House dust mites as potential carriers for IgE sensitization to bacterial antigens.

BACKGROUND: IgE reactivity to antigens from Gram-positive and Gram-negative bacteria is common in patients suffering from respiratory and skin manifestations of allergy, but the routes and mechanisms of sensitization are not fully understood. The analysis of the genome, transcriptome and microbiome of house dust mites (HDM) has shown that Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) species are abundant bacteria within the HDM microbiome. Therefore, our aim was to investigate whether HDM are carriers of bacterial antigens leading to IgE sensitization in patients suffering from atopic dermatitis. METHODS: Plasma samples from patients with AD (n = 179) were analysed for IgE reactivity to a comprehensive panel of microarrayed HDM allergen molecules and to S. aureus and E. coli by IgE immunoblotting. Antibodies specific for S. aureus and E. coli antigens were tested for reactivity to nitrocellulose-blotted extract from purified HDM bodies, and the IgE-reactive antigens were detected by IgE immunoblot inhibition experiments. IgE antibodies directed to bacterial antigens in HDM were quantified by IgE ImmunoCAP™ inhibition experiments. RESULTS: IgE reactivity to bacterial antigens was significantly more frequent in patients with AD sensitized to HDM than in AD patients without HDM sensitization. S. aureus and E. coli antigens were detected in immune-blotted HDM extract, and the presence of IgE-reactive antigens in HDM was demonstrated by qualitative and quantitative IgE inhibition experiments. CONCLUSION: House dust mites (HDM) may serve as carriers of bacteria responsible for the induction of IgE sensitization to microbial antigens.

Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis.

AIM: Introduction of a protocol for broad-range diagnosis of bacterial infections, which remain negative in culture. METHODS AND RESULTS: The new TaqMan real-time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 mul EDTA-blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24.3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. CONCLUSIONS: The introduced broad-range real-time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing-point values and with the Blast result of both the sample and the controls. SIGNIFICANCE AND IMPACT OF THE STUDY: This work introduces a new and well-evaluated broad-range real-time PCR protocol for diagnosis of bacterial infections.

Isolation of Bordetella trematum from a diabetic leg ulcer.

BACKGROUND: The species Bordetella trematum (a Gram-negative, opportunistic bacterium) was established in 1996. To date, 10 cases of human infection/colonization with the pathogen have been described. CASE REPORT: The first case of isolation of B. trematum from a diabetic ulcer is reported. Since there are no commercially available kits for identification of the organism, species diagnosis was based on 16S rDNA sequencing. B. trematum disappeared from the ulcer without antimicrobial therapy. CONCLUSION: In the present case, there was no evidence for a causative role of the organism in the diabetic foot infection, which is in agreement with previously published data. However, B. trematum has to be considered when otherwise unclassified Gram-negative rods are isolated from infected diabetic ulcers.

Application of blood-based polymerase chain reaction for detection of Chlamydia pneumoniae in acute respiratory tract infections.

The aim of this study was to investigate whether blood-based polymerase chain reaction could serve as a diagnostic tool to identify individuals with acute respiratory Chlamydia pneumoniae infection. Respiratory specimens and peripheral blood mononuclear cells of 58 patients were analyzed using nested polymerase chain reaction and cell culture. Fifteen patients were polymerase chain reaction-positive for Chlamydia pneumoniae. Nine patients were positive in only the respiratory specimen; two in both the respiratory and blood sample (time intervals between onset of symptoms and sample collection, 3-10 days and 3-4 weeks, respectively); and four in only the blood sample. Detection of Chlamydia pneumoniae DNA in peripheral blood mononuclear cells does not seem to be a suitable marker for acute respiratory Chlamydia pneumoniae infection.