Positive Control Samples Research Articles

VarAmpliCNV: analyzing variance of amplicons to detect CNVs in targeted NGS data.

Computational identification of copy number variants (CNVs) in sequencing data is a challenging task. Existing CNV-detection methods account for various sources of variation and perform different normalization strategies. However, their applicability and predictions are restricted to specific enrichment protocols. Here, we introduce a novel tool named varAmpliCNV, specifically designed for CNV-detection in amplicon-based targeted resequencing data (Haloplex™ enrichment protocol) in the absence of matched controls. VarAmpliCNV utilizes principal component analysis (PCA) and/or metric dimensional scaling (MDS) to control variances of amplicon associated read counts enabling effective detection of CNV signals. Performance of VarAmpliCNV was compared against three existing methods (ConVaDING, ONCOCNV and DECoN) on data of 167 samples run with an aortic aneurysm gene panel (n = 30), including 9 positive control samples. Additionally, we validated the performance on a large deafness gene panel (n = 145) run on 138 samples, containing 4 positive controls. VarAmpliCNV achieved higher sensitivity (100%) and specificity (99.78%) in comparison to competing methods. In addition, unsupervised clustering of CNV segments and visualization plots of amplicons spanning these regions are included as a downstream strategy to filter out false positives. The tool is freely available through galaxy toolshed and at: https://hub.docker.com/r/cmgantwerpen/varamplicnv. Supplementary Data File S1: https://tinyurl.com/2yzswyhh; Supplementary Data File S2: https://tinyurl.com/ycyf2fb4. Supplementary data are available at Bioinformatics online.

Fungal Metabarcoding Data for Two Grapevine Varieties (Regent and Vitis vinifera ‘Cabernet-Sauvignon’) Inoculated with Powdery Mildew (Erysiphe necator) Under Drought Conditions

Fungal Metabarcoding Data for Two Grapevine Varieties (Regent and <i>Vitis vinifera</i> ‘Cabernet-Sauvignon’) Inoculated with Powdery Mildew (<i>Erysiphe necator</i>) Under Drought Conditions

Long-Read Sequencing Resolves Complex Structural Variants and Identifies Missing Disease-Causing Variants in Unsolved Cases of Hemophilia

Introduction: Despite recent advances in genetic testing, many individuals with inherited bleeding disorders remain unsolved after a complete clinical evaluation. In some cases, no variant is found in a particular gene or genes of interest, while in other cases a large structural variant (SV) is found or suspected that cannot be resolved using existing methods. In the large U.S. hemophilia genotyping initiative MyLifeOurFuture, ~2% of participants with hemophilia A had no F8 variant identified despite exhaustive investigation. Additionally, large SVs were common in severe hemophilia and associated with inhibitor risk but were incompletely characterized in nearly all cases. We hypothesized that long-read sequencing (LRS) would identify missing disease-causing variants and fully characterize SVs associated with hemophilia in these individuals. Methods: We performed targeted or whole-genome LRS on the Oxford Nanopore platform of 25 hemophilia cases with FVIII levels <14% in which prior testing identified no pathogenic variant or identified a suspected SV impacting F8. After basecalling with guppy and alignment to GRCh38 using minimap2, single nucleotide and insertion or deletion variants were identified with Clair3. All variant calls were refined using population frequency and pathogenicity predictions. SVs were called with Sniffles2, SVIM, and CuteSV. We performed de novo assembly of SVs and called methylation in all samples. Methylation differences were identified using an in-house developed pipeline. Results: We identified candidate pathogenic variants and previously undetected SVs in 10/10 cases with no variant found on clinical testing. Candidate intronic variants predicted to affect splicing were validated by exon trap assays. SVs identified included deletions and large inversions. Among 11 cases with suspected SVs, LRS resolved the candidate SV providing detailed genomic structure, orientation, and predicted impact of the variant. Whole genome LRS resolved SVs involving the large segmental duplication within F8 intron 22 (Int22h1), revealing variants that have not previously been observed including unsuspected orientations in the F8 intron 22 positive control sample. We analyzed four females with unexplained low FVIII levels and identified skewed methylation profiles that may help explain their phenotype. Conclusion: In a single genetic test LRS can identify disease-causing variants missed by prior genetic testing, resolve complex SVs, and identify methylation differences that explain hemophilia phenotypes. These comprehensive methods are necessary to fully understand causes of hemophilia and complications such as inhibitor development. We expect this approach will similarly resolve "missing” genetic variation in other inherited disorders. Supported and funded by the BBI at UW, WACBD, HTRS MCRA (Genentech), and NHLBI TOPMed.

A novel technology for disinfecting surfaces infested with Candida auris: the UVC chip

Abstract Background The fungal pathogen Candida Auris is increasingly associated with multidrug-resistant infections that are highly expensive for the Health Care System. The spreading of this pathogen can occur, among others, through contact with infected surfaces or medical instruments. This study evaluated the efficacy of a novel UVC chip, novel alternative to UVC LEDs and lamps, in inactivating Candida auris strain. Methods This experimental study was carried out between July and September 2020 at the University of Siena. Candida auris (ATCC 12372) at two known concentrations (1.5X107 and 1.5x106 CFU/ml) at a fixed distance (7,5 cm) from the chip (5.1mW radiant power) was tested, in triplicates, with three exposure times (5, 10 and 15 minutes). Potato Dextrose Agar (PDA) plates without the plate lid and containing Candida auris were exposed to UVC light. Subsequently, the plates were incubated at 36 °C for 48 h. Log reduction between treated and positive control (not exposed to UVC light) samples was calculated. Results At 15 minutes, we had the highest inactivation result, mean 4.43 log10, starting from a 1.5x106 CFU/mL concentration. At a higher concentration, 1.5X107 CFU/mL, the reduction had a mean of 3.51 log10. Conclusions The results of the experiments showed a significant microbial reduction in relation to the exposure time. The highest level of reduction was reached after 15 minutes of exposure. UVC chip had a relevant biocidal effect on Candida auris and may represent a valuable tool in the prevention of infections caused by this pathogen, which is becoming increasingly prevalent and persistent globally. Key messages • The use of UVC Chip decreases surface contamination. • New technology against healthcare-associated infections.

Protect peanut kernels from Aspergillus spp and their mycotoxins during storage by aqueous extract of carob pulp

Recently, the trend of many studies to replace synthetic antifungal commonly used during storage with natural antifungal; Plant extracts has been one of the most promising strategies for this purpose. Therefore, this study aimed to evaluate the aqueous extract of the carob pulp (AECP) at 1.5, 2.5, and 5 mg/ml on the growth of some toxigenic Aspergillus species, and its effectiveness as an anti-mycotoxigenic (aflatoxins (AFs) and ochratoxin A (OTA) during storage red peanut kernels (RPKs) for 90 days. The results indicate that the increased concentration of AECP, the area of inhibition zone increased with all treatments. As well, the strain of Aspergillus ochraceus was more affected by AECP than Aspergillus aflatoxiformans followed by Aspergillus parasiticus which recorded (15.7 ± 0.4 mm), (13.4 ± 0.5 mm), and (11.9 ± 0.4 mm) at 5 mg/ml. Respectively. While, the lowest inhibition was shown with Aspergillus flavus at three concentrations of AECP. The impact of AECP on the accumulation of total AFs and OTA during storage of RPKs infected by A. parasiticus and A. ochraceus, respectively, for 90 days. The results showed that the active compounds in the AECP at 5 mg/ml can protect RPKs for 45 days without detecting any amounts of AFs and OTA. After 90 days, the inhibition percentages for the four types of aflatoxins (AFB1, AFG1, AFB2, AFG2) and OTA compared with positive control samples were 85.1, 76.5, 86.5, 84.2, and 87.5%, respectively. Finally, AFB1 recorded the lowest inhibition percentages of all treatments; on the contrary, it was OTA.

What do positive and negative Cutibacterium culture results in periprosthetic shoulder infection mean? A multi-institutional control study.

Deep tissue culture specimens obtained at the time of revision shoulder arthroplasty are commonly positive for Cutibacterium. Clinical interpretation of positive cultures can be difficult. This was a multi-institutional study evaluating the accuracy of cultures for Cutibacterium using positive control (PC) and negative control (NC) samples. The relationship between time to culture positivity and strength of culture positivity was also studied. Eleven different institutions were each sent 12 blinded samples (10 PC and 2 NC samples). The 10 PC samples included 2 sets of 5 different dilutions of a Cutibacterium isolate from a failed total shoulder arthroplasty with a probable periprosthetic infection. At each institution, the samples were handled as if they were received from the operating room. Specimen growth, time to culture positivity, and strength of culture positivity (based on semiquantitative assessment) were reported. A total of 110 PC samples and 22 NC samples were tested. One hundred percent of specimens at the 4 highest dilutions were positive for Cutibacterium. At the lowest dilution, 91% of samples showed positive findings. Cutibacterium grew in 14% of NC samples. Cutibacterium grew in PC samples at an average of 4.0±1.3 days, and all of these samples showed growth within 7 days. The time to positivity was significantly shorter (P<.001) and the strength of positivity was significantly higher (P<.001) in true-positive cultures compared with false-positive cultures. This multi-institutional study suggests that different institutions may report highly consistent rates of culture positivity for revision shoulder arthroplasty samples with higher bacterial loads. In contrast, with lower bacterial loads, the results are somewhat less consistent. Clinicians should consider using a shorter time to positivity and a higher strength of positivity as adjuncts in determining whether a tissue culture sample is a true positive.

Involvement of Bacteria in the Pathological Changes Before Achilles Tendon Rupture: A Case Series Investigating 16S rDNA in 20 Consecutive Ruptures.

Background:The source of the pathological changes that occur before an acute Achilles tendon rupture (ATR) is not fully understood. Bacterial DNA has previously been detected in samples from ruptured Achilles tendons, suggesting a pathogenic role of bacteria in ATR.Purpose/Hypothesis:The purpose of this study was to investigate if DNA from bacteria was present in acutely ruptured Achilles tendons. We hypothesized that 20% to 30% of the samples from the rupture site and no samples from healthy tissue would be positive for bacterial DNA.Study Design:Case series; Level of evidence, 4.Methods:This study included 20 consecutive patients scheduled for surgical repair of an acute ATR. Tendon biopsy specimens were taken from the rupture site and from the healthy tendon tissue proximal to the rupture to act as a control. Samples were blinded to the technician and analyzed using polymerase chain reaction targeted to the bacterial 16S rDNA gene and Sanger sequencing to identify the bacterial species present. McNemar test for paired proportions was performed to test for statistically significant differences in the number of samples positive for bacterial DNA between the ruptured and control regions of the Achilles tendon.Results:Of the 20 patients, 1 (5%) had a positive sample with bacterial DNA from the ruptured part of the Achilles tendon. The same patient also had a positive control sample, although with different bacterial DNA. An additional patient had a positive control sample. There was no statistically significant difference in the number of bacterial DNA–positive samples between the ruptured and control regions of the Achilles tendon. The bacteria found (Staphylococcus sp, Micrococcus sp, and Staphylococcus epidermidis) were normal commensal organisms on the human skin.Conclusion:Bacterial DNA was infrequent in tissue from ruptured Achilles tendons and, if identified, likely was a result of contamination. This suggests that bacteria are not involved in the pathological changes occurring before rupture of the Achilles tendon.

A microfluidic diagnostic device with air plug-in valves for the simultaneous genetic detection of various food allergens

The identification of accidental allergen contamination in processed foods is crucial for risk management strategies in the food processing industry to effectively prevent food allergy incidents. Here, we propose a newly designed passive stop valve with high pressure resistance performance termed an “air plug-in valve” to further improve microfluidic devices for the detection of target nucleic acids. By implementing the air plug-in valve as a permanent stop valve, a maximal allowable flow rate of 70 µL/min could be achieved for sequential liquid dispensing into an array of 10 microchambers, which is 14 times higher than that achieved with the previous valve arrangement using single-faced stop valves. Additionally, we demonstrate the simultaneous detection of multiple food allergens (wheat, buckwheat, and peanut) based on the colorimetric loop-mediated isothermal amplification assay using our diagnostic device with 10 microchambers compactly arranged in a 20-mm-diameter circle. After running the assays at 60 °C for 60 min, any combination of the three types of food allergens and tea plant, which were used as positive and negative control samples, respectively, yielded correct test results, without any cross-contamination among the microchambers. Thus, our diagnostic device will provide a rapid and easy sample-to-answer platform for ensuring food safety and security.

A Method for Detecting Antioxidant Activity of Antioxidants by Utilizing Oxidative Damage of Pigment Protein.

A simple and effective method for detecting the antioxidant activity by utilizing oxidative damage of pigment proteins was developed. In this method, phycocyanin and bovine hemoglobin pigment proteins were used as substrates attacked by free radicals; AAPH was used as a free radical initiator; and Trolox as a positive control; and the fermentation products of Lactobacillus plantarum 793, phycocyanin hydrolysates, salmon skin collagen hydrolysates, and synthetic peptides PMRGGYHY and FCVLRP are antioxidants inspected in this study. Because of being attacked by free radicals, the absorbance of the pigment proteins at their characteristic absorption peak changes with time. By recording the time-varying curve at the characteristic absorption peak of the pigment protein in the blank/negative control sample, the Trolox positive control sample, and the samples of inspected antioxidants, the antioxidant activity could be calculated by using the specific equation. The linear detection ranges of Trolox in the phycocyanin assay and the bovine hemoglobin assay were 1-4μM and 4-24μM, respectively. Compared with the ORAC assay, the antioxidant activities of the samples measured by this method were slightly lower. The method proposed in this study reflects the protective effects of inspected antioxidants on pigment proteins, which could potentially serve as new biomarkers of oxidative damage processes.

Function of CTLGA9 Amino Acid Residue Leucine-6 in Modulating Cry Toxicity.

Aedes aegypti is a crucial vector for many arboviral diseases that cause millions of deaths worldwide and thus is of major public health concern. Crystal (Cry) proteins, which are toxins produced by Bacillus thuringiensis, are structurally organized into three-domains, of which domain II is the most variable in terms of binding towards various toxin receptors. The binding of Cry11Aa to putative receptor such as aminopeptidase-N (APN) is explicitly inhibited by midgut C-type lectins (CTLs). The similarity between the domain II fold of Cry11Aa toxin and the carbohydrate recognition domain in the CTLs is a possible structural basis for the involvement of Cry domain II in the recognition of carbohydrates on toxin receptors. In this study, a site-directed point mutation was introduced into the A. aegypti CTLGA9 gene on the basis of molecular docking findings, leading to substitution of the Leucine-6 (Leu-6) residue in the protein with alanine. Subsequently, functional monitoring of the mutated protein was carried out. Unlike the amino acid residues of wild-type CTLGA9, none of the residues of mutant (m) CTLGA9 were competed with Cry11Aa for binding to the APN receptor interface. Additionally, ligand blot analysis showed that both wild-type and mutant CTLGA9 had similar abilities to bind to APN and Cry11Aa. Furthermore, in the competitive ELISA in which labeled mutant CTLGA9 (10 nM) was mixed with increasing concentrations of unlabeled Cry11Aa (0–500 nM), the mutant showed no competition with Cry11Aa for binding to APN., By contrast, in the positive control sample of labeled wild type CTLGA9 mixed with same concentrations of Cry11Aa competition between the two ligands for binding to the APN was evident. These results suggest that Leucine-6 may be the key site involved in the competitive receptor binding between CTLGA9 and Cry11Aa. Moreover, according to the bioassay results, mutant CTLGA9 could in fact enhance the toxicity of Cry11Aa. Our novel findings provide further insights into the mechanism of Cry toxicity as well as a theoretical basis for enhancing the mosquitocidal activity of these toxin through molecular modification strategies.

Qualitative and quantitative analyses of Tripterygium hypoglaucum in Yinning Tablets, a compound traditional Chinese herbal preparation

To conduct qualitative and quantitative analyses of Tripterygium hypoglaucum in Yinning Tablets, a compound preparation of traditional Chinese herbal medicine. Thin-layer chromatography (TLC) was used for qualitative analysis of Tripterygium hypoglaucum in Yining Tablets and the analytical protocols were optimized. High-performance liquid chromatography (HPLC) was used to quantitatively analyze the content of triptolide (the main active ingredient of Tripterygium hypoglaucum) in Yinning Tablets. The results of TLC analysis showed that the test sample of Yinning Tablets and the positive control samples both produced clear, well separated spots without obvious interference in the blank samples. Assessment of the influences of the thin-layer plates from different manufacturers, temperature and humidity on the test results demonstrated good durability of the test. HPLC analysis of triptolide showed a good linear relationship within the concentration range of 1-100 μg/mL (regression equation: A=22.219C-19.165, r=0.9999); the contents of triptolide in 3 batches of Yinning tablets were 0.34, 0.34, and 0.28 μg per tablet, all within the range of 0.28-0.34 μg per tablet. It was finally determined that each Yinning tablet should not contain more than 0.6 μg of triptolide. TLC and HPLC are simple, accurate, durable and specific for qualitative and quantitative analyses of Tripterygium hypoglaucum in Yinning Tablets.

Detection of Post-COVID-19 Patients Using Medical Scent Detection Dogs-A Pilot Study.

There is a growing number of COVID-19 patients experiencing long-term symptoms months after their acute SARS-CoV-2 infection. Previous research proved dogs' ability to detect acute SARS-CoV-2 infections, but has not yet shown if dogs also indicate samples of patients with post-COVID-19 condition (Long COVID). Nine dogs, previously trained to detect samples of acute COVID-19 patients, were confronted with samples of Long COVID patients in two testing scenarios. In test scenario I (samples of acute COVID-19 vs. Long COVID) dogs achieved a mean sensitivity (for acute COVID-19) of 86.7% (95%CI: 75.4–98.0%) and a specificity of 95.8% (95%CI: 92.5–99.0%). When dogs were confronted with Long COVID and negative control samples in scenario IIa, dogs achieved a mean sensitivity (for Long COVID) of 94.4 (95%CI: 70.5–100.0%) and a specificity of 96.1% (95%CI: 87.6–100.0%). In comparison, when acute SARS-CoV-2 positive samples and negative control samples were comparatively presented (scenario IIb), a mean sensitivity of 86.9 (95%CI: 55.7–100.0%) and a specificity of 88.1% (95%CI: 82.7–93.6%) was attained. This pilot study supports the hypothesis of volatile organic compounds (VOCs) being long-term present after the initial infection in post-COVID-19 patients. Detection dogs, trained with samples of acute COVID-19 patients, also identified samples of Long COVID patients with a high sensitivity when presented next to samples of healthy individuals. This data may be used for further studies evaluating the pathophysiology underlying Long COVID and the composition of specific VOC-patterns released by SARS-CoV-2 infected patients throughout the course of this complex disease.

Development and acceptability of value-added products from green mussel (Perna viridis) in Samar, Philippines

Green mussel (Perna viridis) is a bivalve mollusc abundantly produced in the province of Samar, Philippines. The development of value-added products from this resource will increase its utilization and marketability. Thus, this study aimed to develop a green mussel meatloaf, noodles, and spread, and determine the product acceptability. Based on the result of the sensory evaluation, meatloaf containing 70% mussel meat obtained the highest score of 7.9 among other treatments with an adjectival rating of like very much. Noodles with 30% mussel are the most acceptable among other treatments with a score of 8.2, and an adjectival rating of like very much. Spread with 20% mussel meat scored highest at 7.9 with an adjectival rating of like very much. These three most acceptable products showed no significant difference (p&gt;0.05) with their corresponding positive control samples which are the commercial products, indicating that these mussel products could compete in the market. Furthermore, microbial test results revealed that all products were fit for human consumption, showing a lower total plate count than the microbial count limit set by the standards. Overall, this study showed that green mussels can be processed into meatloaf, noodles and spread.

Prussian blue technique is prone to yield false negative results in magnetoreception research

Perls’s Prussian blue staining technique has been used in magnetoreception research to screen tissues for iron-rich structures as proxies for putative magnetoreceptor structures based on magnetic particles. However, seemingly promising structural candidates in the upper beak of birds detected with Prussian blue turned out to be either irreproducible or located in non-neuronal cells, which has spurred a controversy that has not been settled yet. Here we identify possible pitfalls in the previous works and apply the Prussian blue technique to tissues implicated in magnetic-particle-based magnetoreception, in an effort to reassess its suitability for staining single-domain magnetite, i.e., the proposed magnetic substrate for the interaction with the external magnetic field. In the upper beak of night-migratory songbirds, we found staining products in great numbers, but not remotely associated with fiber terminals of the traced ophthalmic branch of the trigeminal nerve. Surprisingly, staining products were absent from the lamina propria in the olfactory rosette of rainbow trout where candidate magnetoreceptor structures were identified with different techniques earlier. Critically, magnetosome chains in whole cells of magnetotactic bacteria remained unstained. The failure to label single-domain magnetite in positive control samples is a serious limitation of the technique and suggests that two most influential but antipodal studies conducted previously stood little chances of obtaining correct positive results under the assumption that magnetosome-like particles were present in the tissues. Nonetheless, the staining technique appears suitable to identify tissue contamination with iron-rich fine dust trapped in epithelia already in vivo.

Evaluation of the Diagnostic Performance of three Rapid Diagnostic Tests for HBsAg screening for blood donation in Cameroon

Background: Rapid Diagnostic Tests have been wildly reported for HBsAg screening in Cameroon. Aims and Objectives: The present study aimed at assessing the diagnostic performance and the limit of detection of three Rapid Diagnostic Tests used for HBsAg screening for blood donation in Cameroon. Study Design: A hospital-based cross-sectional study involving blood donors who met blood banks requirements was done. Setting: The study was carried out at Douala Laquintinie Hospital and Bamenda Regional Hospital. Materials and Methods: Ten mL of blood specimen was collected among blood donors who accepted to partake in the study by signing the inform consent. Laboratory processing was performed at the University of Buea. The limit of detection of the assays under evaluation was checked and the diagnostic performance assessed. The automated Architect HBsAg assay and the ELISA Biorex HBsAg were used as the reference standard. Statistics: Sensitivity, specificity was obtained by comparing the results of each of the assay to those of the reference standard. The limit of detection (LOD) of the three RDTs compared to the ELISA Biorex was assessed by preparing 14-fold Dilution of known positive control samples. Results: The limit of detection of the tests under evaluation was 0.18IU/mL whereas the one of the ELISA Biorex was 0.05IU/mL. Diaspot and Fastep obtained a sensitivity of 88.24% when compared to Architect and respectively 60.53% and 57.89% when compared to Biorex. Abon showed a lower sensitivity of 50.0% as compared to Biorex and 58.82% compare to Architect. Diaspot and Fastep had a specificity &gt; 99% independent on the standard while Fastep had 98.62% using Biorex and 97.54% using Architect. Conclusion: Diaspot and Fastep feature the World Health Organization required specificity independent of the standard used while none of the tests reached the expected sensitivity and limit of detection.

Improved diagnosis of type-1 diabetes mellitus using multiplexed autoantibodies ELISA array

Autoantibodies are currently the most robust biomarkers of type 1 diabetes. However, single autoantibody targeted detection is still limited in diabetes diagnosis with poor performance. Here, we develop a multiplexed Array-ELISA assay that can detect five diabetes-related autoantibodies including glutamic acid decarboxylase antibody (GADA), insulinoma antigen 2 antibody (IA-2A), islet cell antibody (ICA), zinc transporter 8 autoantibody (ZnT8-A) and insulin antibody (IAA) simultaneously. This assay achieved 100% accuracy in identifying the positive and negative control samples with good repeatability (CV<15%). We applied the Array-ELISA assay to 140 clinical serum samples of healthy subjects and diabetes patients and the assay showed improved diagnosis accuracy (sensitivity of 62.5%, specificity of 94.3%) compared with the single target immunoblotting test. These data suggest that the Array-ELISA assay can provide diagnostic and predictive ability in the clinical practice of type 1 diabetes.

Autosterilization Effect of Gamma Radiation in Non-sterile Radiopharmaceuticals

Gamma radiation is commonly used in sterilizing various products because of its microbial-killing property. Several radiopharmaceuticals are processed aseptically, which requires a well-managed GMP-based facility. This study aims to ensure that radiopharmaceutical can behave as an auto sterilizing agent since it contains radionuclide that emits gamma radiation. Sm-153-ethylenediaminetetramethylenephosphonate (Sm-153-EDTMP) and I-131-Hippuran were used as models in this study, in which various radioactivities of these products were added into non-sterile vials in the non-aseptic working area and tested for sterility using direct inoculation method. The result showed that samples containing 7 mCi of Sm-153-EDTMP and less than 2 mCi of I-131-orthoiodohippurate (I-131-Hippuran) changed the clarity of the media, but not for those containing higher radioactivity. The results showed that Sm-153-EDTMP and I-131-Hippuran at certain radioactivity can auto sterilize themselves, whereas the positive control sample and the products diluted with non-sterile water remained unsterile. This study showed that radiopharmaceuticals have auto sterilizing properties at relatively low radioactivity, depending on the products’ bioburden. Therefore, the usual aseptic preparation of some radiopharmaceuticals can be considered terminal sterilization.

Multicentric Evaluation of SeeGene Allplex Real-Time PCR Assays Targeting 28 Bacterial, Microsporidal and Parasitic Nucleic Acid Sequences in Human Stool Samples.

Prior to the implementation of new diagnostic techniques, a thorough evaluation is mandatory in order to ensure diagnostic reliability. If positive samples are scarcely available, however, such evaluations can be difficult to perform. Here, we evaluated four SeeGene Allplex real-time PCR assays amplifying a total of 28 bacteria, microsporidal and parasitic nucleic acid sequence targets in human stool samples in a multicentric approach. In the assessments with strongly positive samples, sensitivity values ranging between 13% and 100% were recorded for bacteria, between 0% and 100% for protozoa and between 7% and 100% for helminths and microsporidia; for the weakly positive samples, the recorded sensitivity values for bacteria ranged from 0% to 100%; for protozoa, from 0% to 40%; and for helminths and microsporidia, from 0% to 53%. For bacteria, the recorded specificity was in the range between 87% and 100%, while a specificity of 100% was recorded for all assessed PCRs targeting parasites and microsporidia. The intra- and inter-assay variations were generally low. Specifically for some helminth species, the sensitivity could be drastically increased by applying manual nucleic acid extraction instead of the manufacturer-recommended automatic procedure, while such effects were less obvious for the bacteria and protozoa. In summary, the testing with the chosen positive control samples showed varying degrees of discordance between the evaluated Allplex assays and the applied in-house reference assays associated with higher cycle threshold values in the Allplex assays, suggesting that samples with very low pathogen densities might be missed. As the targeted species can occur as harmless colonizers in the gut of individuals in high-endemicity settings as well, future studies should aim at assessing the clinical relevance of the latter hint.

Evaluation of the RSR 3 screen ICA™ and 2 screen ICA™ as screening assays for type 1 diabetes in Sweden

AimThe study aim was to evaluate the RSR 3 Screen ICA™ and 2 Screen ICA™ for detection of islet cell autoimmunity in healthy Swedish subjects and patients with newly diagnosed type 1 diabetes (T1D).Methods3 Screen is designed for combined detection of autoantibodies to glutamic acid decarboxylase (GADA), to the islet antigen IA-2 (IA-2A) and to zinc transporter 8 (ZnT8A), while 2 Screen detects GADA and IA-2A. Serum samples from 100 T1D patients at onset and 200 healthy controls were studied.Results3 Screen achieved 93% assay sensitivity and 97.5% specificity, while 2 Screen achieved 91% assay sensitivity and 98.5% specificity. Samples were also tested in assays for individual autoantibodies. There was only one 3 Screen positive healthy control sample (0.5%) that was positive for multiple autoantibodies (IA-2A and ZnT8A). In contrast, most of the 93 3 Screen positive patients were positive for multiple autoantibodies with 72% (67/93) positive for both GADA and IA-2A and 57% (53/93) positive for three autoantibodies (GADA, IA-2A and ZnT8A). Insulin autoantibodies (IAA, measured by radioimmunoassay) were positive in 13 patients and two healthy controls.Conclusion3 Screen achieved high sensitivity and specificity, suitable for islet cell autoimmunity screening in a healthy population. In the case of 3 Screen positivity, further assays for GADA, IA-2A and ZnT8A are required to check for multiple autoantibody positivity, a hallmark for progression to T1D. In addition, testing for IAA in children below two years of age is warranted.

Comprehensive BCMA Expression Profiling in Adult Normal Human Brain Suggests a Low Risk of On-target Neurotoxicity in BCMA-targeting Multiple Myeloma Therapy.

B-cell maturation antigen (BCMA) is a target for the treatment of multiple myeloma with cytolytic therapies, such as chimeric antigen receptor T-cells or T-cell redirecting antibodies. To better understand the potential for "on-target/off-tumor" toxicity caused by BCMA-targeting cytolytic therapies in the brain, we investigated normal brain BCMA expression. An immunohistochemistry (IHC) assay using the E6D7B commercial monoclonal antibody was applied to 107 formalin-fixed, paraffin-embedded brain samples (cerebrum, basal ganglia, cerebellum, brainstem; 63 unique donors). Although immunoreactivity was observed in a small number of neurons in brain regions including the striatum, thalamus, midbrain, and medulla, this immunoreactivity was considered nonspecific and not reflective of BCMA expression because it was distinct from the membranous and Golgi-like pattern seen in positive control samples, was not replicated when a different IHC antibody (D6 clone) was used, and was not corroborated by in situ hybridization data. Analysis of RNA-sequencing data from 478 donors in the GTEx and Allen BrainSpan databases demonstrated low levels of BCMA RNA expression in the striatum of young donors with levels becoming negligible beyond 30 years of age. We concluded that BCMA protein is not present in normal adult human brain, and therefore on-target toxicity in the brain is unlikely.