Potential of hydrogen (pH) is one of the most relevant parameters characterizing aqueous solutions. In biology, pH is intrinsically linked to cellular life since all metabolic pathways are implicated ...into ionic flows. In that way, determination of local pH offers a unique and major opportunity to increase our understanding of biological systems. Whereas the most common technique to obtain these data in analytical chemistry is to directly measure potential between two electrodes, in biological systems, this information has to be recovered in-situ without any physical interaction. Based on their non-invasive optical properties, fluorescent pH-sensitive probe are pertinent tools to develop. One of the most notorious pH-sensitive probes is fluorescein. In addition to excellent photophysical properties, this fluorophore presents a pH-sensitivity around neutral and physiologic domains. This review intends to shed new light on the recent use of fluorescein as pH-sensitive probes for biological applications, including targeted probes for specific imaging, flexible monitoring of bacterial growth, and biomedical applications.
Complement pathway inhibition may provide benefit for severe acute respiratory illnesses caused by viral infections such as COVID-19. We present results from a nonrandomized proof-of-concept study of ...complement C5 inhibitor eculizumab for treatment of severe COVID-19.
All patients (N = 80) with confirmed SARS-CoV-2 infection and severe COVID-19 admitted to our intensive care unit between March 10 and May 5, 2020 were included. Forty-five patients were treated with standard care and 35 with standard care plus eculizumab through expanded-access emergency treatment. The prespecified primary outcome was day-15 survival. Clinical laboratory values and biomarkers, complement levels, and treatment-emergent serious adverse events (TESAEs) were also assessed.
At day 15, estimated survival was 82.9% (95% CI: 70.4%‒95.3%) with eculizumab and 62.2% (48.1%‒76.4%) without eculizumab (log-rank test, P = 0.04). Patients treated with eculizumab experienced a significantly more rapid decrease in lactate, blood urea nitrogen, total and conjugated bilirubin levels and a significantly more rapid increase in platelet count, prothrombin time, and in the ratio of arterial oxygen tension over fraction of inspired oxygen versus patients treated without eculizumab. Eculizumab-associated changes in complement levels, laboratory values, and biomarkers were consistent with terminal complement inhibition, reduced hypoxia, and decreased inflammation. TESAEs of special interest occurring in >5% of patients treated with/without eculizumab were ventilator-associated pneumonia (51%/24%), bacteremia (11%/2%), gastroduodenal hemorrhage (14%/16%), and hemolysis (3%/18%).
Findings from this proof-of-concept study suggest eculizumab may improve survival and reduce hypoxia in patients with severe COVID-19. Randomized studies evaluating the efficacy and safety of this treatment approach are needed.
Programme d'Investissements d'Avenir: ANR-18-RHUS60004.
FosA, a glutathione S-transferase that inactivates fosfomycin, has been reported as the cause of enzymatic resistance to fosfomycin. We show that multiple lineages of FosA-producing extended spectrum ...β-lactamase Escherichia coli have circulated in France since 2012, potentially reducing the efficacy of fosfomycin in treating infections with antimicrobial drug-resistant gram-negative bacilli.
Abstract
Cross-contamination of biological samples during handling and preparation, is a major issue in laboratory setups, leading to false-positives or false-negatives. Sample carryover residue in ...pipette tips contributes greatly to this issue. Most pipette tips on the market are manufactured with hydrophobic polymers that are able to repel high surface tension liquids, yet they lack in performance when low surface tension liquids and viscous fluids are involved. Moreover, hydrophobicity of pipette tips can result in hydrophobic adsorption of biomolecules, causing inaccuracies and loss in precision during pipetting. Here we propose the use of lubricant-infused surface (LIS) technology to achieve omniphobic properties in pipette tips. Using a versatile and simple design, the inner lumen of commercially available pipette tips was coated with a fluorosilane (FS) layer using chemical vapor deposition (CVD). The presence of FS groups on the tips is confirmed by x-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) tests. After lubrication of the tips through a fluorinated lubricant, the omniphobicity and repellent behaviour of the tips drastically enhanced which are revealed via static and hysteresis contact angle measurements. The repellency of the lubricant-infused pipette tips against physical adsorption is investigated through pipetting a food coloring dye as well as human blood samples and are compared to the untreated tips. The results show significantly less amount carryover residue when the lubricant-infused tips are utilized compared to commercially available ones. We also demonstrate the lubricant-infused tips reduce bacteria contamination of the inner lumen by 3 to 6-log (over 99%, depending on the tip size) after pipetting up and down the bacteria solution.
The emergence and quick spread of SARS-CoV-2 has pointed at a low capacity response for testing large populations in many countries, in line of material, technical and staff limitations. The ...traditional RT-qPCR diagnostic test remains the reference method and is by far the most widely used test. These assays are limited to a few probe sets, require large sample PCR reaction volumes, along with an expensive and time-consuming RNA extraction step. Here we describe a quantitative nanofluidic assay that overcomes some of these shortcomings, based on the BiomarkTM instrument from Fluidigm. This system offers the possibility of performing 4608 qPCR end-points in a single run, equivalent to 192 clinical samples combined with 12 pairs of primers/probe sets in duplicate, thus allowing the monitoring of SARS-CoV-2 including the detection of specific SARS-CoV-2 variants, as well as the detection other pathogens and/or host cellular responses (virus receptors, response markers, microRNAs). The 10 nL-range volume of BiomarkTM reactions is compatible with sensitive and reproducible reactions that can be easily and cost-effectively adapted to various RT-qPCR configurations and sets of primers/probe. Finally, we also evaluated the use of inactivating lysis buffers composed of various detergents in the presence or absence of proteinase K to assess the compatibility of these buffers with a direct reverse transcription enzymatic step and we propose several protocols, bypassing the need for RNA purification. We advocate that the combined utilization of an optimized processing buffer and a high-throughput real-time PCR device would contribute to improve the turn-around-time to deliver the test results to patients and increase the SARS-CoV-2 testing capacities.
We performed a multicenter prevalence study of nontuberculous mycobacteria (NTM) involving 1,582 patients (mean age, 18.9 years; male/female ratio, 1.06) with cystic fibrosis in France. The overall ...NTM prevalence (percentage of patients with at least one positive culture) was 6.6% (104/1,582 patients), with prevalences ranging from 3.7% (in the east of France) to 9.6% (in the greater Paris area). Mycobacterium abscessus complex (MABSC; 50 patients) and Mycobacterium avium complex (MAC; 23 patients) species were the most common NTM, and the only ones associated with fulfillment of the American Thoracic Society bacteriological criteria for NTM lung disease. The "new" species, Mycobacterium bolletii and Mycobacterium massiliense, accounted for 40% of MABSC isolates. MABSC species were isolated at all ages, with a prevalence peak between 11 and 15 years of age (5.8%), while MAC species reached their highest prevalence value among patients over 25 years of age (2.2%).
Mycobacterium abscessus is a pathogenic, rapidly growing mycobacterium responsible for pulmonary and cutaneous infections in immunocompetent patients and in patients with Mendelian disorders, such as ...cystic fibrosis (CF). Mycobacterium abscessus is known to transition from a smooth (S) morphotype with cell surface-associated glycopeptidolipids (GPL) to a rough (R) morphotype lacking GPL. Herein, we show that M. abscessus S and R variants are able to grow inside macrophages and are present in morphologically distinct phagosomes. The S forms are found mostly as single bacteria within phagosomes characterized by a tightly apposed phagosomal membrane and the presence of an electron translucent zone (ETZ) surrounding the bacilli. By contrast, infection with the R form leads to phagosomes often containing more than two bacilli, surrounded by a loose phagosomal membrane and lacking the ETZ. In contrast to the R variant, the S variant is capable of restricting intraphagosomal acidification and induces less apoptosis and autophagy. Importantly, the membrane of phagosomes enclosing the S forms showed signs of alteration, such as breaks or partial degradation. Although not frequently encountered, these events suggest that the S form is capable of provoking phagosome–cytosol communication. In conclusion, M. abscessus S exhibits traits inside macrophages that are reminiscent of slow-growing mycobacterial species.
The aims of this study were to identify the predictive factors for microbiological diagnosis through disco-vertebral biopsy (DVB) in patients with pyogenic vertebral osteomyelitis (PVO) and negative ...blood cultures, and compare the performance of DVB under fluoroscopic versus scanographic guidance. We performed a cohort study comparing positive and negative DVB among patients with PVO. All cases of PVO undergoing a DVB for microbiological diagnosis in our center were retrospectively reviewed. Infections due to Mycobacterium tuberculosis, infections on foreign device, and non-septic diseases were excluded. Anamnestic, clinical, biological, microbiological, as well as radiological data were collected from medical charts thanks to a standardized data set. A total of 111 patients were screened; 88 patients were included. Microbiological cultures were positive in 53/88 (60.2%) patients. A thickening of the paravertebral tissue greater than or equai to10 mm on magnetic resonance imaging (MRI) in axial MR scans was a predictive factor of DVB microbiological positivity (52.4% vs. 13.3%; p = 0.006; OR = 5.4). Overall, 51 DVB were performed under fluoroscopic guidance and 37 under scanographic guidance. Considering lumbar DVB, 25/36 (69.4%) of cases yielded positive results under fluoroscopic guidance versus 5/15 (33.3%) under scanographic guidance (p = 0.02; OR = 4.4). No adverse event linked to DVB was notified. Every patient with PVO and negative blood cultures should undergo a DVB. A thickening of the paravertebral tissue greater than or equai to10 mm on MRI is associated with a higher rate of positive DVB culture. A lumbar DVB under fluoroscopic guidance is more sensitive than under scanographic guidance to identify the micro-organism involved.
Establishing the rapid and accurate diagnosis of sepsis is a key component to the improvement of clinical outcomes. The ability of analytical platforms to rapidly detect pathogen-associated molecular ...patterns (PAMP) in blood could provide a powerful host-independent biomarker of sepsis. A novel concept was investigated based on the idea that a pre-bound and fluorescent ligand could be released from lectins in contact with high-affinity ligands (such as PAMPs). To create fluorescent ligands with precise avidity, the kinetically followed TEMPO oxidation of yeast mannan and carbodiimide coupling were used. The chemical modifications led to decreases in avidity between mannan and human collectins, such as the mannan-binding lectin (MBL) and human surfactant protein D (SP-D), but not in porcine SP-D. Despite this effect, these fluorescent derivatives were captured by human lectins using highly concentrated solutions. The resulting fluorescent beads were exposed to different solutions, and the results showed that displacements occur in contact with higher affinity ligands, proving that two-stage competition processes can occur in collectin carbohydrate recognition mechanisms. Moreover, the fluorescence loss depends on the discrepancy between the respective avidities of the recognized ligand and the fluorescent mannan. Chemically modulated fluorescent ligands associated with a diversity of collectins may lead to the creation of diagnostic tools suitable for multiplex array assays and the identification of high-avidity ligands.
Cotrimoxazole (Sulfamethoxazole-Trimethoprim, SXT) has interesting characteristics for the treatment of bone and joint infection (BJI): a broad spectrum of activity with adequate bone diffusion and ...oral and intravenous formulations. However, its efficacy and safety in BJIs are poorly documented and its use remains limited.
We conducted a retrospective study in 2 reference centers for BJIs from 2013 to 2018 among patients treated with SXT for a BJI. Data were collected from patient's medical charts. Outcomes and adverse events were evaluated at day (D)7, D45 and D90.
We analyzed 51 patients with a mean age of 60 ± 20 (SD) years of which 76% presented with an orthopedic device infection (ODI). Gram-negative bacilli (GNB) were involved in 47% of BJIs (n = 24). Moreover, they were often polymicrobial infections (41%). Doses of SXT ranged from 800/160mg bid (61%; n = 31) to 800/160mg tid (39%; n = 20). Median SXT treatment duration was 45 days (IQR 40-45). SXT was part of a dual therapy in 84% of patients (n = 43), associated mainly with fluoroquinolones (n = 17) or rifampicin (n = 14). Outcome was favorable at D7 in 98% (n = 50), at D45 in 88.2% (n = 45) and at D90 in 78.4% (n = 40). The second agent combined with SXT was not an independent factor of favorable outcome (p = 0.97). Adverse events were reported in 8% (n = 4) of patients, with a median of 21 days (IQR 20-30) from SXT initiation and led to discontinuation (n = 3).
SXT appears to be effective for treatment of BJIs as a salvage therapy, even in GNB or polymicrobial infection, including ODI. Further data are needed to confirm SXT efficacy as an alternative oral regimen in BJIs.
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