Assay For Detection Research Articles

Development of RT-PCR Assays for Simple Detection and Identification of Sabin Virus Contaminants in the Novel Oral Poliovirus Vaccines

Background/Objectives: Conventional live oral poliovirus vaccines (OPVs) effectively prevent poliomyelitis. These vaccines are derived from three attenuated Sabin strains of poliovirus, which can revert within the first week of replication to a neurovirulent phenotype, leading to sporadic cases of vaccine-associated paralytic poliomyelitis (VAPP) among vaccinees and their contacts. A novel OPV2 vaccine (nOPV2) with enhanced genetic stability was developed recently; type 1 and type 3 nOPV strains were engineered using the nOPV2 genome as a backbone by replacing the capsid precursor polyprotein (P1) with that of Sabin strains type 1 and type 3, respectively. The nOPV vaccines have a high degree of sequence homology with the parental Sabin 2 genome, and some manufacturing facilities produce and store both Sabin OPV and nOPV. Therefore, detecting Sabin virus contaminations in nOPV lots is crucial. Methods: This study describes the development of pan quantitative reverse transcription polymerase chain reaction (panRT-PCR) and multiplex one-step RT-PCR (mosRT-PCR) assays for the straightforward detection and identification of contaminating Sabin viruses when present in significantly higher amounts of nOPV strains. Results: The two assays exhibit high specificity, reproducibility, and sensitivity to detect 0.0001% and 0.00001% of Sabin viruses in nOPV, respectively. Additionally, an analysis of 12 trivalent nOPV formulation lots using both methods confirmed that the nOPV lots were free from Sabin virus contamination. Conclusions: The results demonstrated that the RT-PCR assays are sensitive and specific. These assays are relevant for quality control and lot release of nOPV vaccines.

Effects of different vaccination regimes on the immunodiagnosis of tuberculosis in goats and evaluation of defined antigens

Tuberculosis (TB) in goats is a chronic infectious disease caused by Mycobacterium tuberculosis complex (MTBC) organisms that pose a great health and economic challenge for the caprine industry in some European and developing countries. It is also a zoonotic disease posing a risk for public health. The control programs of the disease are based on a test-and-slaughter strategy, and vaccination is not feasible with available vaccines due to its interferences with the current TB immunodiagnosis. There is still a need for the development of an effective TB vaccine and, concurrently, diagnostic methods that allow differentiation between infected and vaccinated animals (DIVA approach). In this study, we investigated the interferences caused by the tuberculin (PPD)-based TB diagnostic tests in goats immunized by different mucosal and parenteral vaccination strategies: three single-dose strategies based on intranasal administration of BCG and two heat-inactivated M. bovis (HIMB) vaccines, and two prime-boost strategies based on parenteral BCG or HIMB priming and intranasal HIMB boosting. In addition, the defined antigens ESAT-6, CPF10, and EspC were evaluated as alternative diagnostic reagents to PPDs. At week 14 after prime vaccination of the animals, skin tests, IFN-γ release assay, and antibody detection assays were performed. The two prime-boosted and the single-dose intranasal BCG groups displayed greater cell-mediated immune responses to PPDs than the two single-dose intranasal HIMB vaccines. However, the use of reagents based on the defined antigens eliminated or reduced the vaccine-induced diagnostic interferences in all groups. Based on these results, the use of defined antigens in the current immunodiagnostic tests appears to be suitable in a future goat TB vaccination scenario.

Multiplex one-step RT‒qPCR assays for simultaneous detection of AMDV, MEV and CDV

BackgroundAleutian mink disease, mink viral enteritis and canine distemper are known as the three most serious diseases that cause great economic loss in the mink industry. In clinical practice, aleutian mink disease virus (AMDV), mink enteritis virus (MEV) and canine distemper virus (CDV) are common mixed infections, and they have similar clinical clinical signs, such as diarrhoea. Therefore, a rapid and accurate differential diagnosis method for use on mink ranches is essential for the control of these three pathogens. Here, we developed multiplex one-step real-time quantitative PCR (RT‒qPCR) assays for the simultaneous detection and quantification of AMDV, MEV and CDV by using three primers and probes based on the conserved NS1, VP2 and N genes, respectively.ResultsThe results showed that the established method can not cross-react with other mink pathogens, with a detection sensitivity of 25 copies/µL and a coefficient of variation less than 3.51%. Moreover, the interference experiment showed that the presence of AMDV, MEV and CDV templates at different concentrations would not interfere with the detection results. Furthermore, two hundred clinical samples of mink with diarrhoea were simultaneously analysed using multiplex RT‒qPCR and single RT‒qPCR, the Kappa values were all greater than 0.921, indicating that there was a high degree of coincidence between the two detection methods.ConclusionsIn conclusion, multiplex RT‒qPCR exhibited high specificity, sensitivity, and reproducibility, indicating that this method can be used as a reliable and specific tool for the differential detection and quantification of AMDV, MEV and CDV.

Development of a qPCR assay for early detection and quantification of Phytopythium vexans in kiwifruit plant and soil affected by Vine Decline Syndrome.

Kiwifruit Vine Decline Syndrome (KVDS) is a soilborne disease affecting Actinidia fruit trees in perennial cropping systems. Since its emergence in 2012, studies have increasingly identified the oomycete Phytopythium vexans as a major causative agent of the disease. P. vexans is also implicated in complex soilborne disease systems of woody perennial crops, including replant disease in apple and pear. To date, most molecular assays for the detection of P. vexans target the nuclear ribosomal internal transcribed spacer (ITS), a region which is ill-suited for distinguishing between closely related oomycete species. The cytochrome oxidase subunit I (COI) mitochondrial gene was targeted for the design of new primers because it was previously identified as a better marker for differentiating oomycete species. The FOR2/REV4RCA primer pair gave the best results regarding PCR specificity and was selected for use in a SYBR Green-based qPCR assay. The specificity of the qPCR assay was evaluated using 29 P. vexans strains (including different phylogenetic groups) as well as a wide variety of closely related off-target species associated with pathogenic soil communities of fruit trees. P. vexans strains were successfully quantified down to 20 fg in water and in DNA extracted from kiwifruit roots. P. vexans was also detected in artificially inoculated Actinidia plant roots as well as in a variety of naturally infected field samples of both kiwi and apple trees. These results suggest that the qPCR assay developed in this study is highly sensitive and specific to target pathogen, regardless of sample matrix.

A POCT assay based on commercial HCG strip for miRNA21 detection by integrating with RCA-HCR cascade amplification and CRISPR/Cas12a.

Apoint-of-care testing (POCT) assay based on commercial HCG strip was proposed for miRNA21 detection by integrating RCA-HCR cascaded isothermal amplification with CRISPR/Cas12a. Three modules were integrated in the proposed platform: target amplification module composed of rolling circle amplification (RCA) cascaded with hybridization chain reaction (HCR), signal transduction module composed of CRISPR/Cas12a combined with HCG-agarose gel beads probes, and signal readout module composed of commercial HCG strips. The proposed RCA-HCR-CRISPR/Cas12a-HCG strip assay for miRNA21 detection had high sensitivity, and the limit of detection was as low as 37 fM. The proposed assay showed excellent specificity for miRNA21, as other miRNAs did not caused interference for detection. The recoveries of miRNA21 were ranged from 89.0 to 118.0%. The intra-batch and inter-batch coefficient of variation (CV) were 10.1-13.4% and 11.9-14.5%, respectively, which indicated ahigh accuracy and precision, and the serum matrix did not cause any interference. With the advantages of low-cost, high sensitivity, visualization, and easy popularization, the proposed assay is expected to provide a powerful tool for early diagnosis of tumor disease miRNA, especially in resource-limited areas.

Development of a Duplex PCR-NALFIA Assay for the Simultaneous Detection of Macrophomina phaseolina and Verticillium dahliae Causal Agents of Crown and Root Rot of Strawberry

Strawberry crown and root rot diseases are caused by soil-borne pathogens including Macrophomina phaseolina (Mp) and Verticillium dahliae (Vd). The symptoms caused by these pathogens are very similar and difficult to distinguish, and traditional culture-based detection methods are laborious, time-consuming, and slow in providing results. In this work, we developed a duplex PCR-NALFIA assay using two pairs of species-specific primers labeled at the 5′ end with different molecules for the simultaneous identification of Mp and Vd. For the NALFIA assay, a lateral flow device (LFD) for the detection of two analytes was used. The method was developed by single and duplex PCR (Mp, Vd, Mp + Vd) using increasingly complex biological systems: (i) DNA from pure cultures of the pathogens; (ii) DNA from artificially inoculated cut melon stems; and (iii) DNA from artificially inoculated strawberry plants cv. Aromas. The duplex PCR protocol was effective in detecting the two pathogens within melon tissues and provided good results with strawberry crown tissues only when the DNA samples were purified by removing the PCR inhibitors. The amplicons were used for both agarose gel electrophoresis (AGE) and NALFIA assays and demonstrated the greater sensitivity of the NALFIA assay (10 pg) for simultaneous detection of the two pathogens.

Comparative Evaluation of AI-Based Multi-Spectral Imaging and PCR-Based Assays for Early Detection of Botrytis cinerea Infection on Pepper Plants

Pepper production is a critical component of the global agricultural economy, with exports reaching a remarkable $6.9B in 2023. This underscores the crop’s importance as a major economic driver of export revenue for producing nations. Botrytis cinerea, the causative agent of gray mold, significantly impacts crops like fruits and vegetables, including peppers. Early detection of this pathogen is crucial for a reduction in fungicide reliance and economic loss prevention. Traditionally, visual inspection has been a primary method for detection. However, symptoms often appear after the pathogen has begun to spread. This study employs the Deep Learning algorithm YOLO for single-class segmentation on plant images to extract spatial details of pepper leaves. The dataset included hyperspectral images at discrete wavelengths (460 nm, 540 nm, 640 nm, 775 nm, and 875 nm) from derived vegetation indices (CVI, GNDVI, NDVI, NPCI, and PSRI) and from RGB. At an Intersection over Union with a 0.5 threshold, the Mean Average Precision (mAP50) achieved by the leaf-segmentation solution YOLOv11-Small was 86.4%. The extracted leaf segments were processed by multiple Transformer models, each yielding a descriptor. These descriptors were combined in ensemble and classified into three distinct classes using a K-nearest neighbor, a Long Short-Term Memory (LSTM), and a ResNet solution. The Transformer models that comprised the best ensemble classifier were as follows: the Swin-L (P:4 × 4–W:12 × 12), the ViT-L (P:16 × 16), the VOLO (D:5), and the XCIT-L (L:24–P:16 × 16), with the LSTM-based classification solution on the RGB, CVI, GNDVI, NDVI, and PSRI image sets. The classifier achieved an overall accuracy of 87.42% with an F1-Score of 81.13%. The per-class F1-Scores for the three classes were 85.25%, 66.67%, and 78.26%, respectively. Moreover, for B. cinerea detection during the initial as well as quiescent stages of infection prior to symptom development, qPCR-based methods (RT-qPCR) were used for quantification of in planta fungal biomass and integrated with the findings from the AI approach to offer a comprehensive strategy. The study demonstrates early and accurate detection of B. cinerea on pepper plants by combining segmentation techniques with Transformer model descriptors, ensembled for classification. This approach marks a significant step forward in the detection and management of crop diseases, highlighting the potential to integrate such methods into in situ systems like mobile apps or robots.

Real-time detection of somatostatin release from single islets reveals hypersecretion in type 2 diabetes.

Somatostatin from pancreatic δ-cells is a paracrine regulator of insulin and glucagon secretion, but the release kinetics and whether secretion is altered in diabetes is unclear. This study aimed to improve understanding of somatostatin secretion by developing a tool for real-time detection of somatostatin release from individual pancreatic islets. Reporter cells responding to somatostatin with cytoplasmic Ca2+ concentration ([Ca2+]i) changes were generated by co-expressing somatostatin receptor SSTR2, the G-protein Gα15 and a fluorescent Ca2+ sensor in HeLa cells. Somatostatin induced dose-dependent [Ca2+]i increases in reporter cells with half-maximal and maximal effects at 1.6 ± 0.4 and ~30 nM, respectively. Mouse and human islets induced reporter cell [Ca2+]i elevations that were inhibited by the SSTR2 antagonist CYN154806. Depolarization of islets by high K+, KATP channel blockade or increasing the glucose concentration from 3 to 11 mM evoked concomitant elevations of [Ca2+]i in islets and reporter cells. Exposure of islets to glucagon, GLP-1 and ghrelin also triggered reporter cell [Ca2+]i responses, whereas little effect was obtained by islet exposure to insulin, glutamate, GABA and urocortin-3. Islets from type 2 diabetic human donors induced higher reporter cell [Ca2+]i responses at 11 mM and after K+ depolarization compared with non-diabetic islets, although fewer δ-cells were identified by immunostaining. Type 2 diabetes is associated with hypersecretion of somatostatin, which has implications for paracrine regulation of insulin and glucagon secretion. The new reporter cell assay for real-time detection of single-islet somatostatin release holds promise for further studies of somatostatin secretion in islet physiology and pathophysiology.

Detection and prognostic relevance of PLA2R epitopes in idiopathic membranous nephropathy: a simultaneous quantitative multiplex suspension array detection method

Abstract Objective This study aimed to develop a multiplex suspension assay for the simultaneous quantitative detection of anti-cysteine-rich domain (anti-CysR)/C-type lectin domain 1 (CTLD1)/C-type lectin domain 6–8 (CTLD678)-IgG4 antibodies of M-type phospholipase A2 receptor (PLA2R) in the serum samples of patients to evaluate the clinical application value of PLA2R epitope spreading in disease prognosis. Methods CysR, CTLD1, and CTLD678 antigen domains were coupled to three types of microspheres. The optimal dilution ratio of biotinylated anti-human IgG4 was identified, and a multiplex suspension assay was evaluated in terms of linearity, sensitivity, precision, specificity, and recovery rate. Lastly, the relationship between epitope spreading and the severity of idiopathic membranous nephropathy (IMN) was evaluated. Results The content of all three epitopes could be detected simultaneously within two hours. The intra-assay precision ranged from 4.74% to 9.48% , and the inter-assay precision was between 5.13% and 13.92% . Specific experiments showed that the human IgA antibody did not cause a cross-reaction. The recovery rates ranged between 90% and 100%. The cut-off values of epitopes CysR, CTLD1 and CTLD678 between healthy individuals and patients were 6.30, 12.38, and 10.06 Ru/mL, respectively. Among them, the positive rates of epitopes CysR, CTLD1 and CTLD678 were 100%, 34%, and 75%, respectively. In addition, group 3 (CTLD1 response) accounted for 73% of the twelve patients who were not in remission. Meanwhile, When the concentration of CTLD1 exceeds 42.76 Ru/mL, the patient's prognosis may be poorer. Conclusion A multiplex suspension assay was developed for the simultaneous quantitative detection of anti-CysR/CTLD1/CTLD678-IgG4 antibodies. The epitope migration sequence of PLA2R molecules during disease progression may not follow a simple linear rule. Among them, the epitope CTLD1 is likely to exert the most significant influence on patient remission.

Transforming cancer screening: the potential of multi-cancer early detection (MCED) technologies.

Early cancer detection substantially improves the rate of patient survival; however, conventional screening methods are directed at single anatomical sites and focus primarily on a limited number of cancers, such as gastric, colorectal, lung, breast, and cervical cancer. Additionally, several cancers are inadequately screened, hindering early detection of 45.5% cases. In contrast, Multi-Cancer Early Detection (MCED) assays offer simultaneous screening of multiple cancers from a single liquid biopsy and identify molecular changes before symptom onset. These tests assess DNA mutations, abnormal DNA methylation patterns, fragmented DNA, and other tumor-derived biomarkers, indicating the presence of cancer and predicting its origin. Moreover, MCED assays concurrently detect multiple cancers without recommended screening protocols, potentially revolutionizing cancer screening and management. Large trials have reported promising results, achieving 50-95% sensitivity and 89-99% specificity for multiple cancer types. However, challenges, regarding improving accuracy, addressing ethical issues (e.g., psychosocial impact assessment), and integrating MCED into healthcare systems, must be addressed to achieve widespread adoption. Furthermore, prospective multi-institutional studies are crucial for demonstrating the clinical benefits in diverse populations. This review provides an overview of the principles, development status, and clinical significance of MCED tests, and discusses their potential and challenges.

Comparative Efficacy of Conventional PCR and SYBR Green-based qPCR Assay for Detection of Canine parvovirus-2 in Diarrheic Pups from Nagpur

Comparative Efficacy of Conventional PCR and SYBR Green-based qPCR Assay for Detection of Canine parvovirus-2 in Diarrheic Pups from Nagpur

Solid-phase red cell adherence versus gel column agglutination: Is there any difference for the detection of alloantibodies associated with the JK system?

Detection of alloantibodies associated with the JK system may be tricky. They are nevertheless associated with transfusion reactions and their detection is crucial. Retrospectively and over a period of 7 years, we compared the results obtained using two different assays for antibody detection. We performed antibody detection testing with solid-phase red cell adherence (SPRCA). If positive, the antibody identification was carried out with the SPRCA and confirmed with the gel method in Coombs medium. In case of discrepant results occurring in recently transfused patients, we investigated these patients during the post-transfusion period. Fifty-five patients with anti-JK1 or anti-JK2 were identified out of which 41 were further analyzed. Both techniques clearly identified 10 alloantibodies. In 22 cases, the alloantibody identified by SPRCA was not observed with the gel method because of negative detection tests. In one case, none of the techniques identified the alloantibody and in 8 cases, the final result was inconclusive (1/41 with the SPRCA and 8/41 with the gel method). Two patients having a highly probable delayed hemolytic transfusion reaction (DHTR) were identified among the discrepant results. For both cases, anti-JK2 was clearly identified by the SPRCA but showed inconclusive reactions with the gel method. This study shows that in 29 out of 41 cases, the anti-JK1 or anti-JK2 identified with the SPRCA method were not with the gel method in Coombs medium. Eleven cases (11/29) were clinically significant and among them, 4 likely DHTRs and 2 highly probable DHTRs occurred.

Nanobody-Based Lateral Flow Immunoassay for Rapid Antigen Detection of SARS-CoV-2 and MERS-CoV Proteins.

The COVID-19 pandemic has highlighted the critical need for pathogen detection methods that offer both low detection limits and rapid results. Despite advancements in simplifying and enhancing nucleic acid amplification techniques, immunochemical methods remain the preferred methods for mass testing. These methods eliminate the need for specialized laboratories and highly skilled personnel, making home testing feasible. Here, we developed nanobody-based lateral flow assays (LFAs) for the rapid detection of SARS-CoV-2 and MERS-CoV in single and dual formats as point-of-care diagnostic tools. The developed LFAs are highly sensitive and successfully detected analytes at clinically relevant diagnostic cutoff values. Additionally, our results confirmed that the LFAs have a long shelf life and can be produced cost-effectively and with ease.

Development of recombinant antibody and application of colloidal gold Immunochromatography assay for rapid detection of zearalenone

Development of recombinant antibody and application of colloidal gold Immunochromatography assay for rapid detection of zearalenone

Smartphone-assisted reading result multicolor colorimetric assay for total antioxidant capacity detection based on MnO2NS-TMB mediated etching Au NBPs

Smartphone-assisted reading result multicolor colorimetric assay for total antioxidant capacity detection based on MnO2NS-TMB mediated etching Au NBPs

Development and validation of a one-step SMN assay for genetic testing in spinal muscular atrophy via MALDI-TOF MS.

Spinal muscular atrophy (SMA) is a fatal neuromuscular disorder primarily attributed to the homozygous deletion of the survival motor neuron 1 (SMN1) gene, with disease severity closely correlated to the copy number variations (CNV) of SMN2. Conventional methodologies, however, fail to provide a comprehensive gene overview of SMN and are often both time-intensive and costly. In this study, we present a novel one-step MALDI-TOF MS assay for SMA gene testing. To accurately quantify CNV, we incorporated RPPH1 as an internal control alongside normal samples and competing templates targeting SMN1, SMN2, and RPPH1 for multiple corrections. The CNV assay enables precise quantification of exons 7/8 in both SMN1 and SMN2 genes, achieving a kappa value of 0.935 (P < 0.001) when compared with multiple ligation-dependent probe amplification (MLPA) during its development phase. This accuracy was further corroborated in a cohort comprising 78 individuals. To identify patients harboring compound heterozygous mutations or silent carriers, prevalent pathogenic variants along with sequence variants of SMN1 were integrated into our analysis framework; plasmids were constructed for methodological validation purposes. Utilizing these combinatorial assays for SMN detection, we identified one patient exhibiting a compound heterozygous mutation characterized by genotype [0 + 1d] and another subject presenting genotype [2 + 1], who harbored simultaneous variants of g.27134T > G and g.27706_27707delAT. The CNV assessment combined with pathogenic variants analysis developed through MALDI-TOF MS provides a comprehensive gene profile of SMN within a single analytical run. Given its unparalleled cost-effectiveness and time efficiency, this approach holds significant promise for further application in clinical diagnosis as well as newborn screening for SMA.

A simple and highly sensitive colorimetric assay for the visual detection of lead and chromium using ascorbic acid capped gold nanoparticles.

Lead (Pb2+) and hexavalent chromium (Cr6+) are highly toxic pollutants with no safe exposure levels, posing significant health risks globally, especially in developing countries. Current detection methods for these metals are often complex and inaccessible, highlighting the urgent need for innovative approaches. In this study, we present a rapid, cost-effective colorimetric assay utilizing ascorbic acid-capped gold nanoparticles (AuNPs) for the selective detection of Pb2+ and Cr3+/6+ ions at levels recommended by regulatory bodies such as the WHO and EPA. The synthesis of our AuNPs was achieved by reducing gold(III) chloride with ascorbic acid, resulting in stable, negatively charged nanoparticles, as characterized by dynamic light scattering, UV-vis spectroscopy and high-resolution transmission electron microscopy. Our method demonstrated high sensitivity, with limits of detection (LOD) of 5.4 ± 0.25 ppb for Pb2+, and 6.3 ± 0.23 ppb for Cr6+, confirming specificity towards these ions in various water samples. The assay's efficacy was validated in real-world applications, including testing drinking water from multiple sources and assessing the performance of filtration systems. This straightforward assay offers a promising tool for monitoring water quality, enhancing public health initiatives and accessibility to critical environmental testing.

Multiplexed centrifugal microfluidic system for rapid and non-invasive detection of myocardial fibrosis.

Myocardial fibrosis is a pathological condition characterized by the excessive accumulation of extracellular matrix proteins, primarily collagen, within the myocardium. Early symptoms of myocardial fibrosis are often subtle, leading to late detection. As the disease progresses, it is often associated with life-threatening complications. Therefore, myocardial fibrosis urgently demands a diagnostic system that is rapid, effective, and preferably non-invasive. In this study, we developed a highly sensitive microfluidic light-initiated chemiluminescent assay (LICA) for the simultaneous detection of cTnI, NT-proBNP, HA, LN, PICP, and PIIINP, the key biomarkers involved in the occurrence and development of myocardial fibrosis. The system is highly integrated and compact, incorporating reagent lyophilization and whole blood separation technologies, and is suitable for point-of-care testing. By combining advantages of centrifugal microfluidics and LICA, the system effectively shortens the detection time for key biomarkers associated with MACEs. Moreover, the system provides optimal analytical performance, with LoDs of 0.0018 ng mL-1, 0.0127 ng mL-1, 3.52 ng mL-1, 6.88 ng mL-1, 4.68 ng mL-1, and 0.072 ng mL-1 for cTnI, NT-proBNP, HA, LN, PIIINP and PICP, respectively. Ultimately, the system can achieve an AUC of 0.548, 0.670, 0.772, 0.752, 0.833 and 0.713 for cTnI, NT-proBNP, HA, LN, PIIINP, and PICP, respectively, and an overall combined ROC of 0.946 for the detection of myocardial fibrosis, indicating its excellent efficacy and potential for widespread clinical applications.

Highly-multiplex detection of plasma cell-free human papillomavirus-16 DNA in oropharyngeal carcinoma.

Plasma cell-free Human Papillomavirus DNA (cfHPVDNA) is a biomarker for oropharyngeal carcinoma. Existing diagnostics may be limited by inadequate sensitivity or high cost/complexity for longitudinal monitoring. We hypothesized that sensitive and specific plasma cfHPVDNA detection may be achieved via a highly-multiplex qPCR method. We designed and validated a single-tube one-step genotype-specific qPCR assay for detection of cfHPV16DNA in human plasma using >8,000 genomes spanning 18 genotypes. Amplicons were optimized for cfHPVDNA fragment size. The cfHPV16DNA qPCR amplicons spanned 16 % of the HPV16 genome. Amplicons were conserved in a median of 99.0 % of 3,944 genomes in silico. The 95 % lower limit of detection was 0.35 genome copies/reaction and the limit of blank was 0. Multiplexing achieved a tenfold improvement in sensitivity compared with single amplicons using in silico simulations of cfHPVDNA fragmentation, which was in close agreement with experimental observations. An assay was replicated for HPV18 with similar observations. Among 36 patients with head/neck mucosal carcinomas (26 HPV-positive, 12 HPV-negative), there was 100 % concordance with tissue HPV status and with NavDx digital PCR. Pre-treatment specimens with sub-genomic cfHPVDNA concentration were detected. False negatives were observed with single amplicons but not with this multiplexed method. Among 17 patients with post-treatment landmark specimens, there was 100 % PPV and 100 % NPV for recurrence. This assay is specific for plasma cfHPVDNA detection and prognostic for recurrence. Sub-genomic sensitivity was in close agreement with in silico simulations. The format might be more accessible than dPCR or NGS for longitudinal testing.

Uncovering Cellular Senescence as a Therapeutic Target in NF2-related Vestibular Schwannoma

BackgroundVestibular schwannomas (VS) are complex and heterogeneous human tumors arising from the Schwann cell compartment of the vestibulocochlear nerve. VS cause significant neurological deficit such as hearing loss and vestibular impairment, and in some cases death due to brainstem compression. There is an urgent need to find pharmacotherapies for VS since surgical removal and stereotactic radiosurgery are the only effective treatments. Cancer therapy based in the combination of drug-induced senescence and senolytics may provide an innovative pharmacological alternative for VS management. MethodsSenescence-associated β-galactosidase (SA-β-GAL) activity detection assay, real-time polymerase chain reaction (RT-PCR), western blotting and immunofluorescence, together with viability assays were used to analyze the response to different chemotherapy drugs of the human VS HEI-193 cell line. Human VS tumor paraffin sections were also studied for SA-β-GAL-stained cells. ResultsWe found that chemotherapy compounds induced genotoxic stress and cellular senescence in HEI-193 VS cells, as characterized by increased SA-β-GAL activity, growth arrest, increased levels of the cyclin-dependent kinase inhibitor p21 and the accumulation of DNA damage. These cellular senescence markers were also accompanied by an increase of senescence-associated secretory phenotype (SASP): IL6, IL8, IL1B and MMP1. Induction of senescence by chemotherapy rendered HEI-193 VS cells as druggable targets for senolytic compounds, as navitoclax. Thus, treatment with navitoclax selectively eliminated bleomycin-induced senescent HEI-193 VS cells by activating the extrinsic and intrinsic apoptosis pathways. Our data also show the presence of senescent cells, SA-β-GAL-positive stain, in human VS tumors, which are not present in healthy great auricular nerve sections. ConclusionsThese findings suggest that a one-two punch strategy of pro-senescence therapy induced by chemotherapy treatment followed by senolytic therapy represents a new paradigm for the pharmacological treatment of VS.