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BackgroundDuring the course of a bacterial infection, the rapid identification of the causative agent(s) is necessary for the determination of effective treatment options. We have developed a method based on a modified broad-range PCR and an oligonucleotide microarray for the simultaneous detection and identification of 12 bacterial pathogens at the species level. The broad-range PCR primer mixture was designed using conserved regions of the bacterial topoisomerase genes gyrB and parE. The primer design allowed the use of a novel DNA amplification method, which produced labeled, single-stranded DNA suitable for microarray hybridization. The probes on the microarray were designed from the alignments of species- or genus-specific variable regions of the gyrB and parE genes flanked by the primers. We included mecA-specific primers and probes in the same assay to indicate the presence of methicillin resistance in the bacterial species. The feasibility of this assay in routine diagnostic testing was evaluated using 146 blood culture positive and 40 blood culture negative samples.ResultsComparison of our results with those of a conventional culture-based method revealed a sensitivity of 96% (initial sensitivity of 82%) and specificity of 98%. Furthermore, only one cross-reaction was observed upon investigating 102 culture isolates from 70 untargeted bacteria. The total assay time was only three hours, including the time required for the DNA extraction, PCR and microarray steps in sequence.ConclusionThe assay rapidly provides reliable data, which can guide optimal antimicrobial treatment decisions in a timely manner.

We present a high-throughput roll-to-roll (R2R) manufacturing process for foil-based polymethyl methacrylate (PMMA) chips of excellent optical quality. These disposable, R2R hot embossed microfluidic chips are used for the identification of the antibiotic resistance gene mecA in Staphylococcus epidermidis. R2R hot embossing is an emerging manufacturing technology for polymer microfluidic devices. It is based on continuous feeding of a thermoplastic foil through a pressurized area between a heated embossing cylinder and a blank counter cylinder. Although mass fabrication of foil-based microfluidic chips and their use for biological applications were foreseen already some years ago, no such studies have been published previously.

The rapid identification of microbes responsible for bloodstream infections (BSIs) allows more focused and effective therapies and outcomes. DNA sequence-based methods offer an opportunity for faster, accurate diagnosis and for effective therapy. As our objective of the study, the ability of the Prove-it Sepsis platform, already proven as a rapid PCR-and microarray-based assay for the majority of sepsis-causing bacteria, was extended to also rapidly identify clinically relevant yeasts in blood culture. The performance characteristics of this extended platform are described. We found that the extended diagnostic Prove-it Sepsis platform was found to be highly accurate when analyzing primary isolates, spiked blood cultures, nucleic acid extracts from a retrospective blood culture data set, and primary blood cultures. Comparison of the blood culture results from the Prove-it Sepsis platform with those from conventional culture-based methods or by gene sequencing demonstrated a sensitivity of 99% and a specificity of 98% for fungal targets (based on analysis of a total of 388 specimens). Total assay time was 3 h from DNA extraction to BSI diagnosis. These results extend the performance characteristics of the Prove-it platform for bacteria to the easy, rapid, and accurate detection and species identification of yeasts in positive blood cultures. Incorporation of this extended and rapid diagnostic platform into the tools for clinical patient management would allow possibly faster identification and more focused therapies for BSIs.T he incidence of fungemia, particularly candidemia, continues to increase steadily, especially in hospitalized patients (4,5,8,12,30,34). Candida spp. are the 4th most common causative agents of nosocomial bloodstream infections (BSIs) in the United States and the 5th to 10th most common cause in Europe and Australia (6,8,36). Candida albicans remains the predominant species in these infections, although recent data suggest an increase in the proportion of Candida species other than C. albicans, such as C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei (3, 4, 8, 16, 17, 34).Candidemia is associated with a high mortality rate, a prolonged length of hospital stay, and high hospital costs (13,15,17,26,29,35). Appropriate antifungal therapy administered in a timely fashion impacts morbidity and mortality and costs of treatment (14,15,20,25). Currently, suspected candidemia is diagnosed by blood culture, subsequent Gram staining, and various diagnostic practices, including a germ tube assay and various morphological and biochemical identification methods. These methods are all time-consuming, operator dependent, and costly, delaying effective and focused therapy and leading to avoidable additional mortality (20). In order to respond to the need for improved diagnostic tools, several diagnostic strategies and platforms have emerged for sepsis diagnostics, and some of them take advantage of the combination of PCR with microarray hybridization (19,21,27).In recent work by Tissari and cowor...

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