Evaluation of Real-Time PCR Coupled With Multiplex Probe Melting Curve Analysis for Pathogen Detection in Patients With Suspected Bloodstream Infections

Xiao, Yao; Shen, Xu; Zhao, Qifeng; Yao, Yue; Yang, Tian‐Ci; Niu, Jianjun

doi:10.3389/fcimb.2019.00361

Cited by 7 publications

(5 citation statements)

References 21 publications

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“…While blood culture remains the reference method for BSI diagnosis, new rapid methods have emerged to reduce the delay to identify bacterial and fungal pathogens. They may be realized directly from whole blood (multiplex PCR; T2 magnetic resonance) [61][62][63][64][65] but are mainly performed on positive blood culture bottles using either MALDI-TOF-MS-based techniques (direct identification after purification of bacterial or fungal pellets; identification after short-term incubation on a solid medium) [30][31][32][33]45] or molecular-based techniques (multiplex-PCR, PNA-FISH) [31,[35][36][37]39].…”

Section: Discussionmentioning

confidence: 99%

Evaluation of Rapid Sepsityper® protocol and specific MBT-Sepsityper module (Bruker Daltonics) for the rapid diagnosis of bacteremia and fungemia by MALDI-TOF-MS

Ponderand

Pavèse

Maubon

et al. 2020

Ann Clin Microbiol Antimicrob

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During bloodstream infections, rapid adaptation of empirical treatment according to the microorganism identified is essential to decrease mortality. The aim of the present study was to assess the microbiological performances of a new rapid version of the Sepsityper® kit (Bruker Daltonics) allowing identification of bacteria and yeast by MALDI-TOF mass spectrometry directly from positive blood cultures in 10 min and of the specific MBT-Sepsityper module for spectra analysis, designed to increase identification performance. Identification rates were determined prospectively on 350 bacterial and 29 fungal positive blood cultures, and compared to conventional diagnostic method. Our rapid diagnosis strategy (Rapid Sepsityper® protocol: one spot with and one without formic acid extraction step) combined to MBT-Sepsityper module provided 65.4%, 78.9% and 62% reliable identification to the species level of monomicrobial positive blood cultures growing respectively Gram-positive, Gram-negative bacteria or yeast. Importantly, identification rates of Gram-positive bacteria were higher in anaerobic than in aerobic bottles (77.8% vs 22.2%; p = 0.004), if formic acid extraction step was performed (60.8% vs 39.2%; p = 1.8e−6) and if specific MBT-Sepsityper module was used (76.2% vs 61.9%, p = 0.041) while no significant differences were observed for Gram-negative bacteria. For yeasts identification, formic acid extraction step improved rapid identification rate by 37.9% while the specific MBT-Sepsityper module increased overall performances by 38%, providing up to 89.7% reliable identification if associated with the standard Sepsityper® protocol. These performances, associated with a reduce turnaround time, may help to implement a rapid identification strategy of bloodstream infections in the routine workflow of microbiology laboratories.

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Section: Discussionmentioning

confidence: 99%

Evaluation of Rapid Sepsityper® protocol and specific MBT-Sepsityper module (Bruker Daltonics) for the rapid diagnosis of bacteremia and fungemia by MALDI-TOF-MS

Ponderand

Pavèse

Maubon

et al. 2020

Ann Clin Microbiol Antimicrob

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“…The MCA method can be further developed to distinguish closely related species using species‐specific probes to differentiate species with similar T m or when multiple species detections are intended (e.g. see Xiao et al., 2019). In the case of using species‐specific probes, there is no need to use species‐specific primers (e.g.…”

Section: Discussionmentioning

confidence: 99%

Melting curve analysis for detection and identification of ghost parasitoids in host carcasses a month after host death

Paula

Andow

2021

Methods Ecol Evol

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1. Incidence of parasitism is often underestimated because 'ghost' parasitoids (dead unemerged parasitoids or those that have emerged leaving the host carcasses) are difficult to detect and identify. This study demonstrates that the use of melting curve analysis (MCA) of host carcasses can detect and identify DNA of ghost parasitoids even a month after host death. 2. The coccinellid hosts Cycloneda sanguinea, Eriopis connexa, Harmonia axyridis and Hippodamia convergens were sampled from cole crops in 2017 and 2018 in the Midwest of Brazil, and reared and observed daily for parasitoid emergence. Dead coccinellids were held for 30 days after death before storage and only host carcasses with parasitoid emergence observed were analysed. Species-specific primers were designed for the identification of the parasitoid species that emerged during host rearing: Dinocampus coccinellae, Homalotylus mirabilis, Ho. terminalis, Strongygaster triangulifera and Phalacrotophora sp. The melting temperatures (T m ) of their amplicons were used as positive controls in MCA post-amplification in qPCR.3. Detection of parasitoid DNA in host carcasses was possible for D. coccinellae, Ho. mirabilis and Phalacrotophora sp. with a limit of detection (LOD) for all the parasitoids <1 pg of DNA, except for Phalacroptopthora sp. (LOD = 1.9 ng). Parasitoids were detected in 31 out of 70 host carcasses (44.3%) with detection ranging from 0% to 71%, depending on the parasitoid species.4. Synthesis and applications. This work demonstrates that MCA could be used to detect and rapidly identify the DNA of some parasitoid species (e.g. Ho. mirabilis, D. coccinellae and Phalacrotophora sp.) in host carcasses up to a month after parasitoid emergence with high sensitivity and specificity. MCA can be used on any field-collected host and, for some parasitoid species, it should lead to more accurate estimates of the incidence of parasitism. Our approach can be easily adapted and applied to study other parasitoid-host interactions.

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“…Given the retrospective nature of this study, the requirement for informed consent was waived. Microbial cellular DNA was extracted using a previously described heat‐lysis method (Xiao et al., 2019 ). Briefly, 1.0 mL of the culture suspension was centrifuged at 13,000 g for 2 min to recover the bacteria.…”

Section: Methodsmentioning

confidence: 99%

Direct identification of pathogens via microbial cellular DNA in whole blood by MeltArray

Song,

Lin,

Zhu

et al. 2023

Microbial Biotechnology

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Rapid identification of pathogens is critical for early and appropriate treatment of bloodstream infections. The various culture‐independent assays that have been developed often have long turnaround times, low sensitivity and narrow pathogen coverage. Here, we propose a new multiplex PCR assay, MeltArray, which uses intact microbial cells as the source of genomic DNA (gDNA). The successive steps of the MeltArray assay, including selective lysis of human cells, microbial cell sedimentation, microbial cellular DNA extraction, target‐specific pre‐amplification and multiplex PCR detection, allowed the detection of 35 major bloodstream infectious pathogens in whole blood within 5.5 h. The limits of detection varied depending on the pathogen and ranged from 1 to 5 CFU/mL. Of 443 blood culture samples, including 373 positive blood culture samples and 70 negative blood culture samples, the MeltArray assay showed a sensitivity of 93.8% (350/373, 95% confidence interval [CI] = 90.7%–96.0%), specificity of 98.6% (69/70, 95% CI = 91.2%–99.9%), positive predictive value of 99.7% (95% CI = 98.1%–99.9%), and negative predictive value of 75.0% (95% CI = 64.7%–83.2%). The MeltArray detection results of 16 samples differed from MALDI–TOF and were confirmed by Sanger sequencing. Further testing of 110 whole blood samples from patients with suspected bloodstream infections using blood culture results revealed that the MeltArray assay had a clinical sensitivity of 100% (9/9, 95% CI = 62.8%–100.0%), clinical specificity of 74.5% (70/94, 95% CI = 64.2%–82.7%), positive predictive value of 27.3% (95% CI = 13.9%–45.8%), and negative predictive value of 100.0% (95% CI = 93.5%–100.0%). Compared with metagenomic next‐generation sequencing, the MeltArray assay displayed a positive agreement of 85.7% (6/7, 95% CI = 42.0%–99.2%) and negative agreement of 100.0% (4/4, 95% CI = 39.6%–100.0%). We conclude that the MeltArray assay can be used as a rapid and reliable tool for direct identification of pathogens in bloodstream infections.

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Evaluation of Real-Time PCR Coupled With Multiplex Probe Melting Curve Analysis for Pathogen Detection in Patients With Suspected Bloodstream Infections

Cited by 7 publications

References 21 publications

Evaluation of Rapid Sepsityper® protocol and specific MBT-Sepsityper module (Bruker Daltonics) for the rapid diagnosis of bacteremia and fungemia by MALDI-TOF-MS

Evaluation of Rapid Sepsityper® protocol and specific MBT-Sepsityper module (Bruker Daltonics) for the rapid diagnosis of bacteremia and fungemia by MALDI-TOF-MS

Melting curve analysis for detection and identification of ghost parasitoids in host carcasses a month after host death

Direct identification of pathogens via microbial cellular DNA in whole blood by MeltArray

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