Dataset Information

Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and Candida in Human Blood Samples.

ABSTRACT: The diagnosis of bloodstream infections (BSIs) still relies on blood culture (BC), but low turnaround times may hinder the early initiation of an appropriate antimicrobial therapy, thus increasing the risk of infection-related death. We describe a direct and rapid multiplex PCR-based assay capable of detecting and identifying 16 bacterial and four Candida species, as well as three antibiotic-resistance determinants, in uncultured samples. Using whole-blood samples spiked with microorganisms at low densities, we found that the MicrobScan assay had a mean limit of detection of 15.1 ± 3.3 CFU of bacteria/Candida per ml of blood. When applied to positive BC samples, the assay allowed the sensitive and specific detection of BSI pathogens, including bla KPC-, mecA-, or vanA/vanB-positive bacteria. We evaluated the assay using prospectively collected blood samples from patients with suspected BSI. The sensitivity and specificity were 86.4 and 97.0%, respectively, among patients with positive BCs for the microorganisms targeted by the assay or patients fulfilling the criteria for infection. The mean times to positive or negative assay results were 5.3 ± 0.2 and 5.1 ± 0.1 h, respectively. Fifteen of 20 patients with MicrobScan assay-positive/BC-negative samples were receiving antimicrobial therapy. In conclusion, the MicrobScan assay is well suited to complement current diagnostic methods for BSIs.

SUBMITTER: Liotti FM

PROVIDER: S-EPMC6863929 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and <i>Candida</i> in Human Blood Samples.

Liotti Flora Marzia FM Posteraro Brunella B Mannu Franca F Carta Franco F Pantaleo Antonella A De Angelis Giulia G Menchinelli Giulia G Spanu Teresa T Fiori Pier Luigi PL Turrini Francesco F Sanguinetti Maurizio M

Frontiers in cellular and infection microbiology 20191113

The diagnosis of bloodstream infections (BSIs) still relies on blood culture (BC), but low turnaround times may hinder the early initiation of an appropriate antimicrobial therapy, thus increasing the risk of infection-related death. We describe a direct and rapid multiplex PCR-based assay capable of detecting and identifying 16 bacterial and four <i>Candida</i> species, as well as three antibiotic-resistance determinants, in uncultured samples. Using whole-blood samples spiked with microorganis ...[more]

PMID: 31799215

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Project description:This study developed a new single-tube multiplex real-time PCR method for detecting toxigenic C. difficile directly from fecal samples using tcdA, tcdB, cdtB, and internal gene tpi as targets, which could be performed on kinds of polymerase chain reaction device including point-of-care testing (POCT), with improved detection efficiency. The specificity, sensitivity, and repeatability of each gene was evaluated using 69 C. difficile isolates and 74 fecal samples. Results were compared with established PCR, qPCR, and ELISA methods. Interspecies specificity was 100% based on six common intestinal pathogens (Escherichia coli, Enterococcus Faecium, Enterococcus faecalis, Clostridium perfringens, Bacteroides fragilis, Clostridium botulinum). The lower detection limit (LDL) for tcdA, tcdB, and cdtB with pure C. difficile DNA was 101,100, and 100 copies/μL, respectively, the coefficients of variation among different experimental batches and within each experimental batch were both less than 3%, which shows that this method has strong repeatability. And the LDL of fecal DNA was 5 × 100, 5 × 103, and 5 × 102 colony-forming units (CFU)/g, respectively. In addition, the efficiency for detection of tcdA was compared with established PCR and real-time PCR methods, demonstrating high consistency (98.4%) and similar sensitivity. ELISA was used to confirm inconsistent results, which were identical with our method. The sensitivity and specificity for detecting toxigenic C. difficile in fecal samples were 96.49% and 94.12% compared with the toxigenic culture (TC). This method effectively identified the toxigenic and non-toxigenic strains with high specificity, sensitivity, and repeatability, and could reduce the false positive rate of tcdA, and accurately identify the typical Asian strain RT017, making it potentially contribute to the surveillance of CDI in China.Supplementary informationThe online version contains supplementary material available at 10.1007/s13205-022-03434-6.

Project description:IntroductionWidely used in livestock breeding, residues of antibiotic drugs in milk have become a threat to food safety and human health. Current rapid detection technologies using colorimetric immunochromatographic strip tests (IST) lack the necessary sensitivity for on-site trace monitoring. Fluorescence-based detection in the near-infrared IIa' (NIR-IIa') region (1000 ∼ 1300 nm) has enormous potential due to greatly minimized auto-fluorescence and light scattering.ObjectivesThe aim of this work is to develop an ultrasensitive IST platform using NIR-IIa' fluorescent nanoparticles as labels for multiplex antibiotic residues detection in milk.MethodsNIR-IIa' fluorescent nanoparticles were assembled by encapsulating synthesized NIR-IIa' fluorophores into carboxyl - modified polystyrene nanoparticles. The NIR-IIa' nanoparticles were subsequently used as labels in an IST platform to detect sulfonamides, quinolones, and lincomycin simultaneously in milk. A portable fluorescent reader was fabricated to provide on-site detection. To further validate the developed IST platform, the detection was compared with LC-MS/MS in 22 real milk samples.ResultsFluorescent nanoparticles were synthesized with low energy emission (1030 nm) and large Stokes shift (>250 nm) showing a much higher signal-to-noise ratio compared with fluorophores emitting in the NIR-I region. The developed IST platform yielded a highly sensitive, simultaneous quantification of sulfonamides, quinolones, and lincomycin in milk with detection limits of 46.7, 27.6 and 51.4 pg/mL, respectively, achieving a wide detection range (up to 50 ng/mL). The IST platform showed good accuracy, reproducibility, and specificity with the portable fluorescent reader which could rapidly quantify in 10 s. These results were better than reported immunochromatographic assays using fluorescent labels, and remarkably, showed a higher recognition ability than LC-MS/MS for real samples.ConclusionThe utility of NIR-IIa' fluorescence-based IST platform for the fast, sensitive, and accurate detection of antibiotics in milk was demonstrated, successfully verifying the potential of this platform in detecting trace materials in complex matrices.

Dataset Information

Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and Candida in Human Blood Samples.

Publications

Development of a Multiplex PCR Platform for the Rapid Detection of Bacteria, Antibiotic Resistance, and <i>Candida</i> in Human Blood Samples.

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