Detection Of RNA Research Articles

Abstract 5566: Streamlining tissue sample preparation: A comparative study on the efficiency and reproducibility of the Omni-Stainer™ dewaxing and HIER protocol

Abstract The evolution of life sciences research has entailed a shift toward understanding cellular functions within tissue microenvironments, with an emphasis on cell- and tissue-specific relationships. A key challenge in these endeavors is the need for accurate data acquisition, which can be impeded by variability in the multistep manual assay preparations. In the quest for developing reliable and efficient tissue sample preparation workflows, two protocols using multiple dewaxing agents have been compared: the Omni-Stainer™ Dewaxing and HIER antigen retrieval protocol from Parhelia Biosciences, Inc. and the analogous one on Leica BOND Rx. The objective of this research was to assess the efficiency and reproducibility of these methods in the context of a four-color morphology immunostaining used for GeoMx DSP RNA and protein detection assays (Nanostring). We examined the efficiency of automated dewaxing using various non-toxic xylene alternatives, including C12-15 alkyl Benzoate, diethylene glycol, methyl octanoate or methyl decanoate, and compared them to the proprietary Leica Bond Dewaxing solution. Then, we compared the quality of post-HIER immunostaining using two different four-antibody panels on human jejunum samples. After several rounds of optimization, we found that the V4 of the open-source Omni-Stainer™ dewax-HIER protocol resulted in identical quality of morphology immunostaining compared to proprietary Leica Bond protocol. This study validates the Omni-Stainer™ Dewaxing and HIER protocol as a viable and efficient approach for automation of sample prep for GeoMx DSP studies. Citation Format: Nikolay Samusik, Yury Goltsev, Noemi Kedei. Streamlining tissue sample preparation: A comparative study on the efficiency and reproducibility of the Omni-Stainer™ dewaxing and HIER protocol [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5566.

Abstract 4881: Detecting cancer microbiota using unmapped RNA reads on spatial transcriptomics

Abstract Background: Recently, the study of microbiota has emerged as a novel area in cancer research. While next-generation sequencing (NGS) technologies are becoming more prevalent in the area, the acquisition of spatial information in microbiota is still challenging. Despite the possibility of detecting microbial RNAs on Visium (10X genomic, USA), methods depending on predefined host and microbial references cannot reliably capture microbial RNA reads, since Visium inherently lacks the ability to capture RNAs that are missing poly-A tails. Therefore, a reference-independent microbial RNA detection method needs to be developed for more trustworthy research on cancer microbiota on Visium. Methods: Initially, we reconstructed Visium data from a public raw dataset (Project: PRJNA811533, European Nucleotide Archive) derived from colorectal cancer (CRC) and oral squamous cell carcinoma (OSCC). BAM files were obtained by running Space Ranger on a human reference, GRCh38. These BAM files were firstly separated according to spatial barcodes with samtools and xargs. Then, microbial scores for each barcode were obtained using PathSeq. On the other hand, barcode-wise unmapped reads were extracted from the original BAM files simply by remaining unmapped reads, sorting them by spatial barcodes, and counting the number of reads. Then, the unmapped reads originating from host RNA reads were eliminated by regression analysis, remaining the microbial RNA reads. Subsequently, we conducted a comparison of microbial RNA reads from two different techniques. Results: By filtering out the unmapped reads associated with the host, microbial reads could be isolated. While these remaining microbial RNA reads show a significant correlation with the PathSeq scores, the unmapped reads were significantly higher than the PathSeq scores, increasing the statistical power for the downstream analysis with unmapped reads. Additionally, tissue regions with low PathSeq scores and high volumes of non-human unmapped reads were identified. These regions may indicate the presence of microbial sequences or RNA species that were not detected in the PathSeq analysis. Conclusion: The use of unmapped reads enables us to capture a greater number of RNA transcripts compared to PathSeq to indirectly analyze cancer microbiota. This means that we can observe hard-to-identify RNA variants, such as unstable RNAs or new species originating from the interaction between microbial RNAs and host RNAs. Our methods can provide plausible explanations for the existence of microbial RNAs on Visium, and we suggest a reference-independent detection of cancer microbota reads by utilizing unmapped reads. Citation Format: Jeongbin Park, Seo Hye Park, Dongjoo Lee, Jae Eun Lee, Daeseung Lee, Kwon Joong Na, Hongyoon Choi, Hyung-Jun Im. Detecting cancer microbiota using unmapped RNA reads on spatial transcriptomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4881.

Abstract 5503: Ultrahigh-plex spatial phenotyping of head and neck cancer tissue uncovers multiomic signatures of immunotherapy response

Abstract Background Targeted immune checkpoint inhibitors (ICI) with anti-PD-1/PD-L1 therapy offer durable treatment of mucosal head and neck squamous cell cancer (HNSCC), in both human papillomavirus-positive (HPV+) and negative (HPV-) patients. However, currently available biomarker signatures for targeted ICI therapies have limited predictive value. Our recent ultrahigh-plex profiling of HNSCC tissue with 100+ cancer hallmarks of tumor and immunobiology uncovered distinct spatial domains that serve as defining factors for clinical response and resistance. Methods Our unbiased analysis of whole-slide metastatic HNSCC tumors is from a clinical cohort of patients treated with Pembrolizumab/Nivolumab. The cohort consisted of patients with a range of outcomes from complete vs partial vs progressive disease responses to ICI therapy. We first characterized the tumor microenvironment using our ultrahigh-plex protein panel with 100+ antibodies on the PhenoCycler®-Fusion platform. To expand upon our biomarker discovery, we included multiomic cancer hallmarks with a multimodal protein/RNA detection panel. Targeted spatial RNA detection was performed to complement and augment the microenvironment characterization achieved by our protein panel. To further consolidate the multiomic data, we leveraged MaxFuse, a state-of-the art computational framework that integrates multimodal spatial and single-cell expression data. Results Our multiomic spatial phenotyping uncovered diverse tumor regions, each with distinct biomarker expression that is reflected across modalities including protein, RNA, and metabolic activity, indicating regions likely associated with resistance to immunotherapy. Our multiomic data integration also revealed spatial signatures associated with different tissue compartments, such as the tumor and non-tumor associated tertiary lymphoid structures. Conclusions We demonstrate a multi-pronged approach that incorporated both novel experimental and computational techniques for elucidating tumor microenvironment in HNSCC tissue prior to ICI-based immunotherapy. Our multiomic approach provides deeper characterization of the HNSCC at the transcriptomic and proteomic level incorporating depth across the entire transcriptome and single-cell spatial resolution of key protein determinants for predicting and furthering our understanding of immunotherapy response to ICI therapy. Citation Format: Aditya Pratapa, Lydia Hernandez, Bassem Ben Cheikh, Niyati Jhaveri, Arutha Kulasinghe. Ultrahigh-plex spatial phenotyping of head and neck cancer tissue uncovers multiomic signatures of immunotherapy response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5503.

Abstract 4308: Same-section spatial multiomic analyses using MICS technology for investigating the dynamics of the tumor microenvironment

Abstract The recent increase in image-based, spatially-resolved technologies enables researchers to profile the tumor microenvironment (TME) by capturing gene expression profiles within tissue sections. However, a significant limitation of these technologies is the lack of ability to resolve protein and RNA information in the same section, as well as conveniently analyze multimodal data sets. Here, we report a spatial RNA detection method, RNAsky, using Miltenyi Biotec’s MACSima™ Platform as an automated, multiomic approach. Our method integrates spatial proteomics and transcriptomics data to provide in-depth profiling with single-cell resolution on the same tissue section. We demonstrate these capabilities by characterizing key immune-oncology markers across normal and diseased tissues using our specialized MACS® iQ View analysis software. MACS iQ View provides fast segmentation and intuitive gating and clustering strategies to simultaneously assess protein and RNA data. We investigated the impact of clustering using protein, RNA, or the combination of both to evaluate the contribution of different information modalities on TME spatial dynamics. This cutting-edge approach will enable the identification of valuable parameters and new cell types, furthering the discovery and development of predictive and prognostic biomarkers. Citation Format: Dongju Park, Emily Neil, Rebecca C. Hennessey, Michael DiBuono, Hanna Lafayette, Erica Lloyd, Hsinyi Lo, Julia Femel, Alex Makrigiorgos, Shaina Lu, John Lee, Sameh Soliman, Dominic Mangiardi, Paurush Praveen, Fabian Staubach, Ryan Hindman, Thomas Rothmann, Telma Santos, Stefan Borbe, Hansueli Meyer, Tanya Wantenaar, Jinling Wang, Werner Müller, Robert Pinard, Andreas Bosio. Same-section spatial multiomic analyses using MICS technology for investigating the dynamics of the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4308.

Split activator of CRISPR/Cas12a for direct and sensitive detection of microRNA

CRISPR/Cas12a-based nucleic acid assays have been increasingly used for molecular diagnostics. However, most current CRISPR/Cas12a-based RNA assays require the conversion of RNA into DNA by preamplification strategies, which increases the complexity of detection. Here, we found certain chimeric DNA-RNA hybrid single strands could activate the trans-cleavage activity of Cas12a, and then discovered the activating effect of split ssDNA and RNA when they are present simultaneously. As proof of concept, split nucleic acid-activated Cas12a (SNA-Cas12a) strategy was developed for direct detection of miR-155. By adding a short ssDNA to the proximal end of the crRNA spacer sequence, we realized the direct detection of RNA targets using Cas12a. With the assistance of ssDNA, we extended the limitation that CRISPR/Cas12a cannot be activated by RNA targets. In addition, by taking advantage of the programmability of crRNA, the length of its binding to DNA and RNA was optimized to achieve the optimal efficiency in activating Cas12a. The SNA-Cas12a method enabled sensitive miR-155 detection at pM level. This method was simple, rapid, and specific. Thus, we proposed a new Cas12a-based RNA detection strategy that expanded the application of CRISPR/Cas12a.

A universal molecular control for DNA, mRNA and protein expression

The expression of genes encompasses their transcription into mRNA followed by translation into protein. In recent years, next-generation sequencing and mass spectrometry methods have profiled DNA, RNA and protein abundance in cells. However, there are currently no reference standards that are compatible across these genomic, transcriptomic and proteomic methods, and provide an integrated measure of gene expression. Here, we use synthetic biology principles to engineer a multi-omics control, termed pREF, that can act as a universal molecular standard for next-generation sequencing and mass spectrometry methods. The pREF sequence encodes 21 synthetic genes that can be in vitro transcribed into spike-in mRNA controls, and in vitro translated to generate matched protein controls. The synthetic genes provide qualitative controls that can measure sensitivity and quantitative accuracy of DNA, RNA and peptide detection. We demonstrate the use of pREF in metagenome DNA sequencing and RNA sequencing experiments and evaluate the quantification of proteins using mass spectrometry. Unlike previous spike-in controls, pREF can be independently propagated and the synthetic mRNA and protein controls can be sustainably prepared by recipient laboratories using common molecular biology techniques. Together, this provides a universal synthetic standard able to integrate genomic, transcriptomic and proteomic methods.

Accelerating targeted mosquito control efforts through mobile West Nile virus detection.

Different mosquito control strategies have been implemented to mitigate or prevent mosquito-related public health situations. Modern mosquito control largely relies on multiple approaches, including targeted, specific treatments. Given this, it is becoming increasingly important to supplement these activities with rapid and mobile diagnostic capacities for mosquito-borne diseases. We aimed to create and test the applicability of a rapid diagnostic system for West Nile virus that can be used under field conditions. In this pilot study, various types of adult mosquito traps were applied within the regular mosquito monitoring activity framework for mosquito control. Then, the captured specimens were used for the detection of West Nile virus RNA under field conditions with a portable qRT-PCR approach within 3-4h. Then, positive samples were subjected to confirmatory RT-PCR or NGS sequencing in the laboratory to obtain genome information of the virus. We implemented phylogenetic analysis to characterize circulating strains. A total of 356 mosquito individuals representing 7 species were processed in 54 pools, each containing up to 20 individuals. These pools were tested for the presence of West Nile virus, and two pools tested positive, containing specimens from the Culex pipiens and Anopheles atroparvus mosquito species. As a result of subsequent sequencing, we present the complete genome of West Nile virus and Bagaza virus. The rapid identification of infected mosquitoes is the most important component of quick response adulticide or larvicide treatments to prevent human cases. The conceptual framework of real-time surveillance can be optimized for other pathogens and situations not only in relation to West Nile virus. We present an early warning system for mosquito-borne diseases and demonstrate its application to aid rapid-response mosquito control actions.

Direct Evidence of Powassan Virus Vertical Transmission in Ixodes scapularis in Nature.

Powassan virus (POWV) is a tick-borne flavivirus endemic in North America and Russia. Experimental infections with POWV have confirmed horizontal, transstadial, vertical, and cofeeding transmission routes for potential virus maintenance. In the field, vertical transmission has never been observed. During New York State tick-borne pathogen surveillance, POWV RNA and/or infectious POWV was detected in five pools of questing Ixodes scapularis larvae. Additionally, engorged female I. scapularis adults were collected from hunter-harvested white-tailed deer (Odocoileus virginianus) in a region with relatively high tick infection rates of POWV and allowed to oviposit under laboratory conditions. POWV RNA was detected in three female adult husks and one pool of larvae from a positive female. Infectious virus was isolated from all three RNA-positive females and the single positive larval pool. The detection of RNA and infectious virus in unfed questing larvae from the field and larvae from replete females collected from the primary tick host implicates vertical transmission as a potential mechanism for the maintenance of POWV in I. scapularis in nature, and elucidates the potential epidemiological significance of larval ticks in the transmission of POWV to humans.

Impact of Swabbing Location, Self-Swabbing, and Food Intake on SARS-CoV-2 RNA Detection.

This study compared SARS-CoV-2 RNA loads at different anatomical sites, and the impact of self-swabbing and food intake. Adult symptomatic patients with SARS-CoV-2 or non-SARS-CoV-2 respiratory tract infection were included between 2021 and 2022. Patients performed a nasal and buccal swab before a professionally collected nasopharyngeal/oropharyngeal swab (NOPS). Buccal swabs were collected fasting and after breakfast in a subgroup of patients. SARS-CoV-2 RNA loads were determined by nucleic acid testing. Swabbing convenience was evaluated using a survey. The median age of 199 patients was 54 years (interquartile range 38-68); 42% were female and 52% tested positive for SARS-CoV-2. The majority of patients (70%) were hospitalized. The mean SARS-CoV-2 RNA load was 6.6 log10 copies/mL (standard deviation (SD), ±1.5), 5.6 log10 copies/mL (SD ± 1.9), and 3.4 log10 copies/mL (SD ± 1.9) in the professionally collected NOPS, and self-collected nasal and buccal swabs, respectively (p < 0.0001). Sensitivity was 96.1% (95% CI 90.4-98.9) and 75.3% (95% CI 63.9-81.8) for the nasal and buccal swabs, respectively. After food intake, SARS-CoV-2 RNA load decreased (p = 0.0006). Buccal swabbing was the preferred sampling procedure for the patients. In conclusion, NOPS yielded the highest SARS-CoV-2 RNA loads. Nasal self-swabbing emerged as a reliable alternative in contrast to buccal swabs. If buccal swabs are used, they should be performed before food intake.

A signal-switchable photoelectrochemical biosensor for ultrasensitive detection of long non-coding RNA in cancer cells

Long non-coding RNA (LncRNA) as an emerging tumor biomarker plays a key factor in the early diagnosis of cancer. Herein, an innovative signal-switchable photoelectrochemical (PEC) biosensor based on ZrO2@CuO bimetallic oxides and T7 Exo-assisted signal amplification is reported for the ultrasensitive and selective detection of lncRNA (HOX gene antisense intergenic RNA, HOTAIR) in cancer cells. Firstly, MOFs-derived TiO2 nanodisks as an excellent photoactive material show an anodic background signal. When target lncRNA exists, the abundant auxiliary DNA1 is freed from T7 Exo-assisted cycle signal amplification, and then competitively hybridizes with auxiliary DNA2 on the electrode. Subsequently, bimetallic MOFs-derived ZrO2@CuO octahedra with a high specific surface area and porous structure are introduced into TiO2 nanodisks-modified biosensor, which appears a cathodic photocurrent and achieves a switchable signal. The developed signal-switchable PEC biosensor shows ultrasensitive detection of lncRNA HOTAIR with a detection limit of 0.12 fM, and can eliminate the false interference. Importantly, the established PEC biosensor has good correlation with RT-qPCR analysis (P < 0.05) for the quantification of lncRNA HOTAIR in cancer cells, which has great potential application for biomarker detection in the early diagnosis of cancer.

Selective visual detection of multiplex PCR amplicon using magnetic microbeads

Nucleic acid amplification tests (NATs), such as genetic tests using polymerase chain reaction (PCR), are sensitive methods for detecting pathogens and food contamination. The rapid, easy, and inexpensive detection of amplicons, DNA, or RNA is key to realizing on-site NATs. We have previously developed a novel amplicon detection method using magnetic microbeads based on the hydrophobicity of DNA in deionized water. In this study, we aimed to expand the method for the detection of multiplex DNA amplicons. Tagged primers and probes for selective attachment were used to detect amplicons from two strawberry pathogens. The amplicon-labeled magnetic microbeads were placed in the round-bottom well of a hydrophilic glass substrate. The attachment of amplicons to the magnetic microbeads changed their surface from hydrophilic to hydrophobic. The magnetized microbeads concentrated at the bottom when the substrate was placed on a permanent magnet, and the concentrated microbeads were easily recognizable by the naked eye. Microbeads without amplicons were adsorbed over a broad area of the bottom of the glass well owing to their hydrophilicity. The appropriate tag probe was attached to specific amplicons for detection, and each amplicon from multiplex PCR was selectively detected within approximately 15 min. Notably, this method requires no electric power and contributes to the realization of on-site NAT detection. This study presents a simple and rapid method for the selective detection of multiplex PCR amplicons using DNA–DNA hybridization.

Molecular Detection and Genotyping of Dengue Viruses: Current Techniques and Future Prospect

The Dengue virus is a prevalent mosquito-borne disease, and its global incidence has been steadily increasing due to the favorable environmental conditions that promote mosquito breeding, primarily influenced by rising temperatures. Bangladesh has been particularly hard-hit by an intense and ongoing outbreak, resulting in a surge of cases and fatalities. Effective management of this disease necessitates the implementation of robust public health measures, including rigorous surveillance and early diagnosis. While serological tests are commonly employed in clinical diagnosis, molecular methods hold a critical role in identifying the specific Dengue virus strain, thereby contributing to a more comprehensive understanding of disease severity. This article serves as an extensive review, delving into various molecular testing techniques employed for both surveillance and clinical diagnosis. It offers valuable insights for research and clinical laboratories engaged in the detection of Dengue virus RNA in mosquitoes, environmental samples, and clinical specimens. The methods covered encompass a spectrum of approaches, including conventional PCR, isothermal amplification, real-time RT-PCR, Sanger sequencing, and whole-genome sequencing, providing a holistic overview of the available techniques. These methods play pivotal roles in clinical diagnosis, outbreak analysis, vector surveillance, and vaccine development. Furthermore, the article underscores the importance of integrating these techniques into existing healthcare systems, emphasizing their significance in ensuring precise dengue diagnostics to enhance the efficiency of disease management. These molecular methods are indispensable tools that contribute to accurate diagnosis, enable effective outbreak investigation, facilitate vector surveillance, and support vaccine preparation, thereby enhancing the overall management and control of dengue, ultimately working toward mitigating its impact on public health.&#x0D; Bangladesh J Microbiol, Volume 40, Number 1, June 2023, pp 41-49

The Prevalence of Tick-Borne Encephalitis Virus in Wild Rodents Captured in Tick-Borne Encephalitis Foci in Highly Endemic Lithuania.

Wild rodents are considered to be one of the most important TBEV-amplifying reservoir hosts; therefore, they may be suitable for foci detection studies. To investigate the effectiveness of viral RNA detection in wild rodents for suspected TBEV foci confirmation, we trapped small rodents (n = 139) in various locations in Lithuania where TBEV was previously detected in questing ticks. Murine neuroblastoma Neuro-2a cells were inoculated with each rodent sample to maximize the chances of detecting viral RNA in rodent samples. TBEV RNA was detected in 74.8% (CI 95% 66.7-81.1) of the brain and/or internal organ mix suspensions, and the prevalence rate increased significantly following sample cultivation in Neuro-2a cells. Moreover, a strong correlation (r = 0.88; p < 0.05) was found between the average monthly air temperature of rodent trapping and the TBEV RNA prevalence rate in cell culture isolates of rodent suspensions, which were PCR-negative before cultivation in cell culture. This study shows that wild rodents are suitable sentinel animals to confirm TBEV foci. In addition, the study results demonstrate that sample cultivation in cell culture is a highly efficient method for increasing TBEV viral load to detectable quantities.

Accuracy of Chest Xray in Hospitalized COVID-19 Patients

Introduction: At the end of the year 2019 a novel virus named SARS-CoV-2 caused Coronavirus Disease 19 (COVID-19), manifesting as lung infection which can result in severe pneumonia. The gold standard for diagnosis of the virus is the detection of viral RNA through reverse transcriptase PCR (RT-PCR). Materials and Methods: This study is a retrospective analysis of diagnostic yield of chest imaging modalities in the diagnosis of COVID-19 in patients admitted to the Imam Khomeini hospital, Tehran, Iran during march 2020 until July 2020. Results: 204 hospitalized patients with a mean age of 58.5 years, diagnosed with COVID-19 were enrolled in this study, who had their disease confirmed by PCR. About 20% of patients had normal O2 Saturation (above 93%) and 80% had low O2 Saturation. Also 70% of patients were hospitalized to the ICU. Among investigated patients, 87.6% had abnormal findings in their CXR. Also, 97.1% of patients had abnormal CT-Scan. In this study, the sensitivity of the CXR in the diagnosis of COVID-19 was 87.5% (CI 95%, 83 to 91) and the sensitivity of the chest CT-Scan was 97.1% (CI 95%, 94.8 to 99). Discussion: Utilizing CXR as a first-line imaging modality is recommended in many countries and clinical settings and chest CT-Scan is mainly reserved for other additional roles. This study reveals a CXR sensitivity of 87.5% which is in accordance with the recent literature (69-90%).

Transmission electron microscopy reveals the presence of SARS-CoV-2 in human spermatozoa associated with an ETosis-like response.

Severe acute syndrome coronavirus 2 can invade a variety of tissues, including the testis. Even though this virus is scarcely found in human semen polymerase chain reaction tests, autopsy studies confirm the viral presence in all testicular cell types, including spermatozoa and spermatids. To investigate whether the severe acute syndrome coronavirus 2 is present inside the spermatozoa of negative polymerase chain reaction-infected men up to 3 months after hospital discharge. This cross-sectional study included 13 confirmed moderate-to-severe COVID-19 patients enrolled 30-90 days after the diagnosis. Semen samples were obtained and examined with real-time polymerase chain reaction for RNA detection and by transmission electron microscopy. In moderate-to-severe clinical scenarios, we identified the severe acute syndrome coronavirus 2 inside spermatozoa in nine of 13 patients up to 90 days after discharge from the hospital. Moreover, some DNA-based extracellular traps were reported in all studied specimens. Although severe acute syndrome coronavirus 2 was not present in the infected men's semen, it was intracellularly present in the spermatozoa till 3 months after hospital discharge. The Electron microscopy (EM) findings also suggest that spermatozoa produce nuclear DNA-based extracellular traps, probably in a cell-free DNA-dependent manner, similar to those previously described in the systemic inflammatory response to COVID-19. In moderate-to-severe cases, the blood-testes barrier grants little defence against different pathogenic viruses, including the severe acute syndrome coronavirus 2. The virus could also use the epididymis as a post-testicular route to bind and fuse to the mature spermatozoon and possibly accomplish the reverse transcription of the single-stranded viral RNA into proviral DNA. These mechanisms can elicit extracellular cell-free DNA formation. The potential implications of our findings for assisted conception must be addressed, and the evolutionary history of DNA-based extracellular traps as preserved ammunition in animals' innate defence might improve our understanding of the severe acute syndrome coronavirus 2 pathophysiology in the testis and spermatozoa.

Enhancing COVID-19 Detection Accuracy: Optimal Gene Combinations, Kit Performance, and Reliable Detection Intervals

A significant challenge and threat to public health have been created by the COVID-19 pandemic for the entire global population. The study aimed to compare the SARS-CoV-2 RNA detection capabilities of available primers and probes to identify the most reliable, efficient, and affordable method. From 200 previously detected samples of SARS CoV-2, 94 samples were selected randomly and used for the optimization of our primers and probes. We compared our results with two kits that have been approved by the health authority. In addition, we evaluated the detectability of each gene. The study compared the diagnostic performance of different gene combinations for COVID-19 detection using kits A and B and a novel approach combining RdRp, N, and E genes. Results showed that the combined approach exhibited superior discriminatory power, particularly with the inclusion of the E gene, boasting area under the curve (AUC) values of 83.3%, 79.1%, and 93.7% for the respective genes. Kit B, with Orf1ab and N genes, outperformed Kit A (RdRp and S genes), with AUC values of 81.2% and 90.6% versus 80.2% and 75%, respectively. The chart representation highlighted gene detection frequencies across various cycle threshold (Ct) ranges, demonstrating robust identification within the 20.1–30 Ct range across all kits and genes, emphasizing the reliability of detection within specific intervals. Combining RdRp, N, and E genes showed the highest accuracy for COVID-19 diagnosis, particularly with the E gene. Detection was most reliable within the 20.1–30 Ct range across all gene combinations and kits.

SARS-CoV-2 infection increases airway bleeding risk in patients after tracheostomies

BackgroundAirway bleeding events are a rare incident in SARS-CoV-2-infected patients after tracheostomies. We aimed to explore the correlation between airway bleeding and SARS-CoV-2 infection and evaluate the consistency of SARS-CoV-2 RNA test results in the upper and lower airway samples from patients after tracheostomies.MethodsForty-four patients after temporary or permanent tracheostomy were divided into a positive group (29 patients) and a negative group (15 patients) based on the SARS-CoV-2 RNA test results of their oropharyngeal swabs. The oropharyngeal and tracheal swabs of the positive group were re-collected for SARS-CoV-2 RNA detection. Demographic and clinical characteristics and airway bleeding events were recorded for all enrolled patients.ResultsAirway bleeding was reported in eleven patients of the positive group (11/29), with seven displaying bloody sputum or hemoptysis, and four featuring massive sputum crust formation in the trachea that resulted in dyspnea, and only one patient in the negative group (1/15), with a significant difference in the airway bleeding rate (37.9% vs. 6.7%, p < 0.05). The SARS-CoV-2 RNA test results showed a statistical difference in cycle threshold (Ct) values between oropharyngeal swabs and tracheal swabs (p < 0.05).ConclusionsAfter tracheostomies, patients are more susceptible to airway bleeding if they are infected with SARS-CoV-2. The findings signify that in addition to droplet transmission through tracheostoma, SARS-CoV-2 may infect the oropharynx by airborne and close contact transmission, and that given the higher viral load and longer infection time in the trachea, tracheal swabs are more reliable for SARS-CoV-2 detection in these patients.

Duplex Droplet Digital PCR Assay for Quantification of Hepatitis E Virus in Food.

Hepatitis E virus (HEV) represents an emerging risk in industrialized countries where the consumption of contaminated food plays a pivotal role. Quantitative real-time RT-PCR (RT-qPCR) is one of the most suitable methods for the detection and quantification of viruses in food. Nevertheless, quantification using RT-qPCR has limitations. Droplet digital PCR (ddPCR) provides the precise quantification of nucleic acids without the need for a standard curve and a reduction in the effect on virus quantification due to the presence of inhibitors. The objectives of the present work were (i) to develop a method for the absolute quantification of HEV in swine tissues based on ddPCR technology and provide internal process control for recovery assessment and (ii) to evaluate the performance of the method by analyzing a selection of naturally contaminated wild boar muscle samples previously tested using RT-qPCR. The method was optimized using a set of in vitro synthesized HEV RNA and quantified dsDNA. The limit of detection of the developed ddPCR assay was 0.34 genome copies/µL. The analysis of the wild boar samples confirmed the validity of the ddPCR assay. The duplex ddPCR method showed no reduction in efficiency compared to individual assays. The method developed in the present study could represent a sensitive assay for the detection and absolute quantification of HEV RNA in food samples with the advantage of presenting the co-amplification of internal process control.

Field-effect transistor biosensor with signal amplification by ternary initiation complexes for detection of wide-range RNA concentration

Electrical detection of RNAs using transistor-based biosensors has attracted attention as a strategy for medical diagnosis and environmental monitoring. Herein, we demonstrated a proof-of-concept for specific, sensitive, and label-free RNA detection using a field-effect transistor (FET) biosensor with signal amplification by ternary initiation complexes (SATIC), which is an isothermal one-step nucleic acid amplification initiated by the combination of target RNA, circular DNA template and DNA primer. The SATIC system-applied FET biosensor specifically and quantitatively detected the target RNA with a single-nucleotide difference via the negative charges derived from the amplification products formed by a nucleic acid amplification reaction with φ29 DNA polymerase on the gate surface. In particular, the control of the amplification time allowed the detection of target RNA molecules over a wide concentration range, resulting in a detection limit of up to 6 copies/μL. Therefore, a transistor-based bioassay using the SATIC system could be useful for simple and sensitive nucleic acid analysis.

Liver Insights: Probing Molecular Signatures of Hepatitis C Patients in Hospitals of Lahore- Navigating the Clinical Landscape of Viral Infection and Organ Profiling

Background: Hepatitis C virus (HCV) infection is a major public health problem, leading to chronic liver diseases, cirrhosis, and hepatocellular carcinoma. The disease burden is particularly high in developing countries, with significant gender disparities and varied outcomes across different clinical settings. Objective: The study aimed to assess the prevalence of HCV infections, analyze the clinical correlation of demographic factors with patient outcomes, and examine the age-related duration of hospital stays in a Pakistani cohort. Methods: A cross-sectional retrospective study was conducted on 230 symptomatic liver patients from three major hospitals in Lahore. Inclusion criteria were based on HCV-related liver conditions, excluding post-liver transplant patients. Data on viral load detections were obtained via semi-automated RT-PCR with commercially available kits. Statistical analysis was performed using SPSS version 25 and GraphPad Prism 9.0 to determine frequency distributions, cross-tabulation analyses, and Pearson correlation coefficients. Results: Out of the 230 patients, 69% (158) were male, and 31% (72) were female. Age distribution showed 36.7% (58) of males were over 60 years, and 63.3% (100) under 60 years; 55.6% (40) of females were over 45 years, with 44.4% (32) under 45 years. Most referrals came from the Pulmonary ward (35%), followed by the Surgery (30%) and Gastroenterology (20%) wards. HCV RNA detection was positive in 67% (154) of the patients. Cross-tabulation analysis indicated a mortality rate of 39% in the Pulmonary ward. A significant positive correlation (r=0.494, p&lt;0.000) was observed between patient age and days of hospital stay. Conclusion: The study demonstrates a higher prevalence of HCV in males and a notable age-related impact on the duration of hospitalization. Interdisciplinary approaches and gender-sensitive healthcare interventions are essential for effective HCV management.