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Shiga toxin-producing Escherichia coli (STEC) may cause haemolytic uraemic syndrome (HUS). Age ≤5 years and presence of stx2a and eae are risk factors for the development of HUS. In this study, we investigated STEC isolates for the presence of adhesins, toxins and molecular risk assessment (MRA) factors to identify virulence genes associated with HUS development. We included non-duplicate isolates from all STEC infections (n = 340, HUS = 32) reported to the Norwegian National Reference Laboratory (NRL) for Enteropathogenic Bacteria from 1992 to 2013. The most common STEC were O157:H7/H- (34%) and O103:H2 (14%). We retrospectively screened the isolates by three multiplex polymerase chain reactions (PCRs) for adhesins (n = 11), toxins (n = 5) and MRA (n = 15). We calculated odds ratios (ORs) and adjusted odds ratios (aORs) for associations with HUS development. On average, isolates were positive for 15 virulence genes (range: 1-24); two toxins (range: 0-4), five adhesins (range: 0-8) and eight MRA genes (range: 0-13). The gene combinations were clustered within serotypes. Isolates from HUS cases were positive for eae and IpfA O26, and negative for saa, eibG, astA, cnf, subA and pic. We identified 11 virulence genes with a significant association to HUS development. Multivariable analyses adjusted for age group and Shiga toxin identified nleH1-2 [aOR 8.4, 95% confidence interval (CI); 2.18-32.3] as an independent risk factor for the development of HUS from an STEC infection. This study demonstrated that the non-LEE effector protein nleH1-2 may be an important predictor for elevated risk of developing HUS from STEC infections. We recommend the NRL for Enteropathogenic Bacteria to consider including nleH1-2 screening as part of routine STEC surveillance.

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A real-time polymerase chain reaction (PCR) assay, amplifying the genes encoding lactose permease (lacY) and invasion plasmid antigen H (ipaH), was run on 121 isolates phenotypically classified as Shigella spp., enteroinvasive Escherichia coli (EIEC), or EIEC O nontypable (ONT). The results were compared with data from a generic E. coli multiple-locus variable-number of tandem repeat analysis (MLVA) and a Shigella MLVA. The real-time PCR verified all Shigella spp. (n = 53) as Shigella (lacY negative) and all EIEC O121 (n = 15) and EIEC O124 (n = 2) as EIEC (lacY positive). However, the real-time PCR typed EIEC O164 as either EIEC (n = 2) or Shigella (n = 2) and, thus, was not suited for classifying this group of isolates. Interestingly, the majority (42/47, 89.4%) of the EIEC ONT were classified as Shigella (lacY negative) by the real-time PCR, and in nearly all cases, (92.9%, 39/42) data from both MLVA assays supported these findings. Overall, in 94.7% (114/121) of the isolates, the results from the real-time PCR were substantiated by the results from the MLVA assays. In conclusion, the real-time PCR assay was fast and accurate in differentiating Shigella spp. from EIEC, with the exception of the EIEC O164 group. This molecular assay was particularly pragmatic for the challenging EIEC ONT group.

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AIMS: To compare 167 Norwegian human and nonhuman Escherichia coli O157:H7/NM (nonmotile) isolates with respect to an A/T single nucleotide polymorphism (SNP) in the tir gene and to detect specific SNPs that differentiate STEC O157 into distinct virulence clades (1-3 and 8). METHODS AND RESULTS: We developed a multiplex PCR followed by single base sequencing for detection of the SNPs, and examined the association among SNP genotype, virulence profile (stx and eae status), multilocus variable number of tandem repeats analysis (MLVA) profile and clinical outcome. We found an over-representation of the T allele among human strains compared to nonhuman strains, including 5/6 haemolytic-uraemic syndrome cases. Fourteen strains belonged to clade 8, followed by two clade 2 strains. No clade 1 nor 3 isolates were observed. stx1 in combination with either stx2(EDL933) or stx2c were frequently observed among human strains, whereas stx2c was dominating in nonhuman strains. MLVA indicated that only single cases or small outbreaks with E. coli O157 have been observed in Norway through the years 1993-2008. CONCLUSION: We observed that the tir-255 A/T SNP and the stx status were different between human and nonhuman O157 strains. No major outbreaks were observed, and only a few strains were differentiated into the virulence clades 2 and 8. SIGNIFICANCE AND IMPACT OF THE STUDY: The detection of virulence clade-specific SNPs enables the rapid designation of virulent E. coli O157 strains, especially in outbreak situations.

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Varicella-zoster virus (VZV), the causative agent of chickenpox and herpes zoster, can be life-threatening in prematurely born children and in children with immune defects or who are under immunosuppressive treatment. Therefore agents for passive immunization, such as VZV-specific immunoglobulin preparations (VZIG) derived from convalescent plasma, are crucial in the prophylaxis of VZV infection. This study describes the isolation of human VZV-neutralizing recombinant antibodies. A human single-chain variable fragment (scFv) phage display library was generated from RNA extracted from peripheral blood lymphocytes of a convalescent varicella patient. Specific phage antibodies were selected against VZV-infected human fibroblasts, and eight unique clones were further expressed as soluble scFv in Escherichia coli. They all showed binding characteristics to varicella antigens with affinities in the K(D) range 0.1-0.2 muM. Two of the scFv antibodies, VZV4 and VZV5, showed dose-dependent in vitro neutralization of VZV. VZV39 also showed a neutralizing effect as scFv, an effect that was increased 4000-fold by conversion into IgG and was further increased by the addition of complement. This is possibly the first time that monovalent scFv antibodies have been shown to neutralize VZV in vitro. This finding will have an impact on the production of new prophylactic antibodies, as such antibody fragments can be cost-effectively produced in E. coli. The antibodies isolated bind both complement-dependent and -independent epitopes for neutralization, thus they may prove useful tools for the study of VZV virulence mechanisms.

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Selection of antibodies from large repertoire phage display libraries has become a common technique for isolation of specific antibodies to antigens. Many of these libraries are shown to contain antibodies specific to haptens, but only when these haptens are derivatised or conjugated to an immobilising molecule, such as bovine serum albumin (BSA). There has been little demonstration of the suitability of naive recombinant antibody libraries for isolating antibodies that bind low molecular weight haptens in the absence of a carrier molecule and few have addressed the problems associated with selecting antibodies that only recognize the combination of hapten and the carrier molecule. We have panned two-phage antibody libraries against AflatoxinB1-BSA and screened single-chain antibody fragments for binding to AflatoxinB1-BSA and Aflatoxin-B1. Many of the antibodies isolated specifically bound AflatoxinB1-BSA, but not soluble Aflatoxin-B1 or BSA. Modification of the protocol led to isolation of single-chain fragment variable antibody domain (scFv) antibodies that specifically bound soluble Aflatoxin-B1 with an affinity of 6x10(-9) M.

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