Sensitive Assay Research Articles

Trace amine-associated receptor 1 (TAAR1) agonism for psychosis: a living systematic review and meta-analysis of human and non-human data.

Trace amine-associated receptor 1 (TAAR1) agonism shows promise for treating psychosis, prompting us to synthesise data from human and non-human studies. We co-produced a living systematic review of controlled studies examining TAAR1 agonists in individuals (with or without psychosis/schizophrenia) and relevant animal models. Two independent reviewers identified studies in multiple electronic databases (until 17.11.2023), extracted data, and assessed risk of bias. Primary outcomes were standardised mean differences (SMD) for overall symptoms in human studies and hyperlocomotion in animal models. We also examined adverse events and neurotransmitter signalling. We synthesised data with random-effects meta-analyses. Nine randomised trials provided data for two TAAR1 agonists (ulotaront and ralmitaront), and 15 animal studies for 10 TAAR1 agonists. Ulotaront and ralmitaront demonstrated few differences compared to placebo in improving overall symptoms in adults with acute schizophrenia (N=4 studies, n=1291 participants; SMD=0.15, 95%CI: -0.05, 0.34), and ralmitaront was less efficacious than risperidone (N=1, n=156, SMD=-0.53, 95%CI: -0.86, -0.20). Large placebo response was observed in ulotaront phase-III trials. Limited evidence suggested a relatively benign side-effect profile for TAAR1 agonists, although nausea and sedation were common after a single dose of ulotaront. In animal studies, TAAR1 agonists improved hyperlocomotion compared to control (N=13 studies, k=41 experiments, SMD=1.01, 95%CI: 0.74, 1.27), but seemed less efficacious compared to dopamine D 2 receptor antagonists (N=4, k=7, SMD=-0.62, 95%CI: -1.32, 0.08). Limited human and animal data indicated that TAAR1 agonists may regulate presynaptic dopaminergic signalling. TAAR1 agonists may be less efficacious than dopamine D 2 receptor antagonists already licensed for schizophrenia. The results are preliminary due to the limited number of drugs examined, lack of longer-term data, publication bias, and assay sensitivity concerns in trials associated with large placebo response. Considering their unique mechanism of action, relatively benign side-effect profile and ongoing drug development, further research is warranted. PROSPERO-ID: CRD42023451628.

Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices

BackgroundMelioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence.MethodsAn extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls.ResultsA novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil.ConclusionsThe findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

Severe bradycardia at acute medical take - Think about myxoedema coma.

We report two cases of myxoedema coma that presented to the acute medical take with severe bradycardia. These patients were initially misdiagnosed as bradyarrhythmia of primary cardiac origin. They were then diverted to the cardiology service at another district general hospital (DGH) for admissions. Both cases were subsequently diagnosed with myxoedema coma having screened thyroid function tests on arrival at the cardiology unit. Despite being treated for myxoedema coma, both patients unfortunately succumbed to the disease and later died in the hospital. These cases highlight that clinical suspicion and recognition of myxoedema coma remain significant challenges in a developed world despite readily available and highly sensitive thyroid hormone assays.

Development and validation of ELISA method for quantification of Q-1802 in serum and its application to pharmacokinetic study in ICR Mouse

Q-1802 is a humanized bispecific antibody targeting programmed death-ligand 1 (PD-L1) and Claudin 18.2 (CLDN18.2). It can bind to CLDN18.2 and mediate antibody-dependent cell-mediated cytotoxicity against tumor cells. The Fc segment of the antibody recognizing PD-L1 blocks PD-1 signaling and activates innate immunity and adaptive immunity. In this study, we report the development, validation, and application of sensitive and high-throughput enzyme-linked immunosorbent assays (ELISA) to measure the concentrations of Q-1802 in ICR mouse serum. The assay is sensitive, with a lower limit of quantification of 50 ng/mL, has a broad dynamic range of 50–3200 ng/mL, and exhibits excellent precision and accuracy. These assays were successfully applied to in vitro serum stability and pharmacokinetic (PK) studies. In conclusion, we have developed and validated a highly sensitive and selective method for measuring Q-1802 in ICR mouse serum. The development and validation steps of assays met the required criteria for validation, which suggested that these can be applied to quantify Q-1802, as well as in PK studies.

Xpert MTB/RIF Ultra versus mycobacterial growth indicator tube liquid culture for detection of Mycobacterium tuberculosis in symptomatic adults: a diagnostic accuracy study

Xpert MTB/RIF Ultra (Ultra) is an automated molecular test for the detection of Mycobacterium tuberculosis in sputum. We compared the sensitivity of Ultra to that of mycobacterial growth indicator tube (MGIT) liquid culture, considered the most sensitive assay in routine clinical use. In this prospective, multicentre, cross-sectional diagnostic accuracy study, we used a non-inferiority design to assess whether the sensitivity of a single Ultra test was non-inferior to that of a single liquid culture for detection of Mtuberculosis in sputum. We enrolled adults (age ≥18 years) with pulmonary tuberculosis symptoms in 11 countries and each adult provided three sputum specimens with a minimum volume of 2 mL over 2 days. Ultra was done directly on sputum 1, and Ultra and MGIT liquid culture were done on resuspended pellet from sputum 2. Results of MGIT and solid media cultures done on sputum 3 were considered the reference standard. The pre-defined non-inferiority margin was 5·0%. Between Feb 18, 2016, and Dec 4, 2019, we enrolled 2906 participants. 2600 (89%) participants were analysed, including 639 (25%) of 2600 who were positive for tuberculosis by the reference standard. Of the 2357 included in the non-inferiority analysis, 877 (37%) were HIV-positive and 984 (42%) were female. Sensitivity of Ultra performed directly on sputum 1 was non-inferior to that of sputum 2 MGIT culture (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; 95% CI -2·8 to 1·1). Sensitivity of Ultra performed on sputum 2 pellet was also non-inferior to that of sputum 2 MGIT (MGIT 91·1% vs Ultra 91·9%; difference -0·8 percentage points; -2·7 to 1·0). For the detection of M tuberculosis in sputum from adults with respiratory symptoms, there was no difference in sensitivity of a single Ultra test to that of a single MGIT culture. Highly sensitive, rapid molecular approaches for M tuberculosis detection, combined with advances in genotypic methods for drug resistance detection, have potential to replace culture. US National Institute of Allergy and Infectious Diseases.

Accelerated Development of a COVID-19 Lateral Flow Test in an Academic Setting: Lessons Learned.

The COVID-19 pandemic further demonstrated the need for usable, reliable, and cost-effective point-of-care diagnostics that can be broadly deployed, ideally for self-testing at home. Antigen tests using more-detectable reporter labels (usually at the cost of reader complexity) achieve better diagnostic sensitivity, supporting the value of higher-analytical-sensitivity reporter technologies in lateral flow.We developed a new approach to simple, inexpensive lateral flow assays (LFAs) of great sensitivity, based on the glow stick peroxyoxalate chemistry widely used in emergency settings and in children's toys. At the peak of the COVID-19 pandemic, we had the opportunity to participate in the pandemic-driven NIH Rapid Acceleration of Diagnostics (RADx) initiative aiming to develop a deployable lateral flow diagnostic for SARS-CoV-2 nucleoprotein based on our novel glow stick-inspired light-emitting reporter technology. During this project, we screened more than 250 antibody pairs for analytical sensitivity and specificity directly in LFA format, using recombinant nucleoprotein and then gamma-irradiated virions spiked into negative nasal swab extracts. Membranes and other LFA materials and swabs and extraction reagent components also were screened and selected. Optimization of conjugate preparation and spraying as well as pretreatment/conditioning of the sample pad led to the final optimized LFA strip. Technology development also included optimization of excitation liquid enclosed in disposable droppers, design of a custom cartridge and smartphone-based reader, and app development, even a prototype reader usable with any mobile phone. Excellent preclinical performance was first demonstrated with contrived samples and then with leftover clinical samples. Moving beyond traditional academic focus areas, we were able to establish a quality management system (QMS), produce large numbers of customized LFA cassettes by contract injection molding, build in-house facilities to assemble and store thousands of complete tests for verification and validation and usability studies, and source kitting/packaging services and quality standard reagents and build partnerships for clinical translation, regulatory guidance, scale up, and market deployment. We were not able to bring this early stage technology to the point of commercialization within the limited time and resources available, but we did achieve strong proof-of-concept and advance translational aspects of the platform including initial high-performance LFAs, reading by the iPhone app using only a $2 plastic dark box with no lens, and convenient, usable excitation liquid packaging in droppers manufacturable in very large numbers.In this Account, we aim to provide a concise overview of our 18-month sprint toward the practical development of a deployable antigen lateral flow assay under pandemic conditions and the challenges and successes experienced by our team. We highlight what it takes to coach a technically savvy but commercially inexperienced academic team through the accelerated translation of an early stage technology into a useful product. Finally, we provide a guided tutorial and workflow to empower others interested in the rapid development of translatable LFAs.

KP177R-based visual assay integrating RPA and CRISPR/Cas12a for the detection of African swine fever virus.

Early detection of the virus in the environment or in infected pigs is a critical step to stop African swine fever virus (ASFV) transmission. The p22 protein encoded by ASFV KP177R gene has been shown to have no effect on viral replication and virulence and can serve as a molecular marker for distinguishing field virus strains from future candidate KP177R deletion vaccine strains. This study established an ASFV detection assay specific for the highly conserved ASFV KP177R gene based on recombinase polymerase amplification (RPA) and the CRISPR/Cas12 reaction system. The KP177R gene served as the initial template for the RPA reaction to generate amplicons, which were recognized by guide RNA to activate the trans-cleavage activity of Cas12a protein, thereby leading to non-specific cleavage of single-stranded DNA as well as corresponding color reaction. The viral detection in this assay could be determined by visualizing the results of fluorescence or lateral flow dipstick (LFD) biotin blotting for color development, and was respectively referred to as fluorescein-labeled RPA-CRISPR/Cas12a and biotin-labeled LFD RPA-CRISPR/Cas12a. The clinical samples were simultaneously subjected to the aforementioned assay, while real-time quantitative PCR (RT-qPCR) was employed as a control for determining the diagnostic concordance rate between both assays. The results showed that fluorescein- and biotin-labeled LFD KP177R RPA-CRISPR/Cas12a assays specifically detected ASFV, did not cross-react with other swine pathogens including PCV2, PEDV, PDCoV, and PRV. The detection assay established in this study had a limit of detection (LOD) of 6.8 copies/μL, and both assays were completed in 30min. The KP177R RPA-CRISPR/Cas12a assay demonstrated a diagnostic coincidence rate of 100% and a kappa value of 1.000 (p < 0.001), with six out of ten clinical samples testing positive for ASFV using both KP177R RPA-CRISPR/Cas12a and RT-qPCR, while four samples tested negative in both assays. The rapid, sensitive and visual detection assay for ASFV developed in this study is suitable for field application in swine farms, particularly for future differentiation of field virus strains from candidate KP177R gene-deleted ASFV vaccines, which may be a valuable screening tool for ASF eradication.

Interlaboratory validation and one-year retrospective review of hemp and marijuana decision-point assays

After the passage of the Agriculture Improvement Act of 2018 (Farm Bill) that defined hemp as Cannabis sativa L. if it contained ≤0.3 % Δ9-THC on a dry weight basis, forensic laboratories were faced with the challenge of distinguishing marijuana from hemp in seized drug samples. This study reports the results of an interlaboratory validation of a previously developed qualitative decision-point assay for the differentiation of illegal marijuana from legal hemp, utilizing gas chromatography-mass spectrometry (GC–MS) with a 1 % Δ9-THC threshold. The method was validated in terms of selectivity, linearity, carryover, precision, accuracy, extract stability, dilution integrity, and measurement uncertainty. Other elements such as decarboxylation rate and potential for CBD interference were also evaluated. Specificity and positive predictive value were both 100 % using known cannabis reference materials. A total of 8 false negative results were observed among the 280 analyses, resulting in an overall assay sensitivity of 94 % and a negative predictive value of 95 %. The small number of false negative results near the 1 % threshold were attributed to decarboxylation inefficiency in some laboratories. While qualitative in nature, the measurement uncertainty of the assay at the 0.3 % Δ9-THC cutoff (legal threshold) using a 95.45 % confidence interval (k = 2) ranged from 12.2 % to 21.8 % among laboratories (equivalent to 0.3 % ± 0.04 % to 0.07 % Δ9-THC by weight). A one-year retrospective study across three agencies and fifteen laboratories was undertaken. Among the three agencies participating in the original study, 93–96 % of all suspected cannabis seizures yielded results above the administrative threshold of 1 % Δ9-THC (n = 3,288). The interlaboratory method development highlighted differences in performance between sites, ultimately improving the overall robustness of the method. Observations highlight the need for site-specific validation and vigilance. While multi-agency collaborations and interlaboratory validations are valuable, they do not replace the necessity for full, independent, and rigorous validation.

Diagnostic Accuracy of Blind Bronchial Sample Testing by BioFire Pneumonia plus Panel in Pediatric Intensive Care Unit Patients and Its Impact in Early Adaptation of Antimicrobial Therapy: A Prospective Observational Study.

Community-acquired and nosocomial lower-respiratory-tract infections in critically ill pediatric patients require early appropriate antibiotic therapy to optimize outcomes. Using blind bronchial samples, we assessed the diagnostic performance of the rapid-multiplex polymerase chain reaction (PCR) assay BioFire Pneumonia plus Panel vs. reference standard culturing with antimicrobial susceptibility testing. For this prospective observational study in a single pediatric intensive care unit, we included consecutive patients younger than 18 years admitted for suspected community-, hospital- or ventilator-associated pneumonia in 2021-2022. Sensitivity, specificity, positive predictive value and negative predictive value of the multiplex PCR assay were determined. The kappa coefficient was computed to assess agreement, and univariate analyses were done to identify factors associated with discrepancies between the 2 diagnostic methods. Of the 36 included patients (median age, 1.4 years; interquartile range, 0.2-9.2), 41.7%, 27.8%, and 30.5% had community-, hospital- and ventilator-associated pneumonia, respectively. The overall κ was 0.74, indicating good agreement. Overall, the sensitivity of the multiplex PCR assay was 92% (95% CI: 77%-98%) and specificity 95% (95% CI: 92%-97%), with variations across microorganisms. The median time from sample collection to antimicrobial susceptibility test results was 3.9 (2.5-15) hours with the multiplex PCR assay and 60.5 (47.6-72.2) hours with the reference technique. The BioFire Pneumonia plus Panel used to test blind bronchial samples had satisfactory diagnostic performance in critically ill pediatric patients. The rapid results provided by this test may improve the appropriateness of antimicrobial therapy and help minimize the use of antibiotics.

An in vitro assay for toxicity testing of Clostridium perfringens type C β-toxin.

Veterinary vaccines against Clostridium perfringens type C need to be tested for absence of toxicity, as mandated by pharmacopoeias worldwide. This toxicity testing is required at multiple manufacturing steps and relies on outdated mouse tests that involve severe animal suffering. Clostridium perfringens type C produces several toxins of which the β-toxin is the primary component responsible for causing disease. Here, we describe the successful development of a new cell-based in vitro assay that can address the specific toxicity of the β-toxin. Development of the cell-based assay followed the principle of in vitro testing developed for Cl. septicum vaccines, which is based on Vero cells. We screened four cell lines and selected the THP-1 cell line, which was shown to be the most specific and sensitive for β-toxin activity, in combination with a commercially available method to determine cell viability (MTS assay) as a readout. The current animal test is estimated to detect 100 - 1000-fold dilutions of the Cl. perfringens type C non-inactivated antigen. When tested with an active Cl. perfringens type C antigen preparation, derived from a commercial vaccine manufacturing process, our THP-1 cell-based assay was able to detect toxin activity from undiluted to over 10000-fold dilution, showing a linear range between approximately 1000- and 10000-fold dilutions. Assay specificity for the β-toxin was confirmed with neutralizing antibodies and lack of reaction to Cl. perfringens culture medium. In addition, assay parameters demonstrated good repeatability. Here, we have shown proof of concept for a THP-1 cell-based assay for toxicity testing of veterinary Cl. perfringens type C vaccines that is suitable for all vaccine production steps. This result represents a significant step towards the replacement of animal-based toxicity testing of this veterinary clostridial antigen. As a next step, assessment of the assay's sensitivity and repeatability and validation of the method will have to be performed in a commercial manufacturing context in order to formally implement the assay in vaccine quality control.

Development of a multiplex droplet digital PCR method for detection and monitoring of Mycobacterium tuberculosis and drug-resistant tuberculosis

BackgroundThe prevalence of multidrug-resistant tuberculosis (MDR-TB) among Korean tuberculosis patients is about 4.1%, which is higher than the OECD average of 2.6%. Inadequate drug use and poor patient compliance increase MDR-TB prevalence through selective pressure. Therefore, prompt detection of drug resistance in tuberculosis patients at the time of diagnosis and quantitative monitoring of these resistant strains during treatment are crucial.MethodsA multiplex droplet digital PCR (ddPCR) assay was developed and assessed using DNA material of nine Mycobacterium tuberculosis strains with known mutation status that were purchased from the Korean National Tuberculosis Association. We collected a total of 18 MDR-TB residual samples referred for PCR analysis. Total DNA was extracted from the samples and subjected to the quadruplex ddPCR assay. Their results were compared to those of known resistance phenotypes.ResultsThe analytical sensitivity and specificity of the multiplex ddPCR assay for detecting INH, RIF, EMB, FQ, and SM resistance-causing mutations ranged from 71.43 to 100% and 94.12–100%, respectively. Follow-up sample results showed that the quadruplex ddPCR assay was sensitive enough to detect IS6110 and other mutations even after onset of treatment.ConclusionsWe developed a sensitive and accurate multiplex ddPCR assay that can detect the presence of tuberculosis quantitatively and resistance-conveying mutations concurrently. This tool could aid clinicians in the diagnosis and treatment monitoring of tuberculosis.

Trace Amount of Bi-Doped Core-Shell Pd@Pt Mesoporous Nanospheres with Specifically Enhanced Peroxidase-Like Activity Enable Sensitive and Accurate Detection of Acetylcholinesterase and Organophosphorus Nerve Agents.

The urgent need for sensitive and accurate assays to monitor acetylcholinesterase (AChE) activity and organophosphorus pesticides (OPs) arises from the imperative to safeguard human health and protect the ecosystem. Due to its cost-effectiveness, ease of operation, and rapid response, nanozyme-based colorimetry has been widely utilized in the determination of AChE activity and OPs. However, the rational design of nanozymes with high activity and specificity remains a great challenge. Herein, trace amount of Bi-doped core-shell Pd@Pt mesoporous nanospheres (Pd@PtBi2) have been successfully synthesized, exhibiting good peroxidase-like activity and specificity. With the incorporation of trace bismuth, there is a more than 4-fold enhancement in the peroxidase-like performance of Pd@PtBi2 compared to that of Pd@Pt. Besides, no significant improvement of oxidase-like and catalase-like activities of Pd@PtBi2 was found, which prevents interference from O2 and undesirable consumption of substrate H2O2. Based on the blocking impact of thiocholine, a colorimetric detection platform utilizing Pd@PtBi2 was constructed to monitor AChE activity with sensitivity and selectivity. Given the inhibition of OPs on AChE activity, a biosensor was further developed by integrating Pd@PtBi2 with AChE to detect OPs, capitalizing on the cascade amplification strategy. The OP biosensor achieved a detection limit as low as 0.06 ng mL-1, exhibiting high sensitivity and anti-interference ability. This work is promising for the construction of nanozymes with high activity and specificity, as well as the development of nanozyme-based colorimetric biosensors.

Abstract LB030: Building a platform of validated high throughput screening approaches for molecular glue degrader identification

Abstract Over recent years, Induced proximity therapeutics (IPT) and targeted protein degradation (TPD) approaches have become increasingly popular. Whilst significant initial efforts focused upon heterobifunctional degraders, molecular glue degraders (MGDs) represent the next generation proximity-based drugs, but their rational discovery remains challenging. Here, we describe unbiased and broadly applicable approaches for molecular glue hit ID through the development of a suite of orthogonal and highly complementary screening approaches, relying on Homogeneous Time-Resolved Fluorescence (HTRF), Surface Plasmon Resonance (SPR) and High Content Imaging (HCI). To demonstrate the utility of these assays, we have focussed on the therapeutically relevant protein SOS1 as a model target. SOS1 is the major guanine nucleotide exchange factor (GEF) for KRAS, where inhibition of the SOS1/KRAS interaction reduces tumor growth in preclinical settings, and small molecule inhibitors of the SOS1/KRAS interaction are currently undergoing clinical testing for KRAS driven cancers. Our HTRF approach measures the induced interaction between the ubiquitin ligase CRBN/DDB1 and the SOS1 protein. The assay was successfully developed and validated in a miniaturized format utilizing a bifunctional tool compound as a positive control. Complementing our HTRF based approach, we also developed a highly sensitive SPR assay for detecting compounds that generate a ternary complex with CRBN/DDB1. These assay formats are amenable for large scale HTS screening, and as proof-of-concept we have established workflows and screened compound collections from the Sygnature LeadFinder and fragment libraries, in each of these assay platforms. Our combined biochemical/biophysical approach to glue identification using HTRF and SPR demonstrates the successful and scalable use of these methodologies for HTS, with the exciting potential for identification of novel MGD candidates for SOS1. To further complement these strategies for glue identification, we have additionally developed a functional cell-based approach utilizing HCI. HCI offers a medium-high throughput approach to monitor endogenous protein abundance and localization in complex cellular models. One major advantage of imaging assays is that they are amenable to multiplexing, allowing simultaneous detection of target engagement, biomarker analysis and compound toxicity. Here, we showcase HCI as a robust, high throughput method of screening for induced protein degradation at the endogenous level, again focusing on the KRAS pathway. Using this approach we quantified endogenous protein abundance in response to compound treatment, as well as downstream effects in the RAS pathway. Together, these approaches demonstrate multiple, complementary options for identifying molecular glue degraders. Further, the described assays are also applicable for screening and characterization of bifunctional degrader compounds. This platform is poised to identify high quality lead compounds across a range of oncology targets in areas of unmet clinical need. Citation Format: Allan Jordan, Philip Addis, Toby Allen, Tarun Ayra, Sarah Beck, Roslyn Brant, Roland Hjerpe, Benoit Gourdet, Martin Jennings, Rachel Lawrence, Sigrun Campbell-Maurer, Pei Cing Ng, Lyn Parkinson, Eva Rejnowicz, Gonzalo Robles, Elizabeth Rosethorne, Joshua Shaw, Duncan Smith, Denise Swift, Daniel Tait, Stuart Thomson, Chris Tomlinson, Ailsa Townley, Stephanie Ward, Clare Wilson. Building a platform of validated high throughput screening approaches for molecular glue degrader identification [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB030.

Quantum dot fluorescent microsphere-based immunochromatographic strip for detecting PRRSV antibodies

Porcine reproductive and respiratory syndrome (PRRS) is an immunosuppressive disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). Current vaccine prevention and treatment approaches for PRRS are not adequate, and commercial vaccines do not provide sufficient cross-immune protection. Therefore, establishing a precise, sensitive, simple, and rapid serological diagnostic approach for detecting PRRSV antibodies is crucial. The present study used quantum dot fluorescent microspheres (QDFM) as tracers, covalently linked to the PRRSV N protein, to develop an immunochromatography strip (ICS) for detecting PRRSV antibodies. Monoclonal antibodies against PRRSV nucleocapsid (N) and membrane (M) proteins were both coated on nitrocellulose membranes as control (C) and test (T) lines, respectively. QDFM ICS identified PRRSV antibodies under 10 min with high sensitivity and specificity. The specificity assay revealed no cross-reactivity with the other tested viruses. The sensitivity assay revealed that the minimum detection limit was 1.2 ng/mL when the maximum dilution was 1:2,048, comparable to the sensitivity of enzyme-linked immunosorbent assay (ELISA) kits. Moreover, compared to PRRSV ELISA antibody detection kits, the sensitivity, specificity, and accuracy of QDFM ICS after analyzing 189 clinical samples were 96.7%, 97.9%, and 97.4%, respectively. Notably, the test strips can be stored for up to 6 months at 4 °C and up to 4 months at room temperature (18–25 °C). In conclusion, QDFM ICS offers the advantages of rapid detection time, high specificity and sensitivity, and affordability, indicating its potential for on-site PRRS screening.Key points• QDFM ICS is a novel method for on-site and in-lab detection of PRRSV antibodies• Its sensitivity, specificity, and accuracy are on par with commercial ELISA kits• QDFM ICS rapidly identifies PRRSV, aiding the swine industry address the evolving virus

Laboratory-developed Droplet Digital PCR Assay for Quantification of the JAK2 V617F Mutation.

Precise quantification of the JAK2 V617F mutation using highly sensitive assays is crucial for diagnosis, treatment process monitoring, and prognostic prediction in myeloproliferative neoplasms' (MPNs) patients. Digital droplet polymerase chain reaction (ddPCR) enables precise quantification of low-level mutations amidst a high percentage of wild type alleles without the need for external calibrators or endogenous controls. The objective of this study was to optimize a ddPCR assay for detecting the JAK2 V617F mutation and establish it as a laboratory-developed ddPCR assay in our center. The optimization process involved fine-tuning five key parameters: primer/probe sequences and concentrations, annealing temperature, template amount, and PCR cycles. Our ddPCR assay demonstrated exceptional sensitivity, and the limit of quantification (LoQ) was 0.01% variant allele frequency with a coefficient of variation of approximately 76%. A comparative analysis with quantitative PCR on 39 samples showed excellent consistency (r = 0.988). In summary, through rigorous optimization process and comprehensive analytic performance validation, we have established a highly sensitive and discriminative laboratory-developed ddPCR platform for JAK2 V617F detection. This optimized assay holds promise for early detection of minimal residual disease, personalized risk stratification, and potentially more effective treatment strategies in MPN patients and non-MPN populations.

Antimicrobial profiling of mastitis causing bacteria in buffalo milk

Mastitis is causing huge economic losses to the dairy industry in India. The losses were mainly due to decreased milk production and treatment cost. This study was aimed to detect occurrence of mastitis in buffaloes. A total of 5,665 milk samples from buffaloes were processed for detection of mastitis by white side test (n=4,884) and culture examination (n=781). Overall, 2,808 (57.49%) milk samples were positive by white side test and 96.79% (n=756) samples were found positive by culture examination. Gram-positive and gram-negative bacteria were detected in 54.16% (n=423) and 31.11% (n=243) of the milk samples, respectively. Mixed infection of both was found in 10.62% (n=83) samples. While, Candida spp. was detected in 0.89% (n=7) samples only. Antibiotic sensitivity assay revealed that chloramphenicol was the most sensitive antibiotic against 71.61% samples in both gram-positive and negative bacterial isolates followed by enroloxacin, amikacin, levofloxacin, moxifloxacin and gentamicin in 71.09%, 61.77%, 58.85%, 58.59% and 55.99% samples, respectively. Kanamycin was most resistant against all isolates followed by neomycin (76.36%), norfloxacin (60.99%), amoxicillin+cloxacillin (52.78%), and ceftizoxime (51.85%).

High Frequency of Prior SARS-CoV-2 Infection by Sensitive Nucleocapsid Assays.

Prior infection with SARS-CoV-2 is typically measured by nucleocapsid serology assays. In this study, we show that the Simoa serology assays and T cell intracellular cytokine staining assays are more sensitive than the clinical Elecsys assay for detection of nucleocapsid-specific immune responses. These data suggest that the prevalence of prior SARS-CoV-2 infection in the population may be higher than currently appreciated.

Unstable Angina Risk Stratification Using High-Sensitivity Cardiac Troponin and Coronary Angiography

Patients formerly diagnosed with unstable angina (UA) are being reclassified as non-ST-elevation myocardial infarction (NSTEMI) with the widespread adoption of sensitive troponin assays (hsTn), leading to significant changes in the incidence and prognosis of UA. The main purpose of this study was to evaluate the value of hsTn and the presence of significant obstructive CAD in the risk stratification of patients with unstable angina. We conducted a retrospective, single-centre study of 742 patients hospitalized for UA between 2016-2021. The primary endpoint of this study was all-cause mortality. The secondary outcome (MACE) was defined as a composite of nonfatal myocardial infarction (MI), hospitalization for heart failure (hHF), and repeated coronary angiography due to recurring UA (rUA) after the index event. Outcomes were assessed within one month, one year, and up to 5 years of follow-up. The average follow-up duration was 45±24 months, and 37.2% (N=276) of patients completed a 5-year follow-up. No in-hospital death was observed, and 6.9% of patients died during follow-up, which was more commonly a late event (>12 months). The composite secondary endpoint (MI+hHF+rUA) was observed in 16.7% of the patients. There was 3.2% nonfatal MI, 2.3% hHF, and 11.6% rUA during follow-up. We developed a risk model (UA Mortality Risk) using variables with the highest discriminatory power: age, hsTn, and ST-segment deviation. Our model performed well against the GRACE and TIMI risk scores in predicting death during follow-up. Obstructive CAD on coronary angiography was the only independent predictor of MACE during follow-up. In conclusion, a contemporary cohort of patients with UA presented with favourable prognosis, particularly within the first year after the index event. Non-significant increases in HsTn levels add to the risk stratification of UA patients, and the presence of obstructive CAD was the only independent predictor of MACE, highlighting the potential importance of assessing coronary anatomy.

An ionic liquid-modified PEDOT/Ti3C2TX based molecularly imprinted electrochemical sensor for pico-molar sensitive detection of L-Tryptophan in milk

L-Tryptophan (L-Trp) is essential for the human body and can only be obtained externally. It is important to develop a method to efficiently detect L-Trp in food. In this work, ionic liquid (IL) modified poly(3,4-ethylendioxythiophene)/ Titanium carbide (PEDOT/Ti3C2TX) was used as a substrate material to improve detection sensitivity. Molecular imprinted polymers (MIP) film for specific recognition of L-Trp was fabricated on the surface of modified electrodes using electrochemical polymerization. The monitoring results showed that the molecularly imprinted electrochemical sensors (MIECS) exhibited good linearity ranges (10−6 - 0.1 μM and 0.1–100 μM) with a low detection limit (LOD) of 2.09 × 10−7 μM. In addition, the MIECS exhibited remarkable stability, reproducibility, and immunity to interference. A good recovery (93.54–99.59%) was demonstrated in the detection of milk. The sensor was expected to be developed as a highly selective and sensitive portable assay, and applied to the detection of L-Trp in food.

Long-Term Follow-Up of Patients With Follicular Lymphoma Using Next Generation Sequencing to Detect Minimal Residual Disease

BackgroundFollicular lymphoma (FL) is a highly treatable, indolent non-Hodgkin lymphoma. Although FL is considered incurable, a patient without progression of disease by 24 months after treatment is predicted to have a survival consistent with persons without lymphoma. Using a sensitive assessment of minimal residual disease (MRD), we tested the hypothesis that MRD monitoring can predict long term remissions. MethodsUnselected patients who were in a clinical remission for at least 24 months after their last treatment were enrolled and monitored prospectively for MRD detectability using a sensitive next-generation sequencing assay (clonoSEQ, Adaptive Biotechnologies, Seattle, WA). ResultsForty-seven consecutive patients were monitored. We evaluated the MRD thresholds 10−4, 10−5, and 10−6 for the ability to predict long-term remissions in this cohort and determined that undetectable disease at 10−6 was the best predictor with a specificity and negative predictive value (NPV) of 70% and 100%, respectively. While 3 patients exhibited clinical disease progression during the course of the study, none of the 31 patients with persistent MRD undetectability at 10−6 experienced relapse. ConclusionsA significant proportion (31/47; 66.0%) of FL patients in clinical remission after ≥24 months following last therapy were undetectable at 10−6 by a sensitive assay of MRD. The threshold of sensitivity was 100%, specificity 70%, with a PPV of 19%, but a NPV of 100%. Although longer follow-up is needed for confirmation, many of these patients may continue to have durable complete remissions.