BackgroundThe environmental bacterium Burkholderia pseudomallei causes the infectious disease melioidosis with a high case-fatality rate in tropical and subtropical regions. Direct pathogen detection can be difficult, and therefore an indirect serological test which might aid early diagnosis is desirable. However, current tests for antibodies against B. pseudomallei, including the reference indirect haemagglutination assay (IHA), lack sensitivity, specificity and standardization. Consequently, serological tests currently do not play a role in the diagnosis of melioidosis in endemic areas. Recently, a number of promising diagnostic antigens have been identified, but a standardized, easy-to-perform clinical laboratory test for sensitive multiplex detection of antibodies against B. pseudomallei is still lacking.Methods and Principal FindingsIn this study, we developed and validated a protein microarray which can be used in a standard 96-well format. Our array contains 20 recombinant and purified B. pseudomallei proteins, previously identified as serodiagnostic candidates in melioidosis. In total, we analyzed 196 sera and plasmas from melioidosis patients from northeast Thailand and 210 negative controls from melioidosis-endemic and non-endemic regions. Our protein array clearly discriminated between sera from melioidosis patients and controls with a specificity of 97%. Importantly, the array showed a higher sensitivity than did the IHA in melioidosis patients upon admission (cut-off IHA titer ≥1:160: IHA 57.3%, protein array: 86.7%; p = 0.0001). Testing of sera from single patients at 0, 12 and 52 weeks post-admission revealed that protein antigens induce either a short- or long-term antibody response.ConclusionsOur protein array provides a standardized, rapid, easy-to-perform test for the detection of B. pseudomallei-specific antibody patterns. Thus, this system has the potential to improve the serodiagnosis of melioidosis in clinical settings. Moreover, our high-throughput assay might be useful for the detection of anti-B. pseudomallei antibodies in epidemiological studies. Further studies are needed to elucidate the clinical and diagnostic significance of the different antibody kinetics observed during melioidosis.

BackgroundThe PVL-positive ST772-MRSA-V is an emerging community-associated (CA-) MRSA clone that has been named Bengal Bay Clone since most patients have epidemiological connections to the Indian subcontinent. It is found increasingly common in other areas of the world.MethodsOne isolate of ST772-MRSA-V was sequenced using the Illumina Genome Analyzer System. After initial assembling the multiple sequence contigs were analysed using different in-house annotation scripts. Results were compared to microarray hybridisation results of clinical isolates of ST772-MRSA-V, of related strains and to another ST772-MRSA-V genome sequence.ResultsAccording to MLST e-burst analysis, ST772-MRSA-V belongs to Clonal Complex (CC)1, differing from ST1 only in one MLST allele (pta-22). However, there are several additional differences including agr alleles (group II rather than III), capsule type (5 rather than 8), the presence of the egc enterotoxin gene cluster and of the enterotoxin homologue ORF CM14 as well as the absence of the enterotoxin H gene seh. Enterotoxin genes sec and sel are present. ST772-MRSA-V harbours the genes encoding enterotoxin A (sea) and PVL (lukS/F-PV). Both are located on the same prophage.ConclusionsST772-MRSA-V may have emerged from the same lineage as globally spread CC1 and CC5 strains. It has acquired a variety of virulence factors, and for a CA-MRSA strain it has an unusually high number of genes associated with antibiotic resistance.

No abstract

This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C lostridium difficile is the leading infectious agent of nosocomial diarrhea in humans and causes gastrointestinal infections also in various animal species (e.g., pigs, horses, and rodents) (1, 2). Over the last decade, increasing incidence and changes in the clinical presentation of human C. difficile-associated diarrhea have been reported worldwide (1). Newly emerging C. difficile genotypes (e.g., PCR ribotypes 027 and 078) are involved in these epidemiological changes, which have also been found in companion animals (i.e., calves and piglets), pets (i.e., cats and dogs), and foods (e.g., meat products and vegetables), indicating the possibility of zoonotic transmission (1, 3). Therefore, the genotyping of C. difficile isolates is important for epidemiological and clinical investigations. For genotyping, several molecular methods have been established so far: restriction endonuclease analysis (REA) (4, 5), pulsed-field gel electrophoresis (PFGE) (6, 7), toxinotyping (8), multilocus variable-number tandem repeat (VNTR) analysis (MLVA) (9, 10), multilocus sequence typing (MLST) (11), surface layer protein A typing (slpA typing) (12, 13), and PCR ribotyping (14, 15). PCR ribotyping is the standard typing method used in Europe and is widely used also in the United States and Canada (16). The target for this method is the intergenic spacer region (ISR) between the 16S and 23S rRNA genes (14, 15). The ISR is variable in length and is present up to 10 times in the C. difficile genome. Thus, PCR amplification results in a specific amplicon profile after separation in an agarose gel. However, agarose gel analysis needs a considerable effort in standardization, including a huge number of PCR ribotype reference strains, to correctly assign isolates for interlaboratory comparability (16). Recently, Indra et al. (17) developed a PCR ribotyping method with subsequent capillary gel electrophoresis (seq-PCR ribotyping) and Web database analysis. Compared to the conventional procedure, seq-PCR ribotyping is faster, has a higher resolution, and might be a tool for standardization (17). H...