with Stx-neutralizing monoclonal antibodies, or active immunization with Stx1 And Stx2 toxoids as a preventive procedure. These approaches have been demonstrated to be effective in animal models but clinical trials are lacking.

Diminished response to tick-borne encephalitis vaccination in thymectomized children.

In order to analyze the clinical impact of immunological alterations in thymectomized children after exposure to a new antigen (tick-borne encephalitis virus (TBEV) vaccine), 17 thymectomized children completed a three-dose immunization regimen. Thymectomized children showed significantly lower TBEV IgG antibody levels after the second vaccination when compared to healthy age-matched controls (n=30) (p=0.03), but a normal response after the third vaccination. Age at thymectomy correlated significantly with the TBEV IgG antibody levels (p=0.04). Thymectomized children also showed significantly lower total counts and percentages for naïve T cells correlating with time after thymectomy (p=0.02), than observed for controls. These changes in T cell subsets and the decreased ability to respond to new antigens in thymectomized children, as observed here, may precede more striking effects such as higher infection rates or autoimmune conditions as they age.

Prevalence of Shiga toxin-, intimin- and haemolysin genes in Escherichia coli isolates from drinking water supplies in a rural area of Austria.

Literature harbours several reports of potable water-associated outbreaks. We studied the prevalence of Shiga toxin- (stx1/2), intimin- (eae) and haemolysin (hlyA) genes in Escherichia coli isolates from drinking water of private and public water supplies in a rural area of Upper Austria; 2633 water samples were gained between November 2000 and December 2003. Two hundred and eighty of these water samples were positive for E. coli (10.6%). Of these, 101 samples were drawn from drilled wells (36%), 96 from dug wells (34%), 61 from springs (22%) and 22 from water supplies without available information on technical details (8%); 141 of the samples were from public water supplies, 139 from private water supplies. Eleven of the E. coli isolates were found to be positive for one of the investigated virulence genes (3.9%): one isolate yielded stx2, seven eae, and three isolates had hlyA. The presence of these genes underlines the importance of control of water quality in public and also private water supplies.

The Shiga toxin genotype rather than the amount of Shiga toxin or the cytotoxicity of Shiga toxin in vitro correlates with the appearance of the hemolytic uremic syndrome.

Shiga toxins (Stx) are believed to play a key role in the pathogenesis of diseases caused by Stx-producing Escherichia coli (STEC), including the potentially life-threatening hemolytic uremic syndrome (HUS). In this study, 201 STEC strains collected from patients and environmental sources were investigated with regard to the stx genotypes and pathogenicity. The stx(2) and stx(2c) alleles were associated with high virulence and the ability to cause HUS, whereas stx(2d), stx(2e,)stx(1), and stx(1c) occurred in milder or asymptomatic infections. Quantification of Stx using an enzyme immunoassay and the Vero cell cytotoxicity assay showed no significant differences between the strains associated with HUS and those causing milder diseases. We hypothesize that the stx genotype and perhaps other yet unknown virulence factors rather than the amount of Stx or the in vitro cytotoxicity correlate with the development of HUS.

Comparison of an immunochromatographic rapid test with enzyme-linked immunosorbent assay and polymerase chain reaction for the detection of Shiga toxins from human stool samples.

Rapid detection of enterohemorrhagic Escherichia coli is important for its successful treatment. We have evaluated the immunochromatographic Duopath Verotoxin-test for detection of Shiga toxins, in comparison with enzyme-linked immunosorbent assay and polymerase chain reaction, on 240 clinical human stool samples. The Duopath-test showed a lower sensitivity and specificity.

Variability in tellurite resistance and the ter gene cluster among Shiga toxin-producing Escherichia coli isolated from humans, animals and food.

Tellurite-containing media are widely used for the screening and isolation of Shiga toxin-producing Escherichia coli (STEC) O157:H7, but tellurite resistance among non-O157 STEC is poorly characterized. Therefore, we investigated 202 STEC strains representing 61 different serotypes from humans, animals or food for the presence of ter genes by PCR and their correlation with tellurite resistance, by assessing growth on cefixime-tellurite sorbitol MacConkey agar. All strains were screened for terC, terE and terF as markers for the ter gene cluster. Of the 202 strains, 127 contained terC and terE and were tellurite-resistant, but only 121 of these also contained terF. All 72 non-sorbitol-fermenting O157:H7 and O157:NM (non-motile) strains contained terC, terE and terF and expressed tellurite resistance. In contrast, all eight sorbitol-fermenting STEC O157:NM were terC-, terE- and terF-negative and tellurite-sensitive. Among non-O157 STEC, terC, terE and terF were found in all seven O145:NM, four O111:H8/NM, 17 of 18 O26:H11/NM and in 21 strains of 14 other serotypes. The strong correlation between the presence of ter genes and the ability to grow on tellurite-containing media suggest that the ter genes encode tellurite resistance in the vast majority of these strains. The presence of the ter gene cluster was significantly (P<0.00001) associated with the presence of eae genes. We conclude that the use of tellurite-containing media in screening for STEC will allow the detection of STEC O26, O111, O145 and non-sorbitol-fermenting O157, but most strains (in this study 74.3%) from other serotypes will be missed.

Emerging Shiga toxin-producing Escherichia coli serotypes in Europe: O100:H- and O127:H40.

Novel and as yet rare non-O157 Shiga toxin (Stx)-producing Escherichia coli (STEC) serotypes are emerging in Europe. Two different sorbitol-fermenting STECs, O100:H- carrying the virulence gene stx2 and O127:H40 carrying stx1 and eae genes (found in two related subjects), were isolated from patients' stool samples. Non-O157 STEC infections in humans are currently under-diagnosed. This report highlights the need for, and importance of, screening for Shiga toxins or serotypes other than just O157.

Cytolethal distending toxins in Shiga toxin-producing Escherichia coli: alleles, serotype distribution and biological effects.

To assess the prevalence of cytolethal distending toxin (CDT) among Shiga toxin-producing Escherichia coli (STEC), 202 STEC strains were investigated using PCRs targeting various cdt alleles (cdt-I to cdt-V). Seven of the 202 strains contained cdt-III and an additional seven contained cdt-V. All 14 cdt-positive strains produced biologically active CDT, as demonstrated by a progressive distension of cultured Chinese hamster ovary cells. The CDT-positive STEC belonged to eight different serotypes, including sorbitol-fermenting O157 : NM (non-motile). The data demonstrate that CDT is present in some STEC serotypes only. However, more studies are required to evaluate whether CDT presence is associated with severe disease.