RESUMO
OBJECTIVE: To establish and evaluate a new test system for rapid detection and diagnosis of adenoviral keratoconjunctivitis. DESIGN: After establishment of the molecular assay, 52 conjunctival smears were studied. PARTICIPANTS: Samples were derived from patients with a clinical presentation compatible with keratoconjunctivitis. METHODS: A molecular assay for detection of human adenovirus (HAdV) based on automated nucleic acid extraction and real time polymerase chain reaction was established and evaluated. The new assay included a heterologous internal control. MAIN OUTCOME MEASURES: Statement about the presence or absence of adenoviral DNA in the specimen. RESULTS: The amplification efficiency was found to be 100%. The detection limit was calculated to be 116 copies per LightCycler capillary. When clinical specimens were tested, 15 of 52 conjunctival smears were found to be positive for HAdV DNA. The internal control was detected in all samples. CONCLUSIONS: The new molecular assay proved to be suitable for rapid diagnosis of adenoviral keratoconjunctivitis in the routine diagnostic laboratory.
Assuntos
Infecções por Adenovirus Humanos/diagnóstico , Adenovírus Humanos/isolamento & purificação , Conjuntivite Viral/diagnóstico , DNA Viral/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Infecções por Adenovirus Humanos/virologia , Adenovírus Humanos/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Automação , Criança , Pré-Escolar , Conjuntivite Viral/virologia , Úlcera da Córnea/diagnóstico , Úlcera da Córnea/virologia , Primers do DNA/química , Sondas de DNA/química , Feminino , Amplificação de Genes , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Kit de Reagentes para DiagnósticoRESUMO
The acute disease antigen A (adaA) gene is believed to be associated with Coxiella burnetii strains causing acute Q fever. The detailed analysis of the adaA genomic region of 23 human- and 86 animal-derived C. burnetii isolates presented in this study reveals a much more polymorphic appearance and distribution of the adaA gene, resulting in a classification of C. burnetii strains of better differentiation than previously anticipated. Three different genomic variants of the adaA gene were identified which could be detected in isolates from acute and chronic patients, rendering the association of adaA positive strains with acute Q fever disease disputable. In addition, all adaA positive strains in humans and animals showed the occurrence of the QpH1 plasmid. All adaA positive isolates of acute human patients except one showed a distinct SNP variation at position 431, also predominant in sheep strains, which correlates well with the observation that sheep are a major source of human infection. Furthermore, the phylogenetic analysis of the adaA gene revealed three deletion events and supported the hypothesis that strain Dugway 5J108-111 might be the ancestor of all known C. burnetii strains. Based on our findings, we could confirm the QpDV group and we were able to define a new genotypic cluster. The adaA gene polymorphisms shown here improve molecular typing of Q fever, and give new insights into microevolutionary adaption processes in C. burnetii.
Assuntos
Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Cromossomos Bacterianos/ultraestrutura , Coxiella burnetii/genética , Coxiella burnetii/metabolismo , Evolução Molecular , Febre Q/microbiologia , Antígenos de Bactérias/fisiologia , Proteínas da Membrana Bacteriana Externa , Proteínas de Bactérias/fisiologia , Primers do DNA/genética , DNA Bacteriano/genética , Deleção de Genes , Marcadores Genéticos , Genótipo , Humanos , Tipagem Molecular , Filogenia , Polimorfismo Genético , Modelos de Riscos Proporcionais , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In guided bone regeneration (GBR), a semipermeable membrane is placed over an osseous defect to create a secluded environment in which bone formation can proceed without ingrowth of connective tissue cells from the overlaying soft tissue. Although the cell-occlusive property of GBR membranes appear to be essential to new bone formation, the role of transmembrane tissue fluid diffusion is not known. The objective of this study was to evaluate the degree to which diffusion across commonly used GBR membranes can support functional properties of osteoblast-like cells in vitro. Cells from an established osteoblast-like line (SAOS-2) were cultured on membranes of cross-linked collagen, noncross linked collagen, and ePTFE. The membranes rested on metal grids which allowed the membranes to lightly contact the surface of the culture medium. As a control, cells were directly plated and cultured in control wells. At days 7 and 21, cells were harvested by scraping the membranes or culture wells and analyzed for expression of alkaline phosphatase (ALP), core binding factor 1 (cbfa-1), bone sialoprotein-2 (BSP-2), and osteocalcin (OC). Expression was determined by quantitative real-time PCR. Glucose-6-phosphate dehydrogenase (G6PD) served as a reference gene. The membranes were examined by transmission light microscopy. RT-PCR revealed up-regulation of ALP of up to 60-fold and of cbfa-1 and BSP of up to threefold relative to G6PDH. Expression of OC was less then onefold. The expression profile for each of the four genes tested demonstrated small variations among cells grown on different membranes. Microscopic observations revealed remnants of undisrupted osteoblast-like cells attached to both collagen membranes. Cell morphology and spatial arrangement indicated that vitality was maintained. Diffusion through the three membranes evaluated in this study was sufficient to support osteoblast-like cell differentiation.