Ureaplasma urealyticum is a commensal organism of the lower genital tract of females, but in a subpopulation of individuals, it can invade the upper genital tract. It is a significant cause of chorioamnionitis and neonatal morbidity and mortality. There are 14 recognized serovars of U. urealyticum; these can be divided into two distinct clusters or biovars. Biovar 1 is composed of serovars 1, 3, 6, and 14. Biovar 2 is composed of serovars 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13. We previously identified a surface antigen, the multiple-banded (MB) antigen, which contains both serovar-specific and cross-reactive epitopes. Genotypic characterization of the C-terminal region of the MB antigen of serovar 3 indicates that serovar specificity and MB antigen size variation reside in that domain. In the present study, we used PCR analysis with primers derived from the serovar 3 MB antigen gene DNA sequence to determine if the MB antigen gene was present in the remaining 13 reference serovars as well as in invasive clinical isolates. The results indicated that not only was the MB antigen gene present in all serovars but that the genes' 5' regions were markers of biovar specificity and diversity. Further analysis of this region should reveal the phylogenetic relationship among serovars of U. urealyticum and, possibly, their invasive potential.
Aims: The Cepheid GeneXpert® is a four‐site, automated sample preparation and real‐time PCR detection system. In this study, the capability of the GeneXpert® to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. Methods and Results: A four‐plex, dried‐down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross‐reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross‐reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert® to those run directly on the Cepheid SmartCycler® without sample processing. The GeneXpert® detection capability was three logs lower than the SmartCycler® indicating the benefit of incorporating a nucleic acid extraction procedure. Conclusions: This study demonstrates that the GeneXpert® is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. Significance and Impact of the Study: The GeneXpert® is the only platform currently available that is capable of both nucleic acid purification and real‐time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing.
To compare the diarrheagenic Escherichia coli (DEC) identifications obtained between traditional O serotyping and modern virulence gene detection assays, we developed a multiplex real-time PCR assay by detecting six specific virulence genes for enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), and enteroinvasive E. coli (EIEC). Among 261 clinical diarrheal stool samples, a total of 137 suspected DEC (sDEC) isolates were identified by the use of commercially available antisera. The most prevalent serogroups were O1 (12/137; 8.7%), O25 (9/137; 6.5%), and O44 (9/137; 6.5%). The specific virulence genes for the 137 sDEC isolates were analyzed by the multiplex real-time PCR assay. Fifteen (10.9%) of 137 isolates were confirmed to be true DEC strains, indicating that the serotypic markers did not correlate with the specific virulence genes. ETEC (66.7%) was the most prevalent, followed by EIEC (20%) and EPEC (13.3%). No EHEC strains were identified in the specimens. Four novel serotypes were found in the study: two in EPEC strains (O111:H9 and O63:H6) and two in EIEC strains (O63:H9 and O169:H9). In conclusion, the real-time PCR assay considerably reduces the high false-positive rate from the use of serotyping alone, and thus, it is suggested that serogrouping-based methods are inadequate for the identification of DEC isolates, although they are useful for the identification of a limited number of serogroups. In addition, ETEC, EPEC, and EIEC strains were present in 5. 7% (15/261) of the diarrheal patients in northern Taiwan in 2006.Escherichia coli is a predominant member of the human intestinal flora. Some strains are rendered pathogenic by their ability to possess specific virulence factors, such as enterotoxin or adherent fimbriae, that are genetically encoded by plasmid, chromosome, and bacteriophage DNA (2, 14). These diarrheagenic E. coli (DEC) strains usually play important roles as the causes of endemic and epidemic human diseases, such as severe diarrhea, food poisoning, and similar outbreaks worldwide (9, 15). Infections with these kinds of pathogens have therefore been of increasing concern in the clinical diagnosis of diarrheal disease in recent years. These virulent organisms can be classified into five major categories on the basis of the nature of their infection and pathogenic mechanisms: they are enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), and enteroaggregative E. coli (EAEC) (14, 25). However, the current rates of infections by these important enteric pathogens are probably largely underestimated due to the limitations of existing clinical diagnostic methods to be able to distinguish them from normal nonpathogenic flora. In order to achieve the goal of epidemic prevention and control of DEC in Taiwan, we need a more reliable procedure to identify and categorize DEC isolates so that more reliable studies of DEC incidence rates can be conducted in the future.Phenotypic...
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