Rapid and reliable identification of pathogens is of great importance in healthcare settings and in the control of agonistic and antagonistic disease outbreaks. Broad-range methods for molecular detection of parasites, bacteria and viruses in samples with unknown contents are thus highly desirable. Hyperplex PCR (hpPCR) is a recently invented analysis technique that allows for parallel detection of up to 100 DNA and RNA targets. We present the development and evaluation of a proof-of-concept 16-plex hpPCR method targeting RNA viruses, bacteria and a parasite to enable detection of various gastrointestinal pathogens relevant in food or waterborne disease outbreak scenarios. The panel also includes highly pathogenic microbes such as Bacillus anthracis and Yersinia pestis. The hpPCR method showed high selectivity with detection of all expected targets. None of the tested bacteria (11), parasites (1) or viruses (4) gave false positive signals or cross-reactions for any other target in the panel. The limit of detection was comparable to single-plex qPCR/RT-qPCR assays targeting the same pathogens. HpPCR tolerated high amounts of humic acid, with somewhat better results for the bacteria compared to the viruses. The 16-plex hpPCR method may be supplemented with additional assays to further widen the group of targeted pathogens. A hpPCR method simultaneously targeting a multitude of microbes including bacteria, parasites and viruses may thus provide timely and selective detection and identification of pathogens in an outbreak situation.
Objective Aimed to develop and validate an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 5-Fluorouracil (5-FU), 5,6-dihydro-5-Fluorouracil (5-FUH2), Uracil (U), and Dihydrouracil (UH2) concentrations in human plasma, and to apply 5-FU therapeutic drug monitoring(TDM). Methods UPLC-MS/MS method was adopted using 5-bromouracil (5-Bru) as internal standard (IS).The determination was performed on Waters Acquity UPLC BEH Amide column (100 mm × 2.1 mm,1.7 μm) with mobile phase consisted of acetonitrile and water. An electrospray ion source was used to carry out negative ion scanning with multiple reaction monitoring (MRM).The method was applied to 10 patients receiving FOLFOX-HAIC. 5-FU exposure was estimated as AUC = Css × TCI, and UH2/U ratio was explored as a DPD-related metabolic marker. Results Accuracy, precision, recovery, matrix effect, and stability were examined to meet ICH M10 guidelines for validation of biological methods.All four analytes were quantified over 20-1000 ng/mL with a lower limit of quantification of 20 ng/mL. Mean extraction recovery was approximately 52%-58%, with matrix effects close to 100%.Among 10 patients with advanced hepatocellular carcinoma, the Css of 5-FU ranged from 202.39 to 583.70 ng/mL, the AUC of 5-FU ranged from 9.31 to 26.85 mg h/L, and the plasma concentration of 5-FUH2 ranged from 352.96 to 892.59 ng/mL. The UH2:U concentration ratio ranged from 0.49 to 7.83. Conclusion We developed a rapid 5-min UPLC-MS/MS method for simultaneously determining 5-FU, 5-FUH2, U and UH2 in human plasma and applied it to patients treated with FOLFOX-HAIC. This method can comprehensively evaluate the 5-FU exposure and pyrimidine metabolism of patients with hepatocellular carcinoma.
Bacterial infections, especially those triggered by bacterial biofilms, remain a thorny challenge in antibacterial treatment. Metal-organic frameworks (MOFs), as carriers of metal ions that can sustainably release antibacterial ingredients, provide a promising alternative strategy for combating bacterial infections. Although the introduction of metal ions into MOFs is a relatively uncomplicated process, it remains an arduous task to eradicate all pathogenic bacteria with a limited dosage. Herein, an economical and efficient hollow Co-doped ZIF-L-PDA (HZIF-L-PDA (Co)) nanomaterial was synthesized via a simple dopamine-mediated self-assembly reaction. The uniform incorporation of Co and Zn in the ZIF-L skeleton and the polymerization of dopamine were completed through a one-pot method, avoiding complexity and harsh synthesis conditions. Due to the abundant metal nodes of ZIF-L and the photothermal and adhesion ability of polydopamine (PDA), HZIF-L-PDA (Co) can adhere to the bacterial surface and rely on its chemical antibacterial action and photothermal effect to exhibit a synergistic antibacterial effect, causing cell membrane rupture and invagination in Escherichia coli O157:H7 (E. coli O157:H7) and Staphylococcus aureus (S. aureus), respectively. Antibacterial experiments displayed that the sterilization rates of HZIF-L-PDA (Co) driven by near-infrared (NIR) light at low concentration (75 μg/mL) against E. coli O157:H7 and S. aureus were 96.9% and 97.3%, respectively. The HZIF-L-PDA (Co) realized favorable eradication against E. coli O157:H7 mature biofilms, while the therapeutic effect against S. aureus biofilms was limited at the tested low concentration. Overall, we proposed an easily fabricated MOF-based nanocomposite that fought pathogenic bacteria through chemical and photothermal effects, promising as an alternative to antibiotics.
