Comparison of the diagnostic accuracy of the Pluslife Mini Dock RHAM technology with Abbott ID Now and Cepheid GenXpert: A retrospective evaluation study.

Rapid and sensitive detection of pathogens is critical in interrupting the transmission chain of infectious diseases. Currently, real-time (RT-)PCR represents the gold standard for the detection of SARS-CoV-2. RNase HII-assisted amplification (RHAM) is a promising technology, enabling reliable point-of-care (PoC) testing; however, its diagnostic accuracy has not yet been investigated. The present study compared the Pluslife Mini Dock (RHAM technology), with Abbott ID Now and Cepheid GeneXpert IV. The positive percent agreement (PPA) and negative percent agreement (NPA) were determined in 100 SARS-CoV-2 positive and 210 SARS-CoV-2 negative samples. Further, the reliability of the Pluslife Mini Dock was investigated in different SARS-CoV-2 variants (Delta and Omicron subvariants). The PPA was 99.00% for Pluslife, 100.00% for Abbott ID Now, and 99.00% for Cepheid GeneXpert, with an NPA of 100.00%, 98.90%, and 93.72%, respectively. Abbott ID Now demonstrated the highest rate of invalid results. All SARS-CoV-2 analysed variants were detected by the Pluslife device. Altogether, the Pluslife Mini Dock demonstrated a PPA of 99.16% (235/237) for CT < 36 and an NPA of 100.00% (313/313), respectively. In conclusion, the Pluslife Mini Dock demonstrated better analytical performance than Abbott ID Now and Cepheid GeneXpert IV, representing a highly accurate and rapid PoC alternative to RT-PCR.

System Accuracy and Interference Evaluation of a New Glucose Dehydrogenase-Based Blood Glucose Meter for Patient Self-Testing.

New European medical device regulations require the performance of postmarketing surveillance evaluations for blood glucose meters (BGMs). We conducted an ISO15197:2015-conform system performance evaluation with the approved glucose dehydrogenase (GDH)-based Wellion NEWTON BGM. One hundred subjects were enrolled into the study (44 female, 56 male, 43 healthy subjects, 23 type 1 diabetes, 34 type 2 diabetes, age: 53.7 ± 15.8 years). In addition, manipulated heparinized whole blood was used for a laboratory interference test with ten selected substances (interference definition: substance-induced bias > 10%). The mean absolute relative difference (MARD) was 4.7%, and 100% of the values were in zones A (99.7%) and B (0.3%), respectively, of the consensus error grid. Interference was observed with xylose only, which is a known interfering substance for GDH-based BGMs.

Miniaturization of an Osmotic Pressure-Based Glucose Sensor for Continuous Intraperitoneal and Subcutaneous Glucose Monitoring by Means of Nanotechnology.

The Sencell sensor uses glucose-induced changes in an osmotic pressure chamber for continuous glucose measurement. A final device shall have the size of a grain of rice. The size limiting factor is the piezo-resistive pressure transducers inside the core sensor technology (resulting chamber volume: 70 µL. To achieve the necessary miniaturization, these pressure transducers were replaced by small (4000 × 400 × 150 nm³) nano-granular tunneling resistive (NTR) pressure sensors (chamber volume: 750 nL). For benchmark testing, we filled the miniaturized chamber with bovine serum albumin (BSA, 1 mM) and exposed it repeatedly to distilled water followed by 1 mM BSA solution. Thereafter, we manufactured sensors with glucose testing chemistry (ConcanavalinA/dextran) and investigated sensor performance with dynamic glucose changes between 0 and 300 mg/dL. Evaluation of the miniaturized sensors resulted in reliable pressure changes, both in the BSA benchmark experiment (30-35 mBar) and in the dynamic in vitro continuous glucose test (40-50 mBar). These pressure results were comparable to similar experiments with the previous larger in vitro sensors (30-50 mBar). In conclusion, the NTR pressure sensor technology was successfully employed to reduce the size of the core osmotic pressure chamber by more than 95% without loss in the osmotic pressure signal.

Laboratory Protocol and Pilot Results for Dynamic Interference Testing of Continuous Glucose Monitoring Sensors.

BACKGROUND: Testing the potential influence of interfering substances on the measurement performance of needle sensors for continuous glucose monitoring (CGM) is a challenging task. For proper function, the sensors need an almost stable fluidic environment. Previously published in vitro interference experiments were measuring under static concentration conditons. Our experimental setup allows for interference testing with dynamic changes of the interferent concentrations. METHODS: We designed a macrofluidic test stand that is fueled by several high-pressure liquid chromatography (HPLC) pumps generating programmable glucose and/or interferent gradients in phosphate-buffered saline (PBS). After optimizing experimental parameters (channel dimensions, temperature, flow rates, gradient slopes, buffer, pH etc.), we validated the setup using Dexcom G6 (G6) and Freestyle Libre 2 (L2) sensors with/without interferents, and using YSI 2300 Stat plus as the reference glucose device at room temperature. RESULTS: Both sensors tracked the programmed glucose changes. After calibration, G6 results closely matched glucose reference readings, while L2 routinely showed ~50% to 60% lower readings, most likely because of the factory-based calibration and temperature compensation. Gradients of maltose, acetaminophen, and xylose were employed to further validate the setup. As expected, both sensors were not affected by maltose. We confirmed previous findings regarding susceptibility of G6 readings to acetaminophen and L2 readings to xylose. Signals from both sensors are influenced by temperature in a linear fashion. CONCLUSIONS: Our experimental in vitro setup and protocol may provide a useful method to dynamically test CGM sensors for interfering substances. This may help to improve the accuracy of future CGM sensor generations.

Dynamic electrochemistry corrects for hematocrit interference on blood glucose determinations with patient self-measurement devices.

BACKGROUND: It has been demonstrated that dynamic electrochemistry can be used to correct blood glucose measurement results for potentially interfering conditions, such as humidity, hematocrit (HCT) variations, and ascorbic acid. The purpose of this laboratory investigation was to assess the potential influence of hematocrit variations on a variety of blood glucose meters applying different measurement technologies. METHODS: Venous heparinized whole blood was drawn, immediately aliquoted, and manipulated to contain three different blood glucose concentrations (80, 155, and 310 mg/dl) and five different hematocrit levels (25%, 37%, 45%, 52%, and 60%). After careful oxygenation to normal blood oxygen pressure, each of the resulting 15 different samples was measured 8 times with the following devices: BGStar, Contour, Accu-Chek Aviva, Accu-Chek Aviva Nano, Breeze 2, Precision Xceed, OneTouch Ultra 2, OneTouch Verio, FreeStyle Freedom Lite, Glucocard G+, GlucoMen LX, GlucoMen GM, and StatStrip [point-of-care (POC) device]. Cobas (Roche Diagnostics, glucose hexokinase method) served as laboratory plasma reference method. Stability to hematocrit influence was assumed when less than 10% bias occurred between the highest and lowest hematocrit levels when analyzing mean deviations for all three glucose concentrations. RESULTS: Besides the POC StatStrip device, which is known to measure and correct for hematocrit (resulting in <2% bias), four self-test meters also showed a stable performance in this investigation: dynamic electrochemistry, BGStar (8%), and static electrochemistry, Contour (6%), Glucocard G+ (2%), and OneTouch Verio (6%). The other meters failed this test: colorimetry, FreeStyle Freedom Lite (16%), and static electrochemistry, Accu-Chek Aviva (23%), Accu-Chek Aviva Nano (18%), Breeze 2 (36%), OneTouch Ultra 2 (34%), Precision Xceed (34%), GlucoMen LX (24%), and GlucoMen GM (31%). CONCLUSIONS: As hematocrit variations occur in daily routine (e.g., because of smoking, exercise, hypermenorrhea, pregnancy, stay in mountains, and hemodialysis), our results may encourage use of meters with stable performance under these conditions. Dynamic electrochemistry as used in the BGStar device (sanofi-aventis) appears to be an effective technology to correct for potential hematocrit influence on the meter results.

Determination of nitrotyrosine concentrations in plasma samples of diabetes mellitus patients by four different immunoassays leads to contradictive results and disqualifies the majority of the tests.

BACKGROUND: In the course of type 2 diabetes mellitus, insulin resistance has a severe impact on endothelial function leading to decreased synthesis of nitric oxide (NO). Postprandial hyperglycemia leads to the generation of reactive oxygen species, which counteracts the beneficial NO effects. NO and superoxide combine very fast in solution to form peroxynitrite, which is a potent protein-oxidizing agent. The peroxynitrite concentrations can be indirectly monitored by the detection of nitrotyrosine residues in proteins, reflecting the extent of damage caused by oxidative stress. METHODS: Four commercially available nitrotyrosine-specific immunoassays were evaluated by parallel measurement of nitrotyrosine in 224 serum samples derived from 16 patients with type 2 diabetes and 12 healthy controls (13 male and 15 female, age: 33+/-11 years) following a standardized meal. RESULTS: The available ELISA tests were not applicable for nitrotyrosine determination in human plasma samples due to technical issues and implausible results. However, a competitive luminescence assay was able to provide sufficient sensitivity and lead to clinically meaningful results in our test samples. CONCLUSIONS: All three ELISA methods were disqualified and conclusions previously derived from clinical experiments using these tests should be carefully reconsidered or reconfirmed. In the absence of a liquid tandem chromatography-mass spectrometry reference method, the luminescence test appears to be the method of choice for determination of nitrotyrosine in human plasma.

Differences in the dose accuracy of insulin pens.

BACKGROUND: Modern insulin injection pens provide a convenient and accurate way for diabetes patients to inject insulin. They have widespread use among children and adults with type 1 and type 2 diabetes in the U.S. and Europe. This study compared the dosing accuracy of four commonly available insulin pens (OptiClik and SoloSTAR from sanofi-aventis, FlexPen from Novo Nordisk, and HumaPen LUXURA from Eli Lilly). METHODS: The dosing accuracy was tested for all pens with 24 x 10 IU and 9 x 30 IU injection volumes to investigate whether the pens complied with the acceptable International Organization for Standardization (ISO) limits of 10% (+/- 1 IU) for 10 IU and 5% (+/- 1.5 IU) for 30 IU. The doses were each applied with a new needle strictly according to the instructions for use by the pen manufacturers. A pharmaceutical balance was used for the assessment of the applied volumes, and the results were corrected for the specific density of the insulin formulations. Four insulin pens (two each from different production lots) were used for each of the two volumes, resulting in a total of 192 doses per pen with 10 IU, and 72 doses per pen with 30 IU. RESULTS: FlexPen (mean absolute percent deviation for 10 IU and 30 IU: 1.64 +/- 0.84% and 0.83 +/- 0.26%, respectively) and HumaPen LUXURA (1.10 +/- 0.20% and 0.62 +/- 0.19%; not significant versus FlexPen for both doses) were more accurate than the OptiClik (4.78 +/- 3.31% and 2.97 +/- 2.48%, p <.01) and the SoloSTAR (2.61 +/- 0.92% and 1.70 +/- 0.84%, p <.05). While 6.8% of doses were outside the ISO limit at 10 IU with OptiClik (13.9% at 30 IU), the corresponding figures were 0.5% and 4.1%, respectively, for SoloSTAR. No doses outside the ISO limits were seen with FlexPen or HumaPen LUXURA at 10 IU and only one 30 IU dose (1.4%) was outside the limit for FlexPen. CONCLUSIONS: A direct head-to-head comparison of four insulin pens with a standardized protocol resulted in a more stable dosing accuracy of the FlexPen and the HumaPen LUXURA in comparison to the OptiClik and SoloSTAR. Even though all insulin delivery systems undergo rigorous testing before being approved for sale, there may be reasons to be attentive to the performance of the devices in practical use.

Gamma-adaptin, a novel ubiquitin-interacting adaptor, and Nedd4 ubiquitin ligase control hepatitis B virus maturation.

Hepatitis B virus (HBV) budding from infected cells is a tightly regulated process that requires both core and envelope structures. Here we report that HBV uses cellular gamma2-adaptin and Nedd4, possibly in conjunction with ubiquitin, to coordinate its assembly and release. In search of interaction partners of the viral L envelope protein, we previously discovered gamma2-adaptin, a putative endosomal sorting and trafficking adaptor of the adaptor protein complex family. We now demonstrate that the viral core interacts with the same gamma2-adaptor and that disruption of the HBV/gamma2-adaptin interactions inhibits virus production. Mutational analyses revealed a hitherto unknown ubiquitin-binding activity of gamma2-adaptin, specified by a ubiquitin-interacting motif, which contributes to its interaction with core. For core, the lysine residue at position 96, a potential target for ubiquitination, was identified to be essential for both gamma2-adaptin-recognition and virus production. The participation of the cellular ubiquitin system in HBV assembly was further suggested by our finding that core interacts with the endosomal ubiquitin ligase Nedd4, partly via its late domain-like PPAY sequence. Overexpression of a catalytically inactive Nedd4 mutant diminished HBV egress, indicating that protein ubiquitination is functionally involved in virus production. Additional evidence for a link of HBV assembly to the endosomal machinery was provided by immunolabeling studies that demonstrated colocalization of core and L with gamma2-adaptin in compartments positive for the late endosomal marker CD63. Together, these data indicate that an enveloped DNA virus exploits a new ubiquitin receptor together with endosomal pathway functions for egress from hepatocytes.

Functional incorporation of green fluorescent protein into hepatitis B virus envelope particles.

The envelope of hepatitis B virus (HBV), containing the L, M, and S proteins, is essential for virus entry and maturation. For direct visualization of HBV, we determined whether envelope assembly could accommodate the green fluorescent protein (GFP). While the C-terminal addition of GFP to S trans-dominant negatively inhibited empty envelope particle secretion, the N-terminal GFP fusion to S (GFP.S) was co-integrated into the envelope, giving rise to fluorescent particles. Microscopy and topogenesis analyses demonstrated that the proper intracellular distribution and folding of GFP.S, required for particle export were rescued by interprotein interactions with wild-type S. Thereby, a dual location of GFP, inside and outside the envelope, was observed. GFP.S was also efficiently packaged into the viral envelope, and these GFP-tagged virions retained the capacity for attachment to HBV receptor-positive cells in vitro. Together, GFP-tagged virions should be suitable to monitor HBV uptake and egress in live hepatocytes.