Stakeholder Insights into Czech Performance-Based Managed Entry Agreements: Potential for Transformative Change in Pharmaceutical Access?

Managed Entry Agreements (MEAs) play a pivotal role in addressing the challenges arising from escalating prices of innovative medical technologies, especially in areas like oncology, immunology, and rare diseases. Among MEAs, Performance-Based MEAs (PB MEAs) and Outcome-Based MEAs (OB MEAs) stand out as innovative strategies. This study examines the adoption of PB MEAs in the Czech Republic post a 2022 legislative change. Interviews with key stakeholders, including the Ministry of Health, pharmaceutical companies, insurers, and patient groups, were conducted to explore perceptions and challenges. Stakeholders expressed concerns about legislation completeness, data quality, transparency, and methodology. Interestingly, pharmaceutical companies were less concerned about transparency and methodology, likely due to their multinational experience. Despite legislative progress, challenges persist, especially in data infrastructure, risk-sharing perceptions, and stakeholder readiness. Addressing these issues requires collaboration between pharmaceutical companies and payers. Patient involvement, though mandated, remains limited, potentially due to a lack of awareness. This study emphasizes the need for a comprehensive transformation beyond legislation for a successful PB MEA implementation. Trust, technical infrastructure, and data availability are crucial, necessitating a holistic approach. It contributes to the global discourse on PB MEAs, stressing the adjustment of financial frameworks, embracing value-based healthcare principles, and ensuring high-quality health data metrics. A more holistic, value-based MEA approach could reshape pharmaceutical reimbursement in the future.

Capillary electrophoretic methods for classification of methicillin-resistant Staphylococcus aureus (MRSA) clones.

This study describes differentiation of methicillin-resistant Staphylococcus aureus (MRSA) isolates belonging to different genotype groups by the combination of electrophoretic techniques, transient isotachophoresis and micellar electrokinetic chromatography. MRSA isolates were separated in fused silica capillary with roughened inner surface prepared by etching with supercritical water. Separation temperature together with the rinsing procedure of the capillary turned out to be the key factors of successful analysis. The individual genotype groups were baseline-resolved in 40 min. Partial separation of the individual isolates within the groups was also observed. Relative standard deviations of the migration times of the isolate zones ranged from 0.32 to 0.79%. In addition, capability of the developed CE method to concentrate and separate MRSA isolates in clinical samples was proved by the analysis of blood sample.

Classification of clinical Cutibacterium acnes isolates at phylotype level by capillary electrophoretic methods in roughened fused silica capillary.

Despite being commensal bacterium involved in the maintenance of healthy skin, Cutibacterium acnes is also associated with inflammatory diseases. Since inflammatory and immunogenic properties vary between C. acnes phylotypes, reliable classification of clinical C. acnes isolates is important for determining their pathogenicity. Combination of optimized separation methods, polymer-enhanced transient isotachophoresis and sweeping of the charged bacterial cells in micellar electrokinetic chromatography in the roughened fused silica capillary, was used for the separation of twenty clinical C. acnes isolates. Their correct classification into the individual phylotypes was achieved in 20 min at laboratory temperature. In addition, decrease in the separation temperature to 15 °C led to the separation of the individual isolates of some phylotypes. Relative standard deviations of migration times of both intra- and inter-day analyses did not exceed 1.7%. Linearity of the proposed method in the concentration range from 5 × 105 to 1 × 107 cells mL-1 was characterized by the coefficient of determination R2 = 0.9985. Limit of detection of 5 × 105 cells mL-1 (50 cells in 100 nL of the injected sample) was determined for all the examined bacteria.

Fabrication of monolithic capillary columns with inner diameter 50-530 µm employing a mixture of pentaerythritol tetraacrylate and polyhedral oligomeric silsesquioxane-methacrylate as crosslinkers.

Highly crosslinked monolithic capillary columns with inner diameters in the range of 50-530 µm were prepared by radical polymerization of pentaerythritol tetraacrylate, polyhedral oligomeric silsesquioxane-methacrylate, and n-octadecyl methacrylate in the presence of methanol, dodecyl alcohol, and polyethylene glycol lauryl ether. Columns were evaluated by inverse size-exclusion chromatography employing a set of polystyrene standards of narrow molecular-size distribution and by scanning electron microscopy. Chromatographic performance under reversed-phase conditions was also evaluated. The combination of two effective crosslinkers as pentaerythritol tetraacrylate and polyhedral oligomeric silsesquioxane-methacrylate in the polymerization mixture allows for the preparation of robust and efficient monolithic capillary columns within a fairly wide range of internal diameters.

Characterization and applications of a trioctyl(3/4-vinylbenzyl)phosphonium stationary phase for use in capillary liquid chromatography.

The morphology, composition, and selectivity of a silica-based monolithic stationary phase, grafted by a layer of trioctyl(3/4-vinylbenzyl)phosphonium chloride ([P888VBn]Cl), is presented. The results of elemental analysis confirmed that the prepared stationary phase contains 38.8 at.% of silicon, 60.2 at.% of carbon, and 1.0 at.% of phosphorus. Capillary columns (150 × 0.1 mm) for liquid chromatography were evaluated using alkylbenzenes, monosubstituted benzenes, polyaromatic compounds, substituted benzene regioisomers, and aromatic carboxylic and amino acids. The prepared ionic liquid (IL)-based stationary phase exhibits hydrophobic, hydrophilic, and electrostatic interactions, as confirmed by experiments on the evaluation of the effect of the mobile phase composition (content of acetonitrile and ammonium formate) on the isocratic chromatographic separation. Thus, the IL-based capillary column demonstrates a unique separation selectivity compared to Phenyl-, C8-, and C18-stationary phases, and high efficiency for an expanding number of structurally diverse compounds.

Sensitive identification of milk protein allergens using on-line combination of transient isotachophoresis/micellar electrokinetic chromatography and capillary isoelectric focusing in fused silica capillary with roughened part.

A method for on-line concentration of milk proteins from large sample volumes using combination of transient isotachophoresis (tITP) and micellar electrokinetic chromatography (MEKC) in fused silica capillary with an inner roughened part has been developed. The method utilizes reversible dynamic adsorption of proteins onto a thin layer of PEG 4000 on the roughened surface of the capillary. In addition, the tITP/MEKC method was combined with capillary isoelectric focusing (CIEF) for on-line concentration, separation, identification and sensitive determination of proteins in skimmed milk. The method allows analysis of up to 50 µL of sample. This study has focused on the four important whey proteins, bovine serum albumin (BSA), α-lactalbumin (α-LA), and two genetic variants of ß-lactoglobulin (ß-LG A and ß-LG B). The proteins were identified on the basis of their migration times and characteristic pI values. The pI values of BSA, α-LA, ß-LG A, and ß-LG B were determined as 4.7, 4.4, 5.1, and 5.2, respectively. Limits of detection for BSA, α-LA and both ß-LG variants were found as 1.2, 1.0 and 1.0 pg mL-1, respectively. The linearity of calibration curves was characterized by the R2 = 0.9982. The method provided highly reproducible results as the relative standard deviations of the migration times and peak areas of the examined proteins did not exceed 1.6%.

Bacteriophage replication on permissive host cells in fused silica capillary with nanostructured part as potential of electrophoretic methods for developing phage applications.

Phage therapy could offer a safe and effective alternative to antibiotic treatment of infections caused by Gram-positive bacterium Staphylococcus aureus that have emerged as a significant threat in hospital and community environment and is attracting growing interest among clinicians. The legislation process of approving the phage therapeutics by pharmaceutical authorities requires rapid analytical techniques for assessment of phage activity. Here, we present a three-step method for on-line monitoring the phage effect on bacterial cells dynamically adhered from microliter volumes of high conductivity matrix onto the inner surface of fused silica capillary with a part etched with supercritical water. Phage K1/420 particles of the Kayvirus genus generated by propagation on the host S. aureus cells together with the uninfected cells were concentrated, separated and detected using capillary electrophoretic methods. The phage interactions with selected S. aureus strains exhibiting differences in phage susceptibility were compared. The method allowed determination of the phage burst size and time of phage latent period in analyzed strains. Apart from enumeration of bacteriophages by the plaque assays, the proposed method is suitable for phage activity testing.

Rapid Isolation, Propagation, and Online Analysis of a Small Number of Therapeutic Staphylococcal Bacteriophages from a Complex Matrix.

A method for the fast isolation, propagation, and characterization of very low count bacteriophages active against pathogenic bacterial strains is described in this study. Bacteriophages with a count of 102 phage particles were dynamically adhered from the maximum 10 mL blood plasma sample onto the nanostructured part of the fused silica capillary. One-step propagation of phage particles of genus Kayvirus inside the etched capillary on 104Staphylococcus aureus host cells increased their number to 6 × 104 phage particles. Phage particles were concentrated online and separated by capillary electrophoretic methods. No phage replication occurred when the phage-resistant S. aureus or Escherichia coli cells were used. Two-step phage propagation in the capillary allowed an increase in the total virion count to up to 6 × 105 phage particles and subsequent off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of the phage zone collected after capillary electrophoresis. Relative standard deviations of the phage peak area were at most 2.3%. We expect that the method of isolating bacteriophages from blood plasma and their simultaneous identification will facilitate clinical studies of phage preparations and contribute to pharmacokinetics studies during phage therapy. This approach is also suitable for capturing and enriching new phages from the environment when a susceptible indicator strain is available.

Identification of Aspergillus Conidia in Bronchoalveolar Lavage Using Offline Combination of Capillary Electrophoresis in Supercritical Water-Treated Fused Silica Capillary and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Diagnosis of fungal infection in lung parenchyma is relatively difficult. Bronchoscopy with bronchoalveolar lavage is very useful in its diagnosing. Therefore, a method for rapid online concentration and analysis of Aspergillus conidia in bronchoalveolar lavage fluid using the combination of transient isotachophoresis (tITP) and micellar electrokinetic chromatography (MEKC) with subsequent off-line identification of the separated conidia by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is described in this study. In the proposed procedure, conidia were first dynamically adhered onto the roughened part of the inner surface of a fused silica capillary prepared by etching with supercritical water. Then the adhered conidia were desorbed, concentrated, and separated using a combination of tITP and MEKC. Finally, the fractions containing the separated conidia were collected from the capillary and analyzed by MALDI-TOF MS. The adhesion efficiency under the optimized experimental conditions was about 80%. This rapid diagnosis will contribute to timely initiation of therapy and increase the patient's chances of survival.

Nano-etched fused-silica capillary used for on-line preconcentration and electrophoretic separation of bacteriophages from large blood sample volumes with off-line MALDI-TOF mass spectrometry identification.

The properties of staphylococcal phages from the Siphoviridae, Podoviridae, and Myoviridae families were monitored using capillary electrophoretic methods on fused-silica capillaries with different morphology of surface roughness. Isoelectric points of the examined phages were determined by capillary isoelectric focusing in the original, smooth fused-silica capillary, and they ranged from 3.30 to 3.85. For capillary electrophoresis of phages, fused-silica capillaries with the "pock" and "cone" roughened surface types were prepared by etching a part of the capillary with supercritical water. The best resolution of the individual phages (to range from 3.2 to 4.6) was achieved with the "cone" surface-type fused-silica capillary. Direct application of phage K1/420 at the infection site, represented by human plasma or full blood spiked with Staphylococcus aureus, was on-line monitored by micellar electrokinetic chromatography. The phage particles were dynamically adhered onto the roughened surface of the capillary from 10 µL of the prepared sample at the optimized flow rate of 6.5 µL min-1. The limit of detection was determined to be 104 phage particles. The linearity of the calibration lines was characterized by the regression coefficient, R2 = 0.998. The relative standard deviation (RSD) of the peak area, calculated from ten independent measurements, was (±) 2%. After analysis, viability of the detected phages was verified by the modified "double-layer drop assay" method, and collected phage fractions were simultaneously off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Graphical abstract.

Online Concentration of Bacteria from Tens of Microliter Sample Volumes in Roughened Fused Silica Capillary with Subsequent Analysis by Capillary Electrophoresis and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

This study presents a timely, reliable, and sensitive method for identification of pathogenic bacteria in clinical samples based on a combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In this respect, a part of a single-piece fused silica capillary was etched with supercritical water with the aim of using it for static or dynamic cell-surface adhesion from tens of microliter sample volumes. The conditions for this procedure were optimized. Adhered cells of Staphylococcus aureus (methicillin-susceptible or methicillin-resistant) and of Pseudomonas aeruginosa were desorbed and preconcentrated from the rough part of the capillary surface using transient isotachophoretic stacking from a high conductivity model matrix. The charged cells were swep and separated again in micellar electrokinetic chromatography using a nonionogenic surfactant. Static adhesion of the cells onto the roughened part of the capillary is certainly volumetric limited. Dynamic adhesion allows the concentration of bacteria from 100 µL volumes of physiological saline solution, bovine serum, or human blood with the limits of detection at 1.8 × 102, 1.7 × 103, and 1.0 × 103 cells mL-1, respectively. The limits of detection were the same for all three examined bacterial strains. The recovery of the method was about 83% and it was independent of the sample matrix. A combination of capillary electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry required at least 4 × 103 cells mL-1 to obtain reliable results. The calibration plots were linear (R2 = 0.99) and the relative standard deviations of the peak area were at most 2.2%. The adhered bacteria, either individual or in a mixture, were online analyzed by micellar electrokinetic chromatography and then collected from the capillary and off-line analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without interfering matrix components.

Supercritical water-treated fused silica capillaries in analytical separations: Status review.

Near- and supercritical water (SCW) has recently been shown to provide an unusual but effective tool to roughen the inner surface or manipulate the internal diameter of fused silica capillaries for analytical separation methods In this review, the to-date existing variants of instrumental arrangement for etching the fused silica capillaries with SCW are described, the currently accessible morphologies of SCW-etched capillaries are outlined, and both existing and prospective applications of the SCW-etched capillaries in analytical separations are briefly discussed. Relative merits of SCW and other agents to treat the inner surfaces of fused silica capillaries are also mentioned.

Pre-concentration and separation of bacteria by volume coupling electrophoresis on supercritical water-etched fused silica capillary with two segments of different internal diameters and inner surface roughnesses.

The transient isotachophoretic stacking and sweeping was used for the on-line large-volume sample pre-concentration of bacteria, Escherichia coli and Staphylococcus aureus cells (methicillin-susceptible or methicillin-resistant), in the initial stage of micellar electrokinetic chromatography using a non-ionogenic surfactant or of capillary electrophoresis, respectively. These procedures were employed in single-piece fused silica capillary etched with supercritical water with two different internal diameter segments featuring different inner surface roughness. Large volumes (maximum 2.8 µL) of the high conductivity sample matrices, physiological saline solution, urine or blood (with purification step), spiked with examined cells were injected into the wider end of a capillary with an inlet inner diameter 195 µm. This novel on-line combination of preconcentration strategies for cells produced an up to 680-fold increase in sensitivity for E. coli or S. aureus cells. The average calculated resolutions, R, for five selected methicillin-susceptible or methicillin-resistant strains were found to be 6.3 for the agar-cultivated and 14.9 for the blood-incubated cells. A low number of bacteria similar to those in clinical samples were also tested. The modified surface roughness step helped to significantly narrow the cell zones and to increase resolution. The migration velocities of E. coli agar-cultivated and blood-incubated cells were approximately the same as those of S. aureus, probably due to the minimal differences in their surface properties. This procedure, on-line pre-concentration and separation of bacteria, is rapid and provides good reproducibility and repeatability.

Fused silica capillaries with two segments of different internal diameters and inner surface roughnesses prepared by etching with supercritical water and used for volume coupling electrophoresis.

In this work, single-piece fused silica capillaries with two different internal diameter segments featuring different inner surface roughness were prepared by new etching technology with supercritical water and used for volume coupling electrophoresis. The concept of separation and online pre-concentration of analytes in high conductivity matrix is based on the online large-volume sample pre-concentration by the combination of transient isotachophoretic stacking and sweeping of charged proteins in micellar electrokinetic chromatography using non-ionogenic surfactant. The modified surface roughness step helped to the significant narrowing of the zones of examined analytes. The sweeping and separating steps were accomplished simultaneously by the use of phosphate buffer (pH 7) containing ethanol, non-ionogenic surfactant Brij 35, and polyethylene glycol (PEG 10000) after sample injection. Sample solution of a large volume (maximum 3.7 µL) dissolved in physiological saline solution was injected into the wider end of capillary with inlet inner diameter from 150, 185 or 218 µm. The calibration plots were linear (R2 ∼ 0.9993) over a 0.060-1 µg/mL range for the proteins used, albumin and cytochrome c. The peak area RSDs from at least 20 independent measuremens were below 3.2%. This online pre-concentration technique produced a more than 196-fold increase in sensitivity, and it can be applied for detection of, e.g. the presence of albumin in urine (0.060 µg/mL).

Capillary electrophoresis in a fused-silica capillary with surface roughness gradient.

The electro-osmotic flow, a significant factor in capillary electrophoretic separations, is very sensitive to small changes in structure and surface roughness of the inner surface of fused silica capillary. Besides a number of negative effects, the electro-osmotic flow can also have a positive effect on the separation. An example could be fused silica capillaries with homogenous surface roughness along their entire separation length as produced by etching with supercritical water. Different strains of methicillin-resistant and methicillin-susceptible Staphylococcus aureus were separated on that type of capillaries. In the present study, fused-silica capillaries with a gradient of surface roughness were prepared and their basic behavior was studied in capillary zone electrophoresis with UV-visible detection. First the influence of the electro-osmotic flow on the peak shape of a marker of electro-osmotic flow, thiourea, has been discussed. An antifungal agent, hydrophobic amphotericin B, and a protein marker, albumin, have been used as model analytes. A significant narrowing of the detected zones of the examined analytes was achieved in supercritical-water-treated capillaries as compared to the electrophoretic separation in smooth capillaries. Minimum detectable amounts of 5 ng/mL amphotericin B and 5 µg/mL albumin were reached with this method.

Determination of methicillin-resistant and methicillin-susceptible Staphylococcus aureus bacteria in blood by capillary zone electrophoresis.

Serious bloodstream infections are a significant complication in critically ill patients. The treatment of these infections has become more difficult because of the increasing prevalence of multiresistant strains, especially methicillin-resistant Staphylococcus aureus (MRSA). Rapid differentiation of low number of MRSA from methicillin-susceptible S. aureus (MSSA) cells (10(1)-10(2) cells mL(-1)) in blood is necessary for fast effective antibiotic therapy. Currently, three groups of techniques, phenotyping, genotyping, and mass spectrometry, are used for MRSA and MSSA strains differentiation. Most of these techniques are time-consuming. PCR and other molecular techniques allow the detection and differentiation between MSSA and MRSA directly from blood cultures. These methods alone are rapid and they have good reproducibility and repeatability. Potential disadvantages of the genotyping methods include their discrimination ability, technical complexity, financial costs, and difficult interpretation of the results. Recently, capillary electrophoresis (CZE) was successfully used to differentiate between the agar-cultivated MRSA and MSSA strains in fused silica capillaries etched with supercritical water and modified with (3-glycidyloxypropyl)trimethoxysilane. The possible use of CZE as a fast and low-cost method for distinguishing between the blood-incubated MRSA or MSSA cells has been tested in this manuscript. Our goal was to test low amounts of bacteria (∼10(2) cell mL(-1)) similar to those in clinical samples. The migration times of the purified blood-incubated cells and the agar-cultivated cells were different from each other. However, their isoelectric point was the same for all strains.

Etching of glass microchips with supercritical water.

A novel method of etching channels in glass microchips with the most tunable solvent, water, was tested as an alternative to common hydrogen fluoride-containing etchants. The etching properties of water strongly depend on temperature and pressure, especially in the vicinity of the water critical point. The chips were etched at the subcritical, supercritical and critical temperature of water, and the resulting channel shape, width, depth and surface morphology were studied by scanning electron microscopy and 3D laser profilometry. Channels etched with the hot water were compared with the chips etched with standard hydrogen fluoride-containing solution. Depending on the water pressure and temperature, the silicate dissolved from the glass could be re-deposited on the channel surface. This interesting phenomenon is described together with the conditions necessary for its utilization. The results illustrate the versatility of pure water as a glass etching and surface morphing agent.

Separation of methicillin-resistant from methicillin-susceptible Staphylococcus aureus by electrophoretic methods in fused silica capillaries etched with supercritical water.

Identification and prevention of Staphylococcus aureus-caused infections may benefit from a fast and dependable method to distinguish between the methicillin-resistant (MRSA) and methicillin-susceptible (MSSA) S. aureus strains. The current methods involving polymerase chain reaction and/or other molecular tests are usually laborious and time-consuming. We describe here a fast and low-cost method employing capillary zone electrophoresis (CZE) to distinguish between MRSA and MSSA. The method makes use of a supercritical water-treated fused silica capillary, the inner surface of which has subsequently been modified with (3-glycidyloxypropyl)trimethoxysilane. With optimized proportions of suitable additives to the background electrolyte, a CZE separation of MRSA from MSSA may be completed within 12 min. The cells were baseline-resolved, and resolution was determined to be 3.61. The isoelectric points of MSSA and MRSA were found to be the same for both groups of these strains, pI = 3.4.

Capillary isoelectric focusing of probiotic bacteria from cow's milk in tapered fused silica capillary with off-line matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification.

In this study, combination of capillary isoelectric focusing (CIEF) in tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is presented as an efficient approach for unambiguous identification of probiotic bacteria in real sample. For this purpose, bacteria within genus Lactobacillus were selected as model bioanalytes and cow's milk was selected as a biological sample. CIEF analysis of both the cultivated bacteria and the bacteria in the milk was optimized and isoelectric points characterizing the examined bacteria were subsequently determined independently of the bacterial sample origin. The use of tapered FS capillary significantly enhanced the separation capacity and efficiency of the CIEF analyses performed. In addition, the cell number injected into the tapered FS capillary was quantified and an excellent linearity of the calibration curves was achieved which enabled quantitative analysis of the bacteria by CIEF with UV detection. The minimum detectable number of bacterial cells was 2×10(6) mL(-1). Finally, cow's milk spiked with the selected bacterium was analyzed by CIEF in tapered FS capillary, the focused and detected bacterial cells were collected from the capillary, deposited onto the cultivation medium, and identified using MALDI-TOF MS afterward. Our results have revealed that the proposed procedure can be advantageously used for unambiguous identification of probiotic bacteria in a real sample.

Combination of capillary isoelectric focusing in a tapered capillary with MALDI-TOF MS for rapid and reliable identification of Dickeya species from plant samples.

This study was undertaken to investigate feasibility of a combination of capillary isoelectric focusing (CIEF) in a tapered fused silica (FS) capillary with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and reliable identification of bacteria taken from plant-tissue-containing samples. Eight strains representing different species of the genus Dickeya were selected on the basis of close proximity of their isoelectric points: D. chrysanthemi, D. chrysanthemi bv. parthenii, D. chrysanthemi bv. chrysanthemi, D. dadantii, D. paradisiaca, D. solani, D. diffenbachiae, and D. dianthicola. Because the Dickeya species (spp.) cannot be easily discriminated from each other when CIEF is performed in a cylindrical FS capillary (commonly used in CIEF) even if a narrow pH gradient is used, a tapered FS capillary was employed instead, which enabled satisfactory discrimination of the examined bacteria due to enhanced separation efficiency of CIEF in the tapered FS capillary. CIEF in the tapered FS capillary was also successfully used for the detection and characterization of Dickeya spp. in a plant-tissue-containing sample. Then an off-line combination of CIEF with MALDI-TOF MS was employed for rapid and reliable identification of Dickeya spp. in the plant-tissue-containing sample. It was found that the presence of plant tissue did not affect the results, making the proposed procedure very promising with respect to the fast and reliable detection and identification of bacteria in plant-tissue-containing samples.