Molecular Identification of Bloodstream Pathogens in Patients Presenting to the Emergency Department With Suspected Sepsis

Avolio, Manuela; Diamante, Paola; Zamparo, Silvio; Modolo, Maria Luisa; Grosso, Shamanta; Zigante, Paola; Tosoni, Nilla; Rosa, Rita De; Stano, Paola; Camporese, Alessandro

doi:10.1097/shk.0b013e3181d49299

Cited by 41 publications

(22 citation statements)

References 18 publications

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“…However, not all pathogens detected by blood culture were also found by PCR techniques despite these pathogens being included in the PCR panel [20][21][22][23][24][25][26]. In contrast to increasing amplification cycles, specific enrichment of prokaryotic DNA before multiplex PCR may be able to overcome these limitations of sensitivity without decreasing specificity.…”

Section: A Eubacterial and Panfungal Real-time Pcr That Is Ablementioning

confidence: 98%

Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view

2011

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Polymerase chain reaction (PCR)-based techniques allow more rapid and sensitive detection of pathogens compared with conventional blood culture. Nevertheless, the climate of opinion of relevant studies is that currently PCR can supplement but not replace blood culture. In numerous studies, combined detection rate of both methods was significantly higher compared with PCR or blood culture alone. Also, complete determination of antibiotic resistance can currently be performed only by blood culture. Further increase of the panel of multiplex PCR is complicated, because the vast majority of sepsis pathogens are already included, primer interactions leading to primer heteromers limit the amount of targets detectable within one PCR tube, and an array of too many individual PCR reactions for investigation of a single specimen leads to high cost and workload. Except for diagnostics of patients in whom unusual, not culturable, or fastidious pathogens are detected more often, such as immunosuppressed patients with suspected parasitic infection, etc., it might even not be necessary to further increase the spectrum of detectable species. If the primary aim of PCR diagnostics is to decrease inappropriate empirical treatment and improve patient outcome, detection should focus on those pathogens or resistance determinants that are not covered by guideline-recommended treatment regimens and that have been identified as the major cause of inappropriate treatment according to current studies. In our opinion, such a narrower assay is more cost effective, may achieve higher accuracy due to reduced intratest interference, and would better address current and emerging clinical needs.

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Section: A Eubacterial and Panfungal Real-time Pcr That Is Ablementioning

confidence: 98%

Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view

2011

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“…Recently, polymerase chain reaction (PCR)-based assay have been seen as having the potential to provide an early and accurate diagnosis of diseases caused by bacterial pathogens and have improved the rate of microbial detection [2,[9][10][11]. Target sequences of the primers and probes are species-specific genes and conserved bacterial DNA sequences, such as 16S ribosomal RNA (rRNA), 23S rRNA, and 16S-23S rRNA interspace regions for amplification [2,5].…”

Section: Introductionmentioning

confidence: 99%

The role of a real-time PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples

Obara

Aikawa

Hasegawa

et al. 2011

Journal of Infection and Chemotherapy

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“…Of these techniques, real-time polymerase chain reaction (rtPCR) is the basis for a majority of molecular assays 6 . However, the usefulness of rtPCR can be limited by the specificity of primers and probes, and also by the genetic variability and number of targets that can be simultaneously detected and identified in a single reaction (typically no more than six) due to overlap of fluorescence spectra 3-5, 7 .…”

Section: Introductionmentioning

confidence: 99%

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

Girard

Boissinot

Peytavi

et al. 2015

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The combination of molecular diagnostic technologies is increasingly used to overcome limitations on sensitivity, specificity or multiplexing capabilities, and provide efficient lab-on-chip devices. Two such techniques, PCR amplification and microarray hybridization are used serially to take advantage of the high sensitivity and specificity of the former combined with high multiplexing capacities of the latter. These methods are usually performed in different buffers and reaction chambers. However, these elaborate methods have a high complexity cost related to reagent requirements, liquid storage and the number of reaction chambers to integrate into automated devices. Furthermore, microarray hybridizations have a sequence dependent efficiency not always predictable. In this work, we have developed the concept of a structured oligonucleotide probe which is activated by cleavage from polymerase exonuclease activity. This technology is called SCISSOHR for Structured Cleavage Induced Single-Stranded Oligonucleotide Hybridization Reaction. The SCISSOHR probes enable indexing the target sequence to a tag sequence. The SCISSOHR technology also allows the combination of nucleic acid amplification and microarray hybridization in a single vessel in presence of the PCR buffer only. The SCISSOHR technology uses an amplification probe that is irreversibly modified in presence of the target, releasing a single-stranded DNA tag for microarray hybridization. Each tag is composed of a 3-nucleotidesequence-dependent segment and a unique “target sequence-independent” 14-nucleotide segment allowing for optimal hybridization with minimal cross-hybridization. We evaluated the performance of five (5) PCR buffers to support microarray hybridization, compared to a conventional hybridization buffer. Finally, as a proof of concept, we developed a multiplexed assay for the amplification, detection, and identification of three (3) DNA targets. This new technology will facilitate the design of lab-on-chip microfluidic devices, while also reducing consumable costs. At term, it will allow the cost-effective automation of highly multiplexed assays for detection and identification of genetic targets.

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Molecular Identification of Bloodstream Pathogens in Patients Presenting to the Emergency Department With Suspected Sepsis

Cited by 41 publications

References 18 publications

Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view

Will polymerase chain reaction (PCR)-based diagnostics improve outcome in septic patients? A clinical view

The role of a real-time PCR technology for rapid detection and identification of bacterial and fungal pathogens in whole-blood samples

Structured oligonucleotides for target indexing to allow single-vessel PCR amplification and solid support microarray hybridization

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