Sensitive Assay Research Articles

B-276 Evaluation of the Roche Benzodiazepines II Immunoassay for Urine Drug Testing in Clinical Specimens

Abstract Background Benzodiazepines are one of the most commonly prescribed medications in the United States and are frequently linked to instances of abuse and overdose. Historically, FDA-cleared benzodiazepine urine immunoassays cross-react poorly with glucuronidated metabolites excreted in urine. False negative results are especially prevalent with lorazepam, which is almost exclusively excreted as lorazepam glucuronide. Some clinical laboratories have addressed this problem with the addition of beta-glucuronidase to enhance assay sensitivity as a laboratory developed test (LDT). Roche Diagnostics recently received FDA clearance to offer a benzodiazepine immunoassay that includes beta-glucuronidase. Methods Performance characteristics of two FDA-cleared benzodiazepine urine immunoassays (Benzodiazepines Plus, no glucuronidase and Benzodiazepines II, with glucuronidase; Roche Diagnostics) and a benzodiazepine immunoassay LDT (with glucuronidase) were evaluated using 258 urine specimens. These immunoassays were directly compared to an LC-MS/MS benzodiazepine LDT to determine clinical sensitivity and specificity. Cross-reactivity of all three immunoassays were compared and evaluated based on the measured benzodiazepine concentrations determined by the LC-MS/MS LDT. Cross-reactivity for 7-aminoclonazepam, lorazepam, and lorazepam glucuronide were assessed using drug-free urine spiked with reference materials (Cerilliant) at concentrations ranging from 100 to 1000 ng/mL. The cutoff for positive results was set at 1000 mAbs, corresponding to 200 ng/mL of the nordiazepam calibrator. Results The Benzodiazepines II and LDT immunoassays exhibited greater clinical sensitivity (100% and 95.2%) compared to the Benzodiazepines Plus assay (66.7%). Clinical specificity of 100% was observed for all three assays. Cross-reactivity of the Benzodiazepines II assay was greater across the range of benzodiazepine concentrations tested in comparison to the other immunoassays. In particular, the Benzodiazepines II assay was the most sensitive for 7-aminoclonazepam, as it was the only immunoassay out of the three that detected the presence of specimens containing this metabolite alone. Cross-reactivity analysis shows that both 7-aminoclonazepam and lorazepam yielded positive results when tested using the Benzodiazepines II assay at a concentration of 300 ng/ml. 7-aminoclonazepam tested positive at 800 ng/ml with the LDT and Benzodiazepines Plus assays, whereas lorazepam tested positive at 600 ng/ml with these assays. Additionally, the LDT and Benzodiazepines II assays detected lorazepam glucuronide at concentrations of 400 ng/ml and 250 ng/ml, respectively. However, the Benzodiazepines Plus assay yielded negative results for all concentrations of lorazepam glucuronide tested (up to 1000 ng/mL). Conclusions A comprehensive evaluation of these three immunoassays demonstrates that the Benzodiazepines II immunoassay has increased clinical and analytical sensitivity compared to the Benzodiazepines Plus and LDT immunoassays. The inclusion of a beta-glucuronidase greatly improved the sensitivity of the Benzodiazepines II and LDT immunoassays for lorazepam, which is primarily excreted as a glucuronide metabolite in urine.

A-306 High Sensitivity Capability of an Anti-müllerian Hormone Assay on the Beckman Coulter DxI 9000 Immunoassay Analyzer*, a Next Step Toward Enabling Ovarian Failure Staging?

Abstract Background Measurement of anti-müllerian hormone (AMH) has proven value in the evaluation of ovarian reserve using assays such as the Beckman Coulter Access AMH assay. If improved sensitivity of AMH measurement can be achieved, enhanced clinical utility during the assessment of reproductive aging and ovarian response to enable staging of ovarian failure is possible (Practice Committee of the American Society of Reproductive Medicine 2020, Cappola et al. 2023). The Beckman Coulter DxI 9000 Immunoassay Analyzer* has been designed with new technology that can be leveraged to improve assay sensitivity. Such technological advancements include the new Lumi-Phos PRO chemiluminescent substrate which yields markedly increased signal-to-noise, a new luminometer with increased dynamic range, and improved low-volume pipetting capabilities. Data herein summarize results from analytical verification studies of the Access AMH assay on the DxI 9000 Immunoassay Analyzer. Results demonstrate an example of DxI 9000 technologies enabling sensitivity claims that are up to 20-fold improved. * The official name is DxI 9000 Access Immunoassay Analyzer. Methods A method comparison study was performed based on CLSI guideline EP09c, 3rd ed. to compare results for the Access AMH assay on DxI 9000 to the predicate device, the Access AMH assay on the Access 2 Immunoassay System. Within-laboratory precision was also evaluated following CLSI EP05-A3. Finally, performance at low analyte levels was evaluated following CLSI EP17-A2, and linearity was evaluated following CLSI EP06-Ed2. Results Method comparison of the Access AMH assay on DxI 9000 compared to the Access AMH assay on the Access 2 yielded excellent agreement with a Passing-Bablok slope of 1.02 for N=126 samples. Further, bias estimates for a number of key medical decision points suggest minimal bias (≤ 2%) across the assay range, and minimal non-linearity was observed. 20-day imprecision studies yielded CV’s between 2 and 5% across a broad range of concentrations evaluated. Sensitivity studies yielded estimates of limit of quantitation (LoQ) between 0.001 and 0.003 ng/mL. This represents an approximately 10- to 20-fold improvement in sensitivity claims compared to the AMH assay on the Access 2 and other commercially available automated immunoassay analyzers. Conclusions The data herein present performance data for the Access AMH assay on the DxI 9000 Immunoassay Analyzer. The assay demonstrates excellent accuracy relative to the predicate device, while showing good linearity and imprecision. Of note, the Access AMH assay exhibits exceptional low-end performance on the DxI 9000 analyzer as demonstrated by the ability to enable sensitivity claims that are up to 20-fold improved in comparison to existing immunoassays for the measurement of AMH. The sensitivity capabilities of the Access AMH assay on DxI 9000 suggest an opportunity for the addition of further intended uses for scenarios where extremely low AMH measurements can provide additional value of clinical significance. Further studies are needed to fully understand additional clinical utilities of the Access AMH assay with high sensitivity capability on DxI 9000, including the assessment of reproductive aging for menopause diagnosis or ovarian response for fertility treatment.

Development and Evaluation of a Semi-Nested PCR Method Based on the 18S ribosomal RNA Gene for the Detection of Babesia aktasi Infections in Goats.

We developed and evaluated a semi-nested PCR assay for the detection of Babesia aktasi infection in goats based on the sequence of the B. aktasi 18S ribosomal RNA gene. Following in silico screening, the specificity of the primers was assessed using reference DNA samples, including B. ovis, B. motasi, B. crassa, B. venatorum, B. divergens, B. capreoli, Theileria ovis, and T. annulata. To determine the sensitivity of the method, blood infected with 2% parasitemia of B. aktasi was diluted to 10-fold serial dilutions. The method specifically amplified a 438 bp fragment of B. aktasi DNA, but did not demonstrate cross-amplification with the other hemoparasites tested. The sensitivity assay indicated that this PCR method was able to detect infection at a dilution of 10-8 of 2% parasitemia (0.074 parasites/200 µL). Ninety-seven blood samples collected from goats were used to analyze for B. aktasi, and the infection was detected in 18.5% of the goats. Additionally, the method was also applied to 44 field DNA samples that were detected to be positive for B. aktasi by reverse line blotting (RLB), and showed 84.1% agreement. The findings revealed that newly developed semi-nested PCR can detect B. aktasi infections in goats with high sensitivity and specificity.

Molecular detection and quantification of canine parvovirus 2 using a fast and sensitive SYBR® green-based quantitative polymerase chain reaction assay in dogs affected with gastroenteritis

Background and Aim: Viral gastroenteritis in canines is primarily caused by the canine parvovirus 2 (CPV-2). Infections by this virus can cause severe consequences in dogs, such as fever, vomiting, diarrhea, septicemia, systemic inflammation, and immunosuppression. Therefore, the mortality rate of persistent infections caused by this virus is significantly high. The capsid protein VP2 genome of canine parvovirus has undergone many changes, resulting in the emergence of different genotypes, including CPV-2a, CPV-2b, and CPV-2c. Diagnostic procedures often lack the necessary specificity for early infection diagnosis. Early detection of the infection enhances the likelihood of canine survival because the canine will receive prompt therapy. Hence, this study aimed to develop a quantitative polymerase chain reaction (qPCR)-based diagnostic technique using SYBR Green for the rapid and accurate detection and quantification of CPV-2. Materials and Methods: The assay was specifically designed to identify a portion of the conserved NS gene using primers that amplify a 125-bp fragment. The qPCR method was executed in the fast mode to expedite the process using Power up SYBR Green Master Mix reagent. A standard curve was constructed using the amplified and purified PCR product of the NS gene. Results: The limit of detection and quantification were determined in the one amplified-DNA copy. The standard curve showed an efficiency of 99.5% and inter- and intra-assay coefficients of variation of 0.387%–0.976% and 0.085%–0.430%, respectively. The assay was specific for the amplification of CPV-2, as no amplification was observed for other viral genomes (canine adenovirus II, canine distemper virus, canine coronavirus, and canine astrovirus) or from the negative controls. Inter- and intra-tests for repeatability showed low test variability around the run time. To validate the present assay, 200 samples of fezzes from canines with gastroenteritis and symptoms associated with enteric infection were tested using the qPCR protocol. From the analyzed samples, 136 were positive for CPV-2 by qPCR assay, of which 110 were before diagnostic positive for the virus by endpoint PCR, showing high sensitivity of the current assay. CPV-2 was detected in dogs over 2 weeks old up to dogs 9 years old, where the highest viral concentration found was 16429595 gene copies in dogs aged 2 weeks. Conclusion: In the present study, a rapid, specific, repeatable, and sensitive assay was developed for the detection and quantification of CPV-2. Furthermore, it was demonstrated that in the population of domestic dogs in Ecuador affected with gastrointestinal disease, the virus is presented in dogs of different ages and not only in young dogs. Keywords: canine parvovirus, gastroenteritis, quantitative polymerase chain reaction, SYBR green.

Diversity of Chagas disease diagnostic antigens: Successes and limitations.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, remains a public health issue in endemic regions of the Americas, and is becoming globalised due to migration. In the chronic phase, 2 accordant serological tests are required for diagnosis. In addition to "in-house" assays, commercial tests are available (principally ELISA and rapid diagnostic tests). Herein, we discuss the discovery era of defined T. cruzi serological antigens and their utilisation in commercialised tests. A striking feature is the re-discovery of the same antigens from independent studies, and their overlapping use among commonly reported commercial serological tests. We also consider reports of geographical variation in assay sensitivity and areas for refinement including applications to congenital diagnosis, treatment monitoring, and lineage-specific antigens.

Mass-Tagged Self-Assembled Nanoprobe Reveals the Transport of PD-L1 from Cancer Cells to Tumor-Educated Platelets

BackgroundThe expression level of immune checkpoint proteins detected by tissue biopsy is currently used as a predictive biomarker for immune checkpoint blockade (ICB) therapy. However, tissue biopsy is susceptible to invasive sample collection procedures, significant sampling heterogeneity, and the difficulty of repeated sampling. Therefore, liquid biopsy of blood samples is becoming an alternative choice for immune checkpoint protein detection. Among various vesicles in blood, platelets can obtain cancer information to form a specific group called tumor-educated platelets (TEPs). The platelet-derived proteins in TEPs may have a predictive potential in ICB therapy. ResultsIn this study, a photo-cleavable mass-tagged self-assembled (SAMT) nanoprobe with signal amplification was developed for the quantitative detection of PD-L1. The SAMT probe was assembled by photo-cleavable mass tags, PD-L1 aptamer, and amphiphilic polymer. After binding with PD-L1 on the platelet, the probe can release mass tags with UV light exposure. The amount of the mass tag, representing that of PD-L1, was subsequently determined by mass spectrometry. The assay sensitivity can be greatly improved by up to four orders of magnitude, achieving a detection limit of 10 fM. This assay was subsequently applied to cancer cells and platelet samples from non-small cell lung cancer (NSCLC) patients. The patients with higher tumor stages, higher degrees of lymph node invasion, and better ICB response had higher PD-L1 levels on platelets. Further investigation revealed that PD-L1 on the platelets was transported from cancer cells, providing evidence for the existence of TEPs. SignificanceThe SAMT probe can amplify the signal of the target molecule into that of multiple mass tags, achieving ultrasensitive ICB protein quantitative detection in platelets. Moreover, the employed SAMT assay not only revealed PD-L1 transport from cancer cells to platelets but also confirmed the presence of TEPs.

Evaluation of Multiplex loop-mediated isothermal amplification assay for the detection of Mycobacterium tuberculosis complex from clinically suspected cases of pulmonary tuberculosis

Tuberculosis (TB) is the second leading cause of death from a single infectious agent worldwide. Bangladesh ranks 7th among the 30 high TB burdened countries in the world. Accurate detection of Mycobacterium tuberculosis complex (MTBC) is challenging for developing countries as most of the resource poor settings are not suitable to perform molecular techniques.The purpose of the study was to compare the multiplex TB-LAMP assay with MTB/NTM qPCR, culture, Z-N staining, and fluorescence microscopy in order to assess the effectiveness of the LAMP assay for detecting cases of pulmonary tuberculosis.This research work was done from March 2022 to February 2023. Fulfilling the inclusion criteria 130 sputum samples were collected. TB-LAMP assay, qPCR, culture in L-J media, Z-N staining and fluorescence microscopy were performed.Out of 130 samples qPCR detected MTBC in 56.92% cases and TB-LAMP detected 53.85%. MTBC was detected by culture 46.15%, by Fluorescence microscopy 40.77% and Z-N staining 36.92%. TB-LAMP detected 16.93% more cases than Z-N staining and 13.08% more cases than fluorescence microscopy. The sensitivity, specificity, positive, and negative predictive values of multiplex-LAMP assay were 95%, 81.4%, 81.4% and 95% respectively considering culture as a gold-standard. MTBC negative culture samples (18.57%) showed positivity by LAMP assay as well as by qPCR. This study detected 7.69% non-tuberculous mycobacteria (NTM) by qPCR. All NTM positive samples were negative by TB-LAMP.TB-LAMP is an easy to perform, cost-effective, reliable assay with high sensitivity and specificity. World Health Organization recommended TB-LAMP as a rapid molecular test for rapid detection of tuberculosis and as replacement of microscopy in resource poor settings/hard to reach areas. Bangladesh being a high TB burden country it is essential to implement TB-LAMP to achieve End TB Strategy by 2035.

Lychee-shaped magneto-gold nanohybrid with highly retained magnetic-plasmonic activity for ultrasensitive and multiplex immunochromatographic assay

Utilizing nanomaterials with magnetic and plasmonic properties can significantly enhance the sensitivity of immunochromatographic assay (ICA) through magnetic enrichment, and signal amplification. Herein, we successfully synthesized a lychee-shaped magneto-gold nanohybrid (designated as MSiAuTA). This nanohybrid comprises a magnetic nanoparticle core, a flesh-fiber-like mesoporous silica template hosting numerous gold nanoparticles (AuNPs), and a lychee-exocarp-like tannic acid layer. The resultant MSiAuTA exhibits rapid magnetic response, strong plasmonic activity, and excellent biocompatibility. We further developed a multiplex ICA (mICA) platform utilizing MSiAuTA as labels for target enrichment and colorimetric signal amplification. This platform enables simultaneous and quantitative detection of Escherichia coli O157:H7 and Salmonella typhimurium. The MSiAuTA-mICA supports portable strip reader and smartphone readout and demonstrates exceptional sensitivity, achieving detection limits of 9.8 × 102 CFU/mL for E. coli O157:H7 and 4.9 × 102 CFU/mL for S. typhimurium. Additionally, we validated the accuracy, robustness, and practicality of MSiAuTA-mICA in detecting both spiked and naturally contaminated samples, including water, lettuce, chicken, apple juice, and milk. In conclusion, the lychee-shaped MSiAuTA nanohybrid shows significant promise as a versatile label for enhancing ICA’s detection capabilities in complex sample matrices.

Sensitivity of static and dynamic assays in the confirmation of Cushing's syndrome

Sensitivity of static and dynamic assays in the confirmation of Cushing's syndrome

Construction of a lung cancer 3D culture model based on alginate/gelatin micro-beads for drug evaluation.

Lung cancer is one of the most common malignant tumors worldwide. Despite advances in lung cancer treatment, patients still face challenges related to drug resistance and recurrence. Current methods for evaluating anti-cancer drug activity are insufficient, as they rely on two-dimensional (2D) cell culture and animal models. Therefore, the development of an in vitro drug evaluation model capable of predicting individual sensitivity to anti-cancer drugs would greatly enhance the success rate of drug treatments for lung cancer patients. The purpose of this research is to utilise conditional reprogramming technology to cultivate patient-derived lung cancer cells and to construct an in vitro 3D culture model using sodium alginate (SA) and gelatin. The aim is to study the biological characteristics of cells in the 3D culture model and to further investigate the sensitivity of anti-cancer drugs based on the alginate-gelatin 3D culture model. This approach provides new means and insights for personalized precision anti-cancer therapy and the development of new anti-cancer drugs. Conditional reprogramming technology was used to generate conditionally reprogrammed lung adenocarcinoma cells (CRLCs). Alginate-gelatin hydrogel micro-beads were created to explore their potential use in the assessment of anti-cancer drugs. Cell proliferation was also examined using the MTS assay method. Live/dead staining was performed to estimate cell distribution and viability using calcein acetoxymethyl ester/propidium iodide (calcein-AM/PI) double staining. Protein expression was assessed by Western blot. The cells grown in the three-dimensional (3D) culture were in a state of continuous proliferation, and there was an obvious phenomenon of cell mass growth. The drug sensitivity assay results demonstrated that compared with the 2D-grown cells, the CRLCs grown in the alginate-gelatin hydrogel micro-beads exhibited more resistance to anti-cancer drugs. The results also showed that the 3D-cultured CRLCs showed greater protein expression levels of stem cell hallmarks, such as Nanog Homeobox (NANOG), SRY-Box Transcription Factor 2 (SOX-2), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1), than the 2D-grown cells. These findings suggest that the 3D hydrogel cell culture models more closely mimicked the in vivo biological and clinical behavior of cells, and demonstrated higher innate resistance to anti-cancer drugs than the 2D cell culture models, and thus could serve as valuable tools for diagnosis, drug screening, and personalized medicine.

Highly sensitive β-amyloid1–42 oligomer aptamer-based assay via dual cyclic amplifications of three-way catalytic hairpin assembly and palindrome polymerase reaction

β-amyloid1–42 oligomer (AβO1–42), a neurotoxic protein, is considered the key factor in the progression of Alzheimer's disease (AD). And, high sensitivity detection of AβO1–42 is of considerable importance for the early diagnosis and deterioration prevention of AD. We present here an ultrasensitive fluorescent AβO1–42 detection approach based on aptamer recognition probes and a new dual cyclic signal amplification strategy by combining three-way catalytic hairpin assembly (3W-CHA) with palindromic sequence-based polymerase amplification (PBPA). Once the aptamers bind to target AβO1–42 molecules, ssDNA sequences are released to initiate 3W-CHA between three hairpins to unfold the signal hairpins and to form Y-shaped DNA structures (Y-DNAs). Subsequently, the assistance ssDNAs bind the Y-DNAs to expose the palindromic sequences to yield Y-DNA dimers, which lead to cyclic PBPA reaction with presence of polymerase to further unfold more signal hairpins for yielding considerably magnified fluorescence recovery, enabling sensitive assay of AβO1–42 down to 2.4 pM within a dynamic range of 5 pM∼50 nM. Furthermore, this strategy has been validated for interrogation of low AβO1–42 levels in artificial cerebrospinal fluid (CSF), suggesting its promising potential for application in early diagnosis and prevention of AD.

Type 2 cytokine-JAK1 signaling is involved in the development of dry-skin induced mechanical alloknesis

BackgroundMechanical alloknesis (m-alloknesis) is itch hypersensitivity induced by normally innocuous stimuli. It is sometimes observed in dry skin based itch-related diseases such as atopic dermatitis (AD), and often triggers the vicious itch-scratch cycle. The acetone-ether and water (AEW) mouse model mimics dry skin induced m-alloknesis, yet its underlying mechanism remains unclear. Janus kinase (JAK) inhibitors are used to treat AD, but their effects on m-alloknesis are not fully known. ObjectiveTo reveal the effects of various oral JAK inhibitors on m-alloknesis and their action points, using AEW model. MethodsAEW model was prepared by treatment with a mixture of acetone-ether, and they were orally administrated a JAK1/2 inhibitor baricitinib, a selective JAK1 inhibitor abrocitinib, or a JAK2 selective inhibitor AZ960, and evaluated m-alloknesis score as the total number of scratching responses in 30 mechanical stimulations. To further elucidate the mechanism of action, IL-4, IL-13 or thymic stromal lymphopoietin (TSLP) or their neutralizing antibodies were also applied to mice. In addition, the levels of these cytokines in mouse skin were measured using multiple immunoassays. ResultsAll of JAK inhibitors effectively reduced m-alloknesis, with abrocitinib demonstrating the most significant inhibition. The neutralizing antibodies against IL-4, IL-13, and TSLP inhibited m-alloknesis in AEW mice. Intradermal administration of IL-4, IL-13, or TSLP induced m-alloknesis, and abrocitinib effectively mitigated each cytokine-induced response. Highly sensitive assays detected IL-4, IL-13, IL-31 and TSLP in AEW-treated skin, with TSLP levels significantly increased. ConclusionType 2 cytokine-JAK1 signaling is involved in the development of m-alloknesis in dry skin.

An Investigation of Pharmacokinetic Interaction of Vericiguat with Apigenin based on a Newly Developed Ultra-performance Liquid Chromatography-tandem Mass Spectrometry Assay.

Vericiguat, as a new stimulator of soluble guanylate cyclase (s- GC), was recently approved as a first-in-class treatment for reducing risks in patients with ejection fraction less than 45 percent and heart failure (HF) in the USA. The main aim of the present experiment was to establish an acceptable, sensitive assay based on ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) for quantitatively analyzing the plasma concentration levels of vericiguat in rats, and to further evaluate the effect of apigenin on the metabolism of vericiguat in vivo. In sample processes, acetonitrile was finally chosen for quickly precipitating protein. The levels of vericiguat in plasma were analyzed by a Xevo TQ-S triple quadrupole tandem mass spectrometry (Milford, MA, USA) in a positive ion mode. The scope of the calibration standard for vericiguat ranged from 0.5 to 1000 ng/mL, where a great linearity was acceptable. The lower limit of quantification (also called LLOQ) of vericiguat presented the sensitivity of this assay was evaluated as low as 0.5 ng/mL. Additionally, selectivity, accuracy and precision, extraction recovery, matrix effect, and stability were all verified. Subsequently, this approach also supported to assess the plasmatic concentrations of vericiguat from an interaction survey on herb-- drug, in which oral administration of apigenin (20 mg/kg) obviously increased the plasmatic levels of vericiguat and altered the pharmacokinetics of vericiguat in rats. These results would help us to further understand the pharmacokinetic properties of vericiguat when co-administration with apigenin, and to avoid unexpected clinical risks in the future.

An integrated In-Silico-In-Chemico-In-Vitro (iSiCiV) Approach to identify biomarkers to predict the skin sensitisation potential of phytochemicals

BackgroundSkin sensitisation is one of the important regulatory endpoints used to determine whether a given test chemical can elicit an allergic response in susceptible individuals. The in-vitro approaches for predicting skin sensitisation potential for chemicals have been well-developed and widely accepted. Continuous efforts are being made to identify novel solutions to overcome the limitations of the existing approaches. PurposeThe herbal preparations used in dermal therapeutics are frequently found to elicit skin sensitisation, requiring caution. The herbal preparations tend to contain more than one phytochemical. The expected active ingredient may not possess skin sensitisation. However, the other phytochemical traces could pose synergistic and potentiating effects towards enhanced/suppressed skin sensitisation. In this regard, isolation, purification, yield, and in-vitro / in-vivo evaluation of skin sensitisation for all the traces would be time, cost, and animal-consuming. Hence, the regulatory agencies appreciate using computational tools to substantiate in-vitro methods. The regulators have also endorsed using in-silico approaches like quantitative structure-activity relationships (QSAR) and read-across. These approaches are limited to structure-based predictions and in combination with system-level approaches, which comprehensively facilitate a better understanding of complex biological systems. Study designIn the present study, 603 phytochemicals were screened in-silico using OECD QSAR Toolbox. The limitations of the QSAR Toolbox in predicting skin sensitisers were identified. MethodThe systems biology-based approach to complement the QSAR-based prediction was developed, and 41 biomarkers were identified as a determinant of skin sensitisation. The molecules attributed to these biomarkers were predicted as skin sensitisers. ResultsAs a result, 31 phytochemicals were predicted as skin sensitisers. Acrolein, eugenol, formaldehyde, and glycerol were selected for experimental validation of the in-silico predictions using direct peptide reactivity assay (DPRA), KeratinoSens™, and h-CLAT assays representing each key event in skin sensitisation. Further, identified biomarkers were validated in THP-1 and KeratinoSens cell lines using qRT-PCR. Among all genes, IL-8 and CCL4 were upregulated by 6.4 and 5.6 folds, respectively, in THP-1, while AKR1C2 and AKR1C3 were upregulated by 7.8 and 11.1 folds, respectively, in KeratinoSens. IL-8 level was analysed by ELISA and found to be in line with qRT-PCR results. ConclusionThe study proposes a possible strategy based on the “iSiCiV” approach to enhance the prediction of the dermal sensitisation potential of a test compound. The approach offers a robust platform and highlights an opportunity to shift the paradigm of skin sensitisation assay with a better understanding of toxicology mechanisms.

Development of a graphene field effect transistor-based immersible biosensor for immunodetection of the birch pollen allergen Bet v 1 in air samples

Pollen traps, the current gold standard to determine pollen load and thereby the allergy season, are not sufficient to determine the allergenic risk. Therefore, the establishment of highly sensitive assays for allergen measurement is of highest interest. Herein, a graphene field-effect transistor (GFET) was constructed on an interdigitated electrodes chip to develop an immersible biosensor, which was used to detect the major birch pollen allergen Bet v 1. Graphene was wet-transferred on interdigitated electrodes that contain a reference electrode used as a liquid gate in the GFET. Using a standard ELISA protocol, two different anti-Bet v 1 antibodies were chosen and immobilized on graphene for the specific capture of the target allergen. The sensitivity of the GFET biosensor was evaluated using a standard Ag/AgCl liquid gate electrode and a reference electrode when the chip was immersed in Bet v 1-containing solutions. The results showed a higher performance and sensitivity for Bet v 1 detection compared to a mediator release method, one of the most sensitive assays for allergen detection. Compared with conventional methods of allergen detection, these immersible biosensors significantly improved the speed and level of detection providing the foundation of a point-of-need platform for in-field application. Furthermore, the proposed technique provides both a new biosensor for allergen detection and a strategy for designing low-cost integrated biosensors.

Diagnosis of pleural tuberculosis in the era of thoracoscopic surgery

Background Before thoracoscopic surgery, diagnosing tuberculous (TB) pleurisy was a medical challenge. Thoracoscopy is the most accurate but expensive method for TB pleurisy diagnosis. TB is common in low-income countries, where financial limitations prevent the use of thoracoscopy, motivating the search for a cheaper alternative. Patients and methods A prospective study was done from January 2019 to January 2023 to evaluate diagnostic methods for patients with exudative pleural effusions (PE) of unknown etiologies. The demographic, radiological, procedural, and histological data of exudative PE patients were analyzed. All patients were examined for adenosine deaminase (ADA) and lymphocyte/neutrophil ratio in pleural fluid. Ultrasound-guided Abrams needle biopsy and video-assisted thoracoscopic surgery pleural biopsies were obtained, and histopathological results were assessed. Results Of 250 patients with PE, 161 (64%) had TB PE, 72 (28.8%) had malignant PE, and 17 (6.8%) had idiopathic PE. Sensitivity of ADA (≥40 U/l) was 88%, lymphocyte/neutrophil ratio (≥0.75) was 86.1%, and their overall sensitivity was 91%. They had 93.2, 86.3, and 100% specificity, respectively. For ultrasound-guided Abrams needle biopsy, the sensitivity of histopathology, culture, and combined histopathology/culture was 66, 46.5, and 78.4%, respectively. All were 100% specific. For thoracoscopic biopsy, the sensitivity of histopathology, culture, and combined histopathology/culture was 100, 86.6, and 100%, respectively. All were 100% specific. The assay sensitivity of pleural fluid and tissue Xpert Mycobacterium tuberculosis/rifampin resistance was 12.5 and 49.7%, respectively. Both were 100% specific. Combining ADA more than or equal to 40 U/l, lymphocyte/neutrophil ratio more than or equal to 0.75, and an ultrasound-guided Abrams needle biopsy yielded 92.4% sensitivity and 100% specificity. Conclusion Combining pleural fluid ADA more than or equal to 40 U/l, lymphocyte/neutrophil ratio more than or equal to 0.75, and ultrasound-guided Abrams needle biopsy can accurately detect TB PE in high-TB populations. It may be an affordable alternative to thoracoscopy in countries with limited resources.

Olink proteomics and lipidomics analysis of serum from patients infected with non-tuberculous mycobacteria and Mycobacterium tuberculosis.

Non-tuberculous mycobacterial (NTM) and Mycobacterium tuberculosis (MTB) infections are difficult to diagnose and treat, significantly burdening global health. The host immune status is generally believed to be associated with the onset and progression of NTM and MTB infections, but its specific impact remains unclear. In the present study, proteomics and lipidomics analysis of serum from normal controls (n = 26) and patients with MTB (n = 26), rapidly growing NTM (RGM, n = 15), and slowly growing NTM (SGM, n = 21) were conducted using the Olink technique based on a highly sensitive and specific neighborhood extension assay and the lipidomics technique. IFN-γ, CXCL9, CXCL10, CXCL11, and CXCL13, etc. were simultaneously upregulated in MTB, RGM, and SGM, while lipids FAHFA 22:3, FAHFA 26:4, FAHFA 24:4, FAHFA 20:5, FAHFA 18:2 simultaneously downregulated. IL8, CCL3, CXCL5, and MCP-2, etc. were simultaneously upregulated in RGM and SGM compared to MTB, as well as PCs, LPCs, PEs, and LPEs. Compared with RGM, IL7, CD27, CCL17, CXCL12, and LPC 28:7-SN2 were downregulated in SGM. Pathway analyses revealed that tuberculosis, sphingolipid signaling pathway, and adipocytokine signaling pathway were regulated at the protein level and metabolite level. Diagnostic panels comprising immune-associated proteins and lipids greatly enhance diagnostic specificity and sensitivity. This integrated multi-omics analysis provides a more comprehensive understanding of the molecular landscape of NTM and MTB, which may provide molecular targets for specialized therapies.

Portable detection of Salmonella in food of animal origin via Cas12a-RAA combined with an LFS/PGM dual-signaling readout biosensor.

Ahighly specific and sensitive rapid two-signal assay was developedfor the detection of Salmonella typhimurium in foods of animal origin. TheinvA gene of Salmonella was usedas the biorecognition element and recombinase-assisted amplification (RAA) technology for signal amplification. By utilizing the specific recognition and efficient trans-cleavage activity of CRISPR/Cas12a, point-of-care testing (POCT) for S. typhimurium was achieved via lateral flow strips (LFS) and personal glucometer (PGM) biosensors as dual signal readout systems, with sensitivities of 33CFU/mL and 20CFU/mL, respectively. Users can select the appropriate test system on the basis of specific application requirements: LFSs are ideal for rapid onsite screening, whereas glucometer biosensors offer precise quantitative determination. This approach simplifies the use of large instruments and overcomes site constraints, demonstrating good accuracy and applicability in animal-derived samples, with significant potential for the detection of other pathogens and for use in restricted environments.

Optimizing the detection of N-nitrosamine mutagenicity in the Ames test

Accurately determining the mutagenicity of small-molecule N-nitrosamine drug impurities and nitrosamine drug substance-related impurities (NDSRIs) is critical to identifying mutagenic and cancer hazards. In the current study we have evaluated several approaches for enhancing assay sensitivity for evaluating the mutagenicity of N-nitrosamines in the bacterial reverse mutagenicity (Ames) test. Preincubation assays were conducted using five activation conditions: no exogenous metabolic activation and metabolic activation mixes employing both 10% and 30% liver S9 from hamsters and rats pretreated with inducers of enzymatic activity. In addition, preincubations were conducted for both 60 min and 30 min. These test variables were evaluated by testing 12 small-molecule N-nitrosamines and 17 NDSRIs for mutagenicity in Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537, and Escherichia coli strain WP2 uvrA (pKM101). Eighteen of the 29 N-nitrosamine test substances tested positive under one or more of the testing conditions and all 18 positives could be detected by using tester strains TA1535 and WP2 uvrA (pKM101), preincubations of 30 min, and S9 mixes containing 30% hamster liver S9. In general, the conditions under which NDSRIs were mutagenic were similar to those found for small-molecule N-nitrosamines.

Establishment of a Sensitive and Reliable Droplet Digital PCR Assay for the Detection of Bursaphelenchus xylophilus.

Pine wilt disease (PWD), which poses a significant risk to pine plantations across the globe, is caused by the pathogenic agent Bursaphelenchus xylophilus, also referred to as the pine wood nematode (PWN). A droplet digital PCR (ddPCR) assay was developed for the quick identification of the PWN in order to improve detection sensitivity. The research findings indicate that the ddPCR assay demonstrated significantly higher analysis sensitivity and detection sensitivity in comparison to traditional quantitative PCR (qPCR). However, it had a more limited dynamic range. High specificity was shown by both the ddPCR and qPCR techniques in the diagnosis of the PWN. Assessments of reproducibility revealed that ddPCR had lower coefficients of variation at every template concentration. Inhibition tests showed that ddPCR was less susceptible to inhibitors. There was a strong linear association between standard template measurements obtained using ddPCR and qPCR (Pearson correlation = 0.9317; p < 0.001). Likewise, there was strong agreement (Pearson correlation = 0.9348; p < 0.001) between ddPCR and qPCR measurements in the evaluation of pine wood samples. Additionally, wood samples from symptomatic (100% versus 86.67%) and asymptomatic (31.43% versus 2.9%) pine trees were diagnosed with greater detection rates using ddPCR. This study's conclusions highlight the advantages of the ddPCR assay over qPCR for the quantitative detection of the PWN. This method has a lot of potential for ecological research on PWD and use in quarantines.