Rapid detection of blood using a novel application of RT-RPA integrated with CRISPR-Cas: ALAS2 detection as a model.

A rapid, sensitive and specific test for blood is reported based on a novel application of recombinase polymerase amplification integrated with CRISPR-Cas and lateral flow assay (LFA). The blood specific marker ALAS2 was used as the target to record the presence of blood. The assay used either RNA extracted from a body fluid as a template, or omitting this extraction step and using a direct approach where the questioned body fluid was added directly to the assay. The assay only detected blood (all peripheral blood and some menstrual blood samples) and no other body fluid (semen, saliva, or vaginal fluid). The limit of detection varied from an initial template of 0.195 ng extracted RNA (27 dilution) or 0.0218 µL (26 dilution) liquid peripheral blood. The assay gave the expected result when peripheral blood was mixed with saliva: ratios of peripheral blood/saliva at 19:1, 3:1, 1:1, 1:3 and 1:19 all gave a positive result using extracted RNA. By contrast, only three ratios of peripheral blood and saliva gave a positive result for blood (19:1, 3:1 and 1:1) when adding these two body fluids directly. When peripheral blood was mixed with semen there was a strong inhibition of the assay and ALAS2 could only be detected at ratio of 19:1 using RNA. Using reconstituted peripheral bloodstains gave comparable results to liquid peripheral blood. This is the first application of RT-RPA integrated CRISPR and combined with a LFA assay to detect body fluid-specific RNA. The proposed method opens up the potential to perform this method remote from laboratories such as at crime scenes.

Validation of reliable reference genes for qPCR of CD4+ T cells exposed to compressive strain.

For accurate interpretation of quantitative real-time PCR (qPCR) data, stable reference genes are essential for normalization of target genes. To date, there is no information on reliable housekeeping genes in CD4+ T cells in a three-dimensional (3D) matrix under pressure stimulation. This in vitro study describes for the first time a method for pressure stimulation of CD4+ T cells in a 3D matrix in the context of orthodontic tooth movement (OTM) and identifies a set of reliable reference genes. CD4+ T cells were isolated from murine spleen and activated with anti-CD3/-CD28 Dynabeads (Thermo Fisher, Langenselbold, Germany) on standard cell culture plates or in 3D scaffolds with or without compressive strain. Expression stability of nine potential reference genes was examined using four mathematical algorithms. Gene expression of Il2 was normalized to all potential reference genes to highlight the importance of correct normalization. Cell proliferation and the expression of the surface markers CD25 and CD69 were also determined. The 3D matrix did not inhibit proliferation after immunological activation of T cells and embedded the cells sufficiently to expose them to pressure load. Expression of ubiquitin C (Ubc) and hypoxanthine phosphoribosyltransferase (Hprt) was the most stable under all conditions tested. A combination of these two genes was suitable for normalization of qPCR data. Normalization of Il2 gene expression showed highly variable results depending on the reference gene used. Pressure reduced cell proliferation and the number of CD69-positive T cells. This study provides a basis for performing valid and reliable qPCR experiments with CD4+ T cells cultured in 3D scaffolds and exposed to compressive forces simulating OTM.

The expression of immune response genes in patients with chronic Chagas disease is shifted toward the levels observed in healthy subjects as a result of treatment with Benznidazole.

Introduction: Chagas disease, caused by the Trypanosoma cruzi parasite infection, is a potentially life-threatening neglected tropical disease with a worldwide distribution. During the chronic phase of the disease, there exists a fragile balance between the host immune response and parasite replication that keeps patients in a clinically-silent asymptomatic stage for years or even decades. However, in 40% of patients, the disease progresses to clinical manifestations mainly affecting and compromising the cardiac system. Treatment is recommended in the chronic phase, although there are no early markers of its effectiveness. The aim of this study is to identify differential expression changes in genes involved in the immune response in antigen-restimulated PBMC from chronic patients with Chagas disease due to benznidazole treatment. Methods: Thus, high-throughput real-time qPCR analysis has been performed to simultaneously determine global changes in the expression of 106 genes involved in the immune response in asymptomatic (IND) and early cardiac manifestations (CCC I) Chagas disease patients pre- and post-treatment with benznidazole. Results and discussion: The results revealed that 7 out of the 106 analyzed genes were differentially expressed (4 up- and 3 downregulated) after treatment in IND patients and 15 out of 106 (3 up- and 12 downregulated) after treatment of early cardiac Chagas disease patients. Particularly in CCC I patients, regulation of the expression level of some of these genes towards a level similar to that of healthy subjects suggests a beneficial effect of treatment and supports recommendation of benznidazole administration to early cardiac Chagas disease patients. The data obtained also demonstrated that both in asymptomatic patients and in early cardiac chronic patients, after treatment with benznidazole there is a negative regulation of the proinflammatory and cytotoxic responses triggered as a consequence of T. cruzi infection and the persistence of the parasite. This downregulation of the immune response likely prevents marked tissue damage and healing in early cardiac patients, suggesting its positive effect in controlling the pathology.

RT-RPA-PfAgo detection platform for one-tube simultaneous typing diagnosis of human respiratory syncytial virus.

Human respiratory syncytial virus (HRSV) is the most prevalent pathogen contributing to acute respiratory tract infections (ARTI) in infants and young children and can lead to significant financial and medical costs. Here, we developed a simultaneous, dual-gene and ultrasensitive detection system for typing HRSV within 60 minutes that needs only minimum laboratory support. Briefly, multiplex integrating reverse transcription-recombinase polymerase amplification (RT-RPA) was performed with viral RNA extracted from nasopharyngeal swabs as a template for the amplification of the specific regions of subtypes A (HRSVA) and B (HRSVB) of HRSV. Next, the Pyrococcus furiosus Argonaute (PfAgo) protein utilizes small 5'-phosphorylated DNA guides to cleave target sequences and produce fluorophore signals (FAM and ROX). Compared with the traditional gold standard (RT-qPCR) and direct immunofluorescence assay (DFA), this method has the additional advantages of easy operation, efficiency and sensitivity, with a limit of detection (LOD) of 1 copy/µL. In terms of clinical sample validation, the diagnostic accuracy of the method for determining the HRSVA and HRSVB infection was greater than 95%. This technique provides a reliable point-of-care (POC) testing for the diagnosis of HRSV-induced ARTI in children and for outbreak management, especially in resource-limited settings.

Fabrication of a 3D-Printed Interim Bite Splint for a Hemimandibulectomy Patient: A Case Report.

Mandibular continuity defects can result in varying degrees of cosmetic disfigurement. Restoration of form and function may require surgical reconstruction of the affected area. While surgical reconstruction may improve the overall prognostic outcomes for the patient, the definitive prosthetic phase can commence only after a substantial time lag for adequate hard/soft tissue healing. This interim phase often challenges the patient's masticatory ability. The traditional reconstruction of hemimandibulectomy defects has its own limitations. This case report describes the fabrication of a 3D-printed bite splint for a patient with limited mouth opening and significant malocclusion due to surgical over-correction. The prosthesis given served as an appliance to improve the masticatory ability of the patient.

Screening of reliable reference genes for the normalization of RT-qPCR in chicken liver tissues and LMH cells.

The liver plays a vital role in lipid synthesis and metabolism in poultry. To study the functional genes more effectively, it is essential to screen of reliable reference genes in the chicken liver, including females, males, embryos, as well as the Leghorn Male Hepatoma (LMH) cell line. Traditional reference gene screening involves selecting commonly used housekeeping genes (HKGs) for RT-qPCR experiments and using different algorithms to identify the most stable ones. However, this approach is limited in selecting the best reference gene from a small pool of HKGs. High-throughput sequencing technology may offer a solution to this limitation. This study aimed to identify the most consistently expressed genes by utilizing multiple published RNA-seq data of chicken liver and LMH cells. Subsequently, the stability of the newly identified reference genes was assessed in comparison to previously validated stable poultry liver expressed reference genes and the commonly employed HKGs using RT-qPCR. The findings indicated that there is a higher degree of similarity in stable expression genes between female and male liver (such as LSM14A and CDC40). In embryonic liver, the optimal new reference genes were SUDS3, TRIM33, and ERAL1. For LMH cells, the optimal new reference genes were ALDH9A1, UGGT1, and C21H1orf174. However, it is noteworthy that most HKGs did not exhibit stable expression across multiple samples, indicating potential instability under diverse conditions. Furthermore, RT-qPCR experiments proved that the stable expression genes identified from RNA-seq data outperformed commonly used HKGs and certain validated reference genes specific to poultry liver. Over all, this study successfully identified new stable reference genes in chicken liver and LMH cells using RNA-seq data, offering researchers a wider range of reference gene options for RT-qPCR in diverse situations.

RT-PCR Result of SARS-CoV-2 Viral RNA in Cadavers and Viral Transmission Risk to Handlers.

During the onset of the pandemic, a common research question was asked by the hospital staff, and family members who were handling COVID-19-infected cadavers, "does COVID-19-positive dead body harbor SARS-CoV-2 viral RNA?" Several research findings were reported but due to the lack of proper research findings, the question remained unanswered. The present study was planned to observe the virus transmission risk from cadavers to the handlers. A pilot study was conducted on 54 cadavers who died in COVID-ICU (SARS-CoV-2-positive diagnosed by RT-PCR) during 2021-2022. Skin swab sample from 54 dead bodies and 54 glove samples of handlers were taken within 1 hour of death for the RT-PCR test. Viability results from RT-PCR show that the infection risk was 50% in cadavers, whereas the transmission risk to handlers while handling was 7%, which is minimal. The SARS-CoV-2 viability was high in cases of those died after a long time of infection. Based on the RT-PCR result and data analysis the interpretation of the study was that the SARS-CoV-2 transmission risk from dead bodies to the handlers is minimal but the SARS-CoV-2 viability persists in the cadavers. This fact is helpful for the people who will conduct funeral activities, autopsy staff, and hospital staff handling dead bodies. How to cite this article: Panda B, Singh N, Singh G, Patro ARK, Mohanty AP, Patnaik PK, et al. RT-PCR Result of SARS-CoV-2 Viral RNA in Cadavers and Viral Transmission Risk to Handlers. Indian J Crit Care Med 2024;28(6):614-616.

PI primers increase the efficacy of LAMP and RT-LAMP for SARS-CoV-2 and MS2 phage detection.

LAMP (Loop-mediated isothermal amplification) is a popular method for the molecular diagnostics of numerous pathogens, specifically useful for point-of-care testing. However, the efficacy and sensitivity of LAMP still need to be maximised for the best performance in clinical settings. Adding a novel fourth primer pair is a promising way to accelerate the LAMP speed. Here, we report PI primers that are part of inner primers and can be used in LAMP without a specific design. PI primers were tested in quantitative LAMP detecting SARS-CoV-2 and MS2. The new primers have increased the speed and sensitivity of quantitative LAMP, RT-LAMP, and duplex LAMP with artificial templates and RNA samples from nasal swabs. Adding PI primers could become a valuable option for LAMP optimisation, especially when a desirable LAMP target is a highly variable DNA sequence with a few conservative sites for primers.

Cyt b gene as a valid molecular authentication marker of cow leathers.

Authentication of true (genuine) cow leathers is in high demand to promote merchandise and economic growth. The present study employs RT-PCR-based TaqMan assay to facilitate the identification. Species-specific primers and probes were designed utilizing the existing NCBI data on mitochondrial DNA (mtDNA) genes, particularly the cytochrome b region (Cyt b). Mitochondrial DNA extracted from leather samples of both Bos taurus and Bos indicus and analyzed following the appropriate procedures. The RT-PCR results showed the designed primers and probes are exceptionally precise for cow leather samples. The established detection limit for the assay is estimated as 0.1 ng of DNA. In summary, the amplifiable mtDNA extracted from finished leather enables the identification of authentic cow leathers using the RT-PCR TaqMan assay, representing a pioneering report in this field.

Spatiotemporal evolution of the distribution of Chronic bee paralysis virus (CBPV) in honey bee colonies.

Chronic bee paralysis virus (CBPV) is a Apis mellifera viral infectious disease, exhibiting dark and hairless abdomen in workers with tremors and ataxita. Clinical signs are also typically linked to adverse weather conditions and overcrowding in the hive. The disease occurs in spring but recently it has been observed cases increase and seasonality loss of the disease incidence. This study analyses the evolution of CBPV in Italy, through data collected from 2009 to 2023 within three monitoring projects comprising nationwide extended detection networks, aimed to investigate the evolution of the CBPV spatial distribution, identifying high-risk areas for the virus spread. This study highlights an increased risk over years. Prevalence increased from 4.3% during 2009-2010 to 84.7% during 2021-2023 monitoring years. CBPV outbreaks were irregular between investigated seasons, highlighting Spring and Autumn as the most susceptible seasons. Risk of CBPV infection has increased, reaching high-risk in last years of monitoring. Sequence analysis showed a high similarity to other isolated Italian CBPVs. The study offers an epidemiological insight into the aetiology of this disease. CBPV distribution is a prerequisite to predict its future spread and factors involved in its propagation not only in honey bees but also in other pollinators and environments.

Semiparametric inference of effective reproduction number dynamics from wastewater pathogen surveillance data.

Concentrations of pathogen genomes measured in wastewater have recently become available as a new data source to use when modeling the spread of infectious diseases. One promising use for this data source is inference of the effective reproduction number, the average number of individuals a newly infected person will infect. We propose a model where new infections arrive according to a time-varying immigration rate which can be interpreted as an average number of secondary infections produced by one infectious individual per unit time. This model allows us to estimate the effective reproduction number from concentrations of pathogen genomes, while avoiding difficulty to verify assumptions about the dynamics of the susceptible population. As a byproduct of our primary goal, we also produce a new model for estimating the effective reproduction number from case data using the same framework. We test this modeling framework in an agent-based simulation study with a realistic data generating mechanism which accounts for the time-varying dynamics of pathogen shedding. Finally, we apply our new model to estimating the effective reproduction number of SARS-CoV-2, the causative agent of COVID-19, in Los Angeles, CA, using pathogen RNA concentrations collected from a large wastewater treatment facility.

Evaluation of a multigenomic liquid biopsy (PROSTest) for prostate cancer detection and follow-up in a Caribbean population.

INTRODUCTION: The PROSTest is a novel machine learning-based liquid biopsy assay that functions as a diagnostic and prognostic tool in prostate cancer (PCa). The algorithm outcome (scored 0-100) has a cutoff of >50 to indicate PCa. In this study, we evaluated the screening utility of the test in comparison with the commonly used PSA test. MATERIALS AND METHODS: 509 men (median age: 62 [40-88]), predominantly Afro-Caribbean (n=449, 88.2 %) were enrolled in this study at a referral centre for prostatic cancer screening and management. These included 417 symptomatic patients with urinary tract (UT) problems, of which 12 were diagnosed with PCa, 53 with benign prostatic hyperplasia (BPH) and 352 were free from prostatic diseases, prior to PROSTest (Cohort 1). The remaining 92 of the 509, with relatively young age (median 49 and range 40-75 years) were screened with the PROSTest first and included 28 with UT symptoms and 64 asymptomatic but with a family history of cancer (Cohort 2). PROSTest and PSA was performed on all subjects. RESULTS: In Cohort 1, the PROSTest was positive in 11 (91.7 %) PCa, 9 (17.0 %) of BPH, and 29 (8.2 %) of subject with UT symptoms but no disease. In Cohort 2, PROSTest was positive in 4 of 28 (14.3 %) symptomatic patients and in 5 of 64 (7.8 %) asymptomatic subjects. A total of 111 individuals (from both cohorts) were identified as "at risk" (PSA >4 ng/mL). Sixteen of 111 (14.4 %) were PROSTest-positive. Thirty-nine percent (43/111) have completed a 1-year follow-up. Of the 43 with elevated PSA, 2 out of 2 (100 %) who were also PROSTest-positive, were diagnosed with PCa. CONCLUSION: PROSTest is a more sensitive and specific biomarker than PSA in the diagnosis and, potentially, screening for PCa. The seemingly false positive PROSTest results in BPH or symptomatic UT require a longer follow-up of these patients.

SARS-CoV-2 RNA Detection in Wastewater and Its Effective Correlation with Clinical Data during the Outbreak of COVID-19 in Salamanca.

Wastewater treatment plants (WWTPs) are the final stage of the anthropogenic water cycle where a wide range of chemical and biological markers of human activity can be found. In COVID-19 disease contexts, wastewater surveillance has been used to infer community trends based on viral abundance and SARS-CoV-2 RNA variant composition, which has served to anticipate and establish appropriate protocols to prevent potential viral outbreaks. Numerous studies worldwide have provided reliable and robust tools to detect and quantify SARS-CoV-2 RNA in wastewater, although due to the high dilution and degradation rate of the viral RNA in such samples, the detection limit of the pathogen has been a bottleneck for the proposed protocols so far. The current work provides a comprehensive and systematic study of the different parameters that may affect the detection of SARS-CoV-2 RNA in wastewater and hinder its quantification. The results obtained using synthetic viral RNA as a template allow us to consider that 10 genome copies per µL is the minimum RNA concentration that provides reliable and consistent values for the quantification of SARS-CoV-2 RNA. RT-qPCR analysis of wastewater samples collected at the WWTP in Salamanca (western Spain) and at six pumping stations in the city showed that below this threshold, positive results must be confirmed by sequencing to identify the specific viral sequence. This allowed us to find correlations between the SARS-CoV-2 RNA levels found in wastewater and the COVID-19 clinical data reported by health authorities. The close match between environmental and clinical data from the Salamanca case study has been confirmed by similar experimental approaches in four other cities in the same region. The present methodological approach reinforces the usefulness of wastewater-based epidemiology (WBE) studies in the face of future pandemic outbreaks.

Characterization and protein engineering of a novel UDP-glycosyltransferase involved in pseudoginsenoside Rt5 biosynthesis from Panax japonicus.

As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.

Real-Time RT-PCR Assays for Typing of Epizootic Hemorrhagic Disease Virus.

Real-time RT-PCR for the detection of epizootic hemorrhagic disease virus (EHDV) in clinical samples is a fast and sensitive tool for the diagnosis and confirmation of disease. Several real-time RT-PCR methods have been reported over the last 10 years. In this chapter, we describe seven duplex real-time RT-PCR assays to amplify part of genome segment 2 of EHDV to enable serotype identification. The assay includes the detection of an endogenous control gene-beta-actin.

End-Point RT-PCR Assays for Detection and Typing of Epizootic Hemorrhagic Disease Virus.

The emergence of EHDV in Europe during the autumn of 2022 reinforces the need for molecular tools (RT-PCR) for rapid detection of animals infected with this virus. Viral genome testing can be performed on whole blood under anticoagulant, spleen, and bloody organ homogenates from ruminants. It can also be performed on cell culture following viral isolation tests. Various so-called classical or end-point RT-PCRs will be described, which permit the amplification of a part of the viral genome (targeting segment 7) allowing the detection of EHDV whatever the serotype (pan-RT-PCR) and also to amplify a portion of the gene coding the viral protein (VP) 2 enabling serotyping. The PCR amplification products are visualized by agarose gel electrophoresis. Sequencing of the type-specific RT-PCR amplification products allows for the serotype of the virus to be determined.

Real-Time RT-PCR for the Diagnosis of Epizootic Hemorrhagic Disease Virus.

Real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) has become an essential tool in rapid and reliable detection of animal diseases such as epizootic hemorrhagic disease (EHD). Here we provide a protocol for the detection of epizootic hemorrhagic disease virus (EHDV) genetic material in blood and tissue samples, using a real-time RT-PCR that targets a conserved region in segment 9 of the EHDV genome. This protocol can be used to detect up to approximately 90 samples in a single run and can be completed in less than 4 h.

Characterization of the dominant strain (G-VII) of Newcastle disease viruses isolated from commercial chickens in Bangladesh during recent outbreaks.

Objective: Newcastle disease virus genotype VII (NDV-GVII), an extremely infectious pathogen, has been causing severe economic consequences for the chicken industry. The current study aimed to isolate and characterize NDV-GVII from commercial chickens in Bangladesh during a recent outbreak. Materials and Methods: From clinically suspected chickens from 70 commercial poultry farms, a total of 420 samples (trachea, lungs, and brain tissue) were collected. The samples were cultivated in 9-10 day-old seronegative embryonated chicken eggs (ECEs) after evaluating them using the rapid Newcastle disease virus (NDV) antigen detection kit. The hemagglutination (HA) inhibition test, agar gel immune diffusion (AGID) test, molecular detection by reverse transcription-polymerase chain reaction (RT-PCR), and phylogenetic studies using gene sequences of fusion (F) protein. The HA pattern of isolated NDV was determined using different avian and mammalian red blood cells (RBCs). The pathogenicity of the isolated virus was evaluated using mean death time (MDT), intravenous pathogenicity index (IVPI), and intracerebral pathogenicity index (ICPI). Results: The study found 87 NDV samples positive using the rapid NDV Ag detection kit and then 60 positives for virus isolation in ECEs. All 60 isolates were positive for NDV by HI, AGID, and RT-PCR. Phylogenetic tree analysis indicated that recent NDV isolates belong to genotype VII and exhibit a similarity of 99.7%-98.5% with isolates from Bangladesh, Iran, and India. The new isolates, identified as velogenic strains of NDV, possess an F protein cleavage site with 112-R-T-K-R-F-117 amino acid motifs. The isolated NDV showed diversified HA activity while using RBCs from birds and mammals. The results of ICPI, IVPI, and MDT indicated that the recent NDV isolates were very virulent. Conclusion: This study concluded that NDV-GVII is prevalent in commercial poultry farms in Bangladesh.

Monkeypox-related ophthalmic disease (MPXROD): Monitoring the antiviral effect of tecovirimat with monkeypox virus detection in tear samples.

INTRODUCTION: Although monkeypox-related ophthalmic disease (MPXROD) is rare, visual impairing complications have been reported. At present, tecovirimat is the standard-of-care antiviral treatment. In this MPXROD case, the effect of tecovirimat was assessed with PCR analysis of tear samples and concurrent monitoring of inflammation with laser flare photometry (LFP). CASE DESCRIPTION: The patient presented with a palpebral lesion and a corneal ulcer in his right eye, with complete absence of the corneal epithelium, high intraocular pressure and anterior uveitis. MPXV-DNA was detected in tear samples with real-time PCR (RT-PCR). A total volume of 0.5 ml tear-wash was aspirated from the inferior fornix, following instillation of saline onto the ocular surface. In addition, LFP was used to quantify inflammation in both eyes. Viral load in tear samples was detected prior to treatment initiation. In the left eye, tear samples tested negative for MPXV-DNA one week post-treatment while MPXV-DNA was still detected in the right eye, before reaching undetectable levels four weeks post-treatment. Objective quantification of anterior chamber inflammation through LFP demonstrated gradual decrease that was more pronounced in the affected right eye and coincided with the clinical improvement of the corneal ulcer. CONCLUSIONS: This case of Mpox related corneal ulcer with associated uveitis manifests the feasibility of monitoring the antiviral effect of tecovirimat with virus detection in tear samples and LFP. Our observations indicate that tecovirimat resulted in viral load reduction in both eyes. RT-PCR MPXV detection in tear samples and LFP represent noninvasive tools that could assist with treatment response monitoring.