Detection of in vivo hepatitis B virus surface antigen mutations-A comparison of four routine screening assays.

An important requirement for a state-of-the-art hepatitis B surface antigen (HBsAg) screening assay is reliable detection of mutated HBsAg. Currently, there is a striking shortage of data regarding the detection rates of in vivo HBsAg mutations for these clinically important assays. Therefore, we compared the detection rates of four commercial HBsAg screening assays using a global cohort of 1553 patients from four continents with known HBV genotypes. These samples, which represent the broadest spectrum of known and novel HBsAg major hydrophilic region (MHR) mutations to date, were analyzed for the presence of HBsAg using the Roche Elecsys® HBsAg II Qualitative, Siemens ADVIA Centaur XP HBsAg II, Abbott Architect HBsAg Qualitative II and DiaSorin Liaison® HBsAg Qualitative assays, respectively. Of the 1553 samples, 1391 samples could be sequenced; of these, 1013 (72.8%) carried at least one of the 345 currently known amino acid substitutions (distinct HBsAg mutation) in the HBsAg MHR. All 1553 patient samples were positive for HBsAg using the Elecsys® HBsAg II Qual assay, with a sensitivity (95% confidence interval) of 99.94% (99.64%-100%), followed by the Abbott Architect 99.81% (99.44%-99.96%), Siemens ADVIA 99.81% (99.44%-99.96%) and DiaSorin Liaison® 99.36% (98.82%-99.69%) assays, respectively. Our results indicate that the Elecsys® HBsAg II Qual assay exhibits the highest sensitivity among the commercial HBsAg screening assays, and demonstrate that its capacity to detect HBV infection is not compromised by HBsAg MHR mutants.

Spatial organisation of microbiota in quiescent adenoiditis and tonsillitis.

BACKGROUND: The reasons for recurrent adenotonsillitis are poorly understood. METHODS: The in situ composition of microbiota of nasal (5 children, 25 adults) and of hypertrophied adenoid and tonsillar tissue (50 children, 20 adults) was investigated using a broad range of fluorescent oligonucleotide probes targeted to bacterial rRNA. None of the patients had clinical signs of infection at the time of surgery. RESULTS: Multiple foci of ongoing purulent infections were found within hypertrophied adenoid and tonsillar tissue in 83% of patients, including islands and lawns of bacteria adherent to the epithelium, with concomitant marked inflammatory response, fissures filled with bacteria and pus, and diffuse infiltration of the tonsils by bacteria, microabscesses, and macrophages containing phagocytosed microorganisms. Haemophilusinfluenzae mainly diffusely infiltrated the tissue, Streptococcus and Bacteroides were typically found in fissures, and Fusobacteria,Pseudomonas and Burkholderia were exclusively located within adherent bacterial layers and infiltrates. The microbiota were always polymicrobial. CONCLUSIONS: Purulent processes persist during asymptomatic periods of adenotonsillitis. Most bacteria involved in this process are covered by a thick inflammatory infiltrate, are deeply invading, or are located within macrophages. The distribution of the bacteria within tonsils may be responsible for the failure of antibiotic treatment.

Direct detection of methicillin-resistant Staphylococcus aureus in clinical specimens by a nucleic acid-based hybridisation assay.

The occurrence of methicillin-resistant Staphylococcus aureus (MRSA) is still increasing worldwide and is associated with significant morbidity, mortality and hospital costs. Screening for MRSA plays a key role in limiting further nosocomial spread of this organism. Control measures require a rapid and sensitive test for direct detection of MRSA carriage. This study evaluated an easy-to-use PCR-hybridisation assay for the direct detection of MRSA in clinical swab specimens. In total, 508 pairs of swabs from 242 patients at risk for MRSA carriage were analysed by the standard culture method and the PCR assay. One swab was used for PCR and culture, while the second was used for culture only. Of the 508 pairs tested, 37 were positive by culture and 35 were positive by PCR. Among the 471 culture-negative specimens, 465 were negative by PCR and six were PCR-positive. The PCR assay had a sensitivity of 94.59%, a specificity of 98.73%, a positive predictive value of 85.37%, and a negative predictive value of 99.57%. The PCR-hybridisation assay enabled reliable detection of MRSA carriage in c. 4 h, thereby allowing its effective use in an MRSA control strategy.

Evaluation of a rapid direct assay for identification of bacteria and the mec A and van genes from positive-testing blood cultures.

We performed the first evaluation of a DNA strip assay (GenoType blood culture; Hain Lifescience, Nehren, Germany) for the detection of the most relevant bacterial sepsis pathogens directly from positive BACTEC blood culture bottles (Becton Dickinson, Heidelberg, Germany). The test comprises two panels, one for the direct species identification of important gram-positive cocci and the other for gram-negative rods. Additionally, detection of the mec A and the van genes are implemented. The GenoType assay was validated regarding its analytical sensitivity with blood cultures spiked with reference strains. Approximately 10(4) CFU per ml were detected. Analytical specificity was calculated with a test panel of 212 reference strains. Of the strains tested, 99% were correctly identified. Additionally, 279 consecutive blood cultures signaled positive by BACTEC were processed directly, in comparison to conventional methods. The GenoType assays were performed according to Gram stain morphology. A total of 243 (87.1%) of the 279 organisms isolated were covered by specific probes. A total of 152 organisms were gram-positive cocci, of which 148 (97.4%) were correctly identified by the GenoType assay. Ninety-one organisms were gram-negative rods, of which 89 (97.8%) were correctly identified. Concerning mec A gene detection, GenoType assay correctly detected 12 of 13 methicillin-resistant Staphylococcus aureus isolates. One Enterococcus faecium isolate with a positive van A gene isolated was correctly differentiated by the assay. All results were available 4 h after the results of microscopic analysis. The evaluated GenoType blood culture assay showed fast and reliable results in detecting the most important sepsis pathogens and the mec A and van genes directly from positive blood culture bottles.

Evaluation of a new molecular system for simultaneous identification of four Enterococcus species and their glycopeptide resistance genotypes.

We compared the performances of a new DNA-based strip assay and the VITEK 2 system for identification of 105 enterococcal strains and differentiation of their van gene-associated resistance levels. Both methods provided excellent results. The molecular assay showed advantages in time to result for identification of van-associated genes.

Prevalence of hepatitis G in patients on chronic hemodialysis.

Hepatitis G virus (HGV) is a newly described RNA virus from the family of flaviviridae. It is closely related to the hepatitis C Virus (HCV) but is more common than HCV among healthy blood donors. The pathogenicity of HGV in immunosuppressed patients such as those undergoing hemodialysis is unclear. We measured the incidence of HGV in 105 patients undergoing hemodialysis in a chronic outpatient hemodialysis facility. HGV-RNA was detected using a RT-PCR method with primers directed against the 5' non-coding region and the NS5a gene of HGV. Nine (8.6%) patients were HGV RNA positive, eleven (10.5%) were anti-HCV positive, three (2.9%) were positive for hepatitis B surface antigen. Four patients were positive for both HGV and HCV; three of them had normal liver enzymes while one showed elevated ALT levels but no other signs of exacerbation of preexisting hepatitis. The prevalence of HGV among dialysis patients is comparable to that of HCV. The transmission route for HCV is nosocomial transmission during dialysis, whereas HGV shows both ways of transmission: blood transfusion mediated by a high prevalence of HGV among healthy blood donors and nosocomial transmission. HGV appears to play a minor role in acute hepatitis, even in immunosuppressed patients.

A closer look at the insertion within helix 54 of the 23S-rRNA of the genus Corynebacterium (and related taxa).

Direct sequencing of the insertion in helix 54 of the 23S-rRNA of different strains of 8 species of the genus Corynebacterium was performed in order to determine the extent of variations of these strains. The results demonstrated that there is considerable variation within different strains of a single species. While this clearly jeopardizes the usefulness of the insertion with regard to species-specific diagnostic probes, the variations were found to concentrate within two clearly defined regions comprising the ascending and descending parts of the distal helix of a putative stemlike secondary structure. The remainder of the insertion sequences seem to be fairly constant within a single species while displaying considerable differences toward sequences of other species of the same genus.

Bacterial phylogeny based on comparative sequence analysis.

Comparative sequence analysis of small subunit rRNA is currently one of the most important methods for the elucidation of bacterial phylogeny as well as bacterial identification. Phylogenetic investigations targeting alternative phylogenetic markers such as large subunit rRNA, elongation factors, and ATPases have shown that 16S rRNA-based trees reflect the history of the corresponding organisms globally. However, in comparison with three to four billion years of evolution the phylogenetic information content of these markers is limited. Consequently, the limited resolution power of the marker molecules allows only a spot check of the evolutionary history of microorganisms. This is often indicated by locally different topologies of trees based on different markers, data sets or the application of different treeing approaches. Sequence peculiarities as well as methods and parameters for data analysis were studied with respect to their effects on the results of phylogenetic investigations. It is shown that only careful data analysis starting with a proper alignment, followed by the analysis of positional variability, rates and character of change, testing various data selections, applying alternative treeing methods and, finally, performing confidence tests, allows reasonable utilization of the limited phylogenetic information.

Phylogenetic evidence for the transfer of Eubacterium suis to the genus Actinomyces as Actinomyces suis comb. nov.

The 16S rRNA primary structures of Eubacterium suis DSM 20639T (T = type strain) and Bifidobacterium bifidum DSM 20456T were determined by sequencing in vitro amplified rDNA. Sequence comparisons indicated that B. bifidum is moderately related to representatives of the genera Actinomyces and Mobiluncus. The closest relative of E. suis is Actinomyces pyogenes. E. suis and A. pyogenes are more closely related phylogenetically to one another than to the other Actinomyces species that have been investigated by using comparative 16S rRNA analysis. Therefore, we propose that E. suis should be transferred to the genus Actinomyces as Actinomyces suis comb. nov.

The phylogenetic position of Peptococcus niger based on 16S rRNA sequence studies.

A 1330 base-pair fragment of a 16S rRNA gene has been amplified, cloned and sequenced. Comparison to other 16S rRNA sequences of eubacteria showed that P. niger represents a deep branch within the subdivision "Gram-positive with Gram-negative cell walls". It is not related to peptostreptococci, representatives of this genus studied so far are more closely related to clostridia.

Complete nucleotide sequences of seven eubacterial genes coding for the elongation factor Tu: functional, structural and phylogenetic evaluations.

The nucleotide sequences of cloned genes coding for the elongation factor Tu of seven eubacteria have been determined. These genes were from Anacystis nidulans, Bacillus subtilis, Bacteroides fragilis, "Deinonema" spec., Pseudomonas cepacia, Shewanella putrefaciens and Streptococcus oralis. The primary structures of the genes were compared to the available sequences of prokaryotic elongation factors Tu and eukaryotic elongation factors 1 alpha. A conservation profile was determined for homologous amino acid residues. Sites of known or putative functions are usually located at highly conserved positions or within highly conserved sequence stretches. The aligned 24 amino acid sequences were used as basis for a phylogenetic analysis. The phylogenetic tree corroborates the kingdom as well as phylum concept deduced from 16S rRNA data.

Comparative 16S rRNA oligonucleotide analyses and murein types of round-spore-forming bacilli and non-spore-forming relatives.

The phylogenetic incoherency of the genus Bacillus as presently described is demonstrated by analysis of both published and new data from comparative 16S rRNA cataloguing of nine Bacillus species and a number of related non-Bacillus taxa, i.e. Caryophanon latum, Filibacter limicola and Planococcus citreus. While the ellipsoidal-spore-forming bacilli, e.g. B. subtilis and allied species, formed a coherent cluster, the round-spore-forming bacilli showed a higher degree of relationship to the non-spore-forming organisms than these bacilli show among each other. Thus B. sphaericus clustered with C. latum, B. globisporus grouped with F. limicola, B. pasteurii with Sporosarcina ureae, and 'B. aminovorans' with P. citreus, respectively. These organisms formed two related subclusters which, in their phylogenetic depth, are comparable to that of the B. subtilis subline. With the exception of 'B. aminovorans', the 16S rRNA phylogeny was entirely consistent with the distribution of murein types. Even more distantly related to and grouping outside the main Bacillus cluster was B. stearothermophilus, which displayed a moderate relationship to Thermoactinomyces vulgaris. Taxonomic problems arising from the new insights into the intrageneric relationships of Bacillus are discussed.