A European multicentre evaluation of detection and typing methods for human enteroviruses and parechoviruses using RNA transcripts.

Polymerase chain reaction (PCR) detection has become the gold standard for diagnosis and typing of enterovirus (EV) and human parechovirus (HPeV) infections. Its effectiveness depends critically on using the appropriate sample types and high assay sensitivity as viral loads in cerebrospinal fluid samples from meningitis and sepsis clinical presentation can be extremely low. This study evaluated the sensitivity and specificity of currently used commercial and in-house diagnostic and typing assays. Accurately quantified RNA transcript controls were distributed to 27 diagnostic and 12 reference laboratories in 17 European countries for blinded testing. Transcripts represented the four human EV species (EV-A71, echovirus 30, coxsackie A virus 21, and EV-D68), HPeV3, and specificity controls. Reported results from 48 in-house and 15 commercial assays showed 98% detection frequencies of high copy (1000 RNA copies/5 µL) transcripts. In-house assays showed significantly greater detection frequencies of the low copy (10 copies/5 µL) EV and HPeV transcripts (81% and 86%, respectively) compared with commercial assays (56%, 50%; P = 7 × 10-5 ). EV-specific PCRs showed low cross-reactivity with human rhinovirus C (3 of 42 tests) and infrequent positivity in the negative control (2 of 63 tests). Most or all high copy EV and HPeV controls were successfully typed (88%, 100%) by reference laboratories, but showed reduced effectiveness for low copy controls (41%, 67%). Stabilized RNA transcripts provide an effective, logistically simple and inexpensive reagent for evaluation of diagnostic assay performance. The study provides reassurance of the performance of the many in-house assay formats used across Europe. However, it identified often substantially reduced sensitivities of commercial assays often used as point-of-care tests.

Emergence and molecular characterisation of non-toxigenic tox gene-bearing Corynebacterium diphtheriae biovar mitis in the United Kingdom, 2003-2012.

Non-toxigenic Corynebacterium diphtheriae have become increasingly recognised as emerging pathogens across Europe causing severe invasive disease. A subset of non-toxigenic C. diphtheriae are 'non-toxigenic tox gene-bearing' (NTTB) strains; these strains are genotypically toxpositive, but do not express the protein. The circulation of NTTB strains was first observed during the 1990s upsurge of diphtheria in Eastern Europe but has not been reported in other European countries. Circulation of NTTB strains could be considered an increased risk for diphtheria and other related diseases, given their possible role as a tox gene reservoir with the theoretical risk of re-emerging toxin expression. Here we report the characterisation of 108 non-toxigenic C. diphtheriae biovar mitis isolates submitted to the World Health Organization (WHO) Global Reference Centre for Diphtheria at Public Health England, London, between 2003 and 2012, in order to determine the presence of NTTB strains. Using molecular methods, five NTTB isolates were identified; four human isolates (MLST type 212) and one isolate from a companion cat (MLST type 40). The emergence of these strains could indicate continuation of the circulation of potentially toxigenic strains and appropriate laboratory diagnostic methods should be used for detection. Given the complacency that currently exists in Europe awareness with regards to diphtheria diagnostics must be enhanced.

Infection of human enteroendocrine cells with Chlamydia trachomatis: a possible model for pathogenesis in irritable bowel syndrome.

BACKGROUND: Irritable bowel syndrome (IBS) is a widespread gastrointestinal disorder of unknown etiology. Recently, our group detected chlamydial antigens in enteroendocrine cells (EEC) of jejunum biopsies from patients with IBS. Impairment of EEC secretion upon Chlamydia infection might lead to disturbances of gut functions. We have therefore studied the interaction between Chlamydia and EEC in vitro. METHODS: Two different human enteroendocrine cell lines were studied: LCC-18 from a neuroendocrine colonic tumour and CNDT2 from a small intestinal carcinoid. Cell lines were infected with C. trachomatis serovar LGV II strain 434. We used Penicillin G for inducing persistent infection. The ultrastructure of infected cells was studied using transmission electron microscopy and immunofluorescence and we used RT-PCR analysis for studying changes in gene expression at different stages of infection. KEY RESULTS: We found that both cell lines could be infected with C. trachomatis yielding productive infections and persistence could be induced using penicillin G. Immunofluorescence showed different cellular distributions of serotonin and chromogranin A in non-infected (cytoplasmatic distribution) compared with infected cells (serotonin and chromogranin mostly in chlamydial inclusions). In line with the microscopical findings, we found a significant down-regulation of the gene coding for the vesicular monoamine transporter (VMAT1) in infected compared with non-infected EEC (P<0.05). CONCLUSIONS & INFERENCES: Altered protein distributions together with down-regulation of VMAT1 suggest that chlamydial infection may influence vesicular transport. It is therefore possible that such an infection in vivo could lead to disturbances in the regulation of gut functions.

Increase in invasive Streptococcus pyogenes and Streptococcus pneumoniae infections in England, December 2010 to January 2011.

Increases in invasive Streptococcus pyogenes and S. pneumoniae above the seasonally expected levels are currently being seen in England. Preliminary analyses suggest that the high level of influenza activity seen this winter may be contributing to an increased risk of concurrent invasive bacterial and influenza infections in children and young adults.

[Characterization of the interaction between Candida albicans and host cells: In vitro model using reconstituted human skin and mucosa]. / Charakterisierung der Interaktion zwischen Candida albicans und Wirtszellen. In-vitro-Modelle auf der Basis von rekonstituierter humaner Haut/Schleimhaut.

Basic research on the biology and immunology of microbial infection requires appropriate model systems. Currently most such studies involve animal studies which are a focus of ethical controversy. Possible alternatives, especially for localized infections, are provided by models using in vitro reconstituted human epithelium or epidermis (RHE). In recent years, these model systems have been successfully established to evaluate the effectiveness of topical anti-infectives, to characterize the role of fungal virulence factors, and to study the immune responses during localized Candida albicans infections. Most recently, these models have been supplemented with immune cells such as lymphocytes, polymorphonuclear leukocytes, mast cells or dendritic cells, to study their role during the course of infection and to characterize the interaction between the skin barrier and accessory immune cells. Although the most experience is with Candida albicans RHE infections, such model systems can also be used to study infections with other fungi or bacteria.