Real-time RT-PCR Research Articles

Emergence and spread of the SARS-CoV-2 omicron (BA.1) variant across Africa: an observational study.

In mid-November, 2021, the SARS-CoV-2 omicron variant (B.1.1.529; BA.1 sublineage) was detected in southern Africa, prompting international travel restrictions. We aimed to investigate the spread of omicron BA.1 in Africa. In this observational study, samples from patients infected with SARS-CoV-2 from 27 laboratories in 24 African countries, collected between June 1, 2021 and April 14, 2022, were tested for omicron BA.1 and delta (B.1.617.2) variants using real-time RT-PCR. Samples that tested positive for BA.1 by RT-PCR and were collected before estimated BA.1 emergence according to epidemiological properties were excluded from downstream analyses. The diagnostic precision of the assays was evaluated by high-throughput sequencing of samples from four countries. The observed spread of BA.1 was compared with mobility-based mathematical simulations and entries for SARS-CoV-2 in the Global Initiative on Sharing All Influenza Data (GISAID) genomic database. We estimated the effective reproduction number (Rt) at the country level considering the BA.1 fraction and the reported numbers of infections. Phylogeographical analyses were done in a Bayesian framework. Through testing of 13 294 samples from patients infected with SARS-CoV-2, we established that, by November-December, 2021, omicron BA.1 had replaced the delta variant of SARS-CoV-2 in all African subregions, following a south-north gradient, with a median Rt of 2·60 (95% CI 2·46-2·71). This south-north spread, established on the basis of PCR data, was substantiated by phylogeographical reconstructions, ancestral state reconstructions, and GISAID data. PCR-based reconstructions of country-level BA.1 predominance and the availability of BA.1 genomic sequences in GISAID correlated significantly in time (p=0·0002, r=0·78). The first detections of BA.1 in high-income settings beyond Africa were predicted accurately in time by mobility-based mathematical simulations (p<0·0001). Comparing PCR-based reconstructions with mobility-based mathematical simulations suggested that SARS-CoV-2 infections in Africa were under-reported by approximately ten times. Inbound travellers infected with BA.1, departing from five continents, were identified in six African countries by early December, 2021. Omicron BA.1 was widespread in Africa when travel bans were implemented, limiting their effectiveness. Combined with genomic surveillance and mobility-based mathematical modelling, PCR-based strategies can inform Rt and the geographical spread of emerging pathogens in a cost-effective and timely manner, and can guide evidence-based, non-pharmaceutical interventions such as travel restrictions or physical distancing. Bill & Melinda Gates Foundation. For the French, Portugese and Spanish translations of the abstract see Supplementary Materials section.

Severe acute respiratory syndrome coronavirus 2 variants in patients with coronavirus disease 2019 and environmental sampling from the hospital and market during the coronavirus disease 2019 pandemic in Thailand

Limited genomic surveillance data is available for SARS-CoV-2 in Thailand during the second and third wave outbreaks, including both patient and environmental samples. This study investigated the presence of SARS-CoV-2 RNA in patient samples, on frequently touched surfaces, and in environmental swab samples (EVSs) collected from urban markets in Bangkok between April 2021 and August 2022. A total of 78,159 nasopharyngeal swab samples from patients and 327 environmental swab samples from hospital and market settings were collected. SARS-CoV-2 RNA was detected in 3,706 of 78,159 patient samples and one of 327 environmental samples using real-time RT-PCR. In total, 54 patient samples and an environmental sample were subjected to whole-genome sequencing and mass array genotyping, respectively. Only 46 samples passed the quality assessment based on the analysis criteria. The lineages detected included B.1.1.529 (2 samples), B.1.1.7 (15 samples), B.1.351 (3 samples), B.1.36.16(6 samples), B.1.617.2 (1 sample), AY.102 (1 sample), AY.4 (11 samples), AY.25 (1 sample), BA.1 (1 sample), BA.1.1 (3 samples), and BA.2 (2 samples). The phylogenetic analysis of the viral genome sequences revealed similar lineages during this study period.

Mechanisms of immune responses in Acanthopagrus latus to Streptococcus agalactiae infection revealed by transcriptomic analysis.

Acanthopagrus latus (yellowfin seabream) is an economically important fish in the southeast coastal sea of China. Its slower growth rate makes it more prone to diseases in the cultivation period, leading to substantial economic losses. Epidemiological investigations have indicated that Streptococcus agalactiae is one of the most common Gram-positive pathogens, which have garnered increasing attention due to its high contagion and lethality rates in A. latus. In this work, an infection model of yellowfin seabream was established with an intraperitoneal injection of S. agalactiae. Clinical sign observations and various analyses, including histological examination, serum biochemical index assessment, immune-related enzyme level measurement, and transcriptome analysis of tissues (liver and intestine) with obvious clinical signs, were conducted for revealing the effects of S. agalactiae infection and immune response mechanisms in yellowfin seabream. The results indicate that evident clinical signs and multi-tissue damages with the notable changes in indices and significant increase in immune-related enzyme levels in the serum occurred in infected fish. RNA sequencing analysis identified 1130 differentially expressed genes (DEGs) in the liver and 1218 DEGs in the intestine, which were involved in multiple immune- and metabolism-related pathways via KEGG enrichment analysis. The transcriptomic results were further corroborated by quantitative real-time RT-PCR (qRT-PCR) tests of some specific immune-related genes. These findings provide new insights into the molecular immune mechanisms in yellowfin seabream following S. agalactiae infection and offer valuable reference data for disease prevention and molecular breeding (i.e., selective breeding through developing molecular markers of key genes).

Performance evaluation of TaqMan™ Arbovirus Triplex Kit (ZIKV/DENV/CHIKV) for detection and differentiation of Dengue and Chikungunya viral RNA in serum samples of symptomatic patients

IntroductionGlobal outbreaks of mosquito-transmitted arbovirus infections, such as dengue (DENV) and chikungunya (CHIKV), are increasing. Differentiating these infections is challenging due to non-specific symptoms and serology limitations. PCR-based approaches offer higher sensitivity and specificity. This study evaluated the performance of TaqMan™ Arbovirus Triplex Kit (ZIKV/DENV/CHIKV) (TaqMan™ Kit) to detect DENV and CHIKV in clinical samples from patients in south India. MethodsIn total, 280 serum samples with 90 DENV-positive, 90 CHIKV-positive, and 100 negative samples were tested with TaqMan™ Kit and CDC Trioplex Real-Time RT-PCR assay. No Zika virus was detected. The sensitivity and specificity of viral RNA detection were determined, and discordant results were resolved using comparator PCRs, dengue NS1 antigen detection, virus-specific antibody results, or previously de-identified in-house PCR results. ResultsTaqMan™ Kit showed 100% agreement with the comparator for DENV detection in 92 positive samples. Among 188 samples negative for DENV by the comparator, 30 showed positive results with the TaqMan™ kit, and 23 of those were confirmed as true positives. The resulting sensitivity and specificity for DENV detection were 100% and 95.1%, respectively. For CHIKV, 77 positive and 195 negative results were concordant. Eight samples showed discordant results, but upon resolution testing, sensitivity and specificity for CHIKV were 93.9% and 100.0%, respectively. ConclusionTaqMan™ Arbovirus Triplex Kit demonstrated high sensitivity and specificity (>93%) for detecting circulating DENV and CHIKV strains. Multiplex PCR testing can improve case detection, surveillance, and public health responses while optimizing laboratory resources for outbreak control.

Concurrent transmission of Zika virus during the 2023 dengue outbreak in Dhaka, Bangladesh.

During the 2023-dengue outbreak in Bangladesh, a diagnostic evaluation study was conducted to investigate concurrent Zika virus (ZIKV) and dengue virus (DENV) transmission in Dhaka in 2023. The study explored to simultaneously detect the presence of ZIKV, DENV, and/or CHIKV while considering relevant clinical and epidemiological risk factors, using a real-time multiplex RT-PCR system. Following this, it was planned to sequence the selected samples to identify genetic variations of the ZIKV infections within the population. This study was designed as a diagnostic evaluation, where participants meeting the inclusion criteria were prospectively recruited with written informed consent. A total of 399 febrile individuals were screened, with 185 meeting the inclusion criteria of having a fever onset within 2-5 days, along with one of the following clinical features, e.g. headache, myalgia, arthralgia or bone pain, rash, nausea, vomiting, or diarrhea and 152 undergoing real-time RT-PCR testing. Five ZIKV-positive cases were identified, including one DENV-ZIKV co-infection. Phylogenetic analysis revealed the ZIKV strains were part of the Asian lineage, closely related to Cambodian and Chinese strains from 2019. All ZIKV-positive cases were male, residing within a one-kilometer radius, with no prior travel history, suggesting community-level transmission. This study marks the first identification of ZIKV in Dhaka city and the first report of ZIKV-DENV co-infection in Bangladesh that highlights the diagnostic challenges posed by the symptomatic similarities between ZIKV and other arboviruses and underscores the need for enhanced surveillance and public health interventions to mitigate the spread and impact of ZIKV in dengue-endemic regions.

P-217. Risk Factors Associated with Clostridioides difficile Infection and Mortality in a Referral Hospital in Nicaragua

Abstract Background Clostridioides difficile infection (CDI) is one of the main problems among healthcare-associated infections. Some studies report an increase in the incidence of this infection causing enterocolitis in hospitalized patients and eventually being associated with various complications, mortality, and increased hospital costs. The data about the epidemiology and outcomes of CDI in Nicaragua are insufficient. The aim of this study was to determine risk factors associated with CDI infection and mortality in patients admitted to a referral hospital (Dr. Fernando Vélez Paiz Hospital) in Managua, Nicaragua.Table 1.Clinical Factors Associated in Patients with and without Clostridioides difficile Infection Methods It is an analytical nested cohort study. A total of 150 patients hospitalized in different services of the hospital between January 2020 and December 2022 and who started with in-hospital diarrhea (defined as the absence of pre-existing diarrhea prior to hospitalization and onset 72 hours after admission to the hospital) were included. Of the150 patients, 50 were defined as CDI cases and 100 as controls without CDI. The diagnosis of CDI was made by molecular method (real-time RT-PCR) using Xpert C. difficile®. Differences between cases and controls were analyzed.Figure 1.Comparative Mortality Rate in Patients with and without Clostridioides difficile Infection Results The mean age of the patients was 52.8±18.7 years-old, 52% were men, and 50% had an associated chronic disease. A higher proportion of patients with CDI had a history of antibiotic use (98% vs 52%, p&amp;lt; 0.001), use of proton pump inhibitors (46% vs 25%, p=0.009), previous hospitalization (94% vs 46%, p&amp;lt; 0.001), and previous surgery (64% vs 25%, p&amp;lt; 0.001) (Table 1). Mortality in patients with CDI was 20% vs. 11% in controls (Figure 1). In the univariate analysis, the risk factors for CDI were the classic ones, such as previous use of antibiotics (ceftriaxone, quinolones, clindamycin), previous hospitalization, previous surgery, and use of proton pump inhibitors. The multivariate analysis found that previous use of ceftriaxone (OR: 6.40; 95%CI: 2.09–19.59) and clindamycin (OR: 3.0; 95%CI: 1.01–8.92) were significantly associated with CDI (Table 2). Regarding mortality, a significant association was found with age over 60 years-old (OR: 4.33; 95%CI: 1.02-18.41).Table 2.Risk Factors in Multivariate Analysis for Clostridioides difficile Infection and Mortality Conclusion Prior use of ceftriaxone and clindamycin are independent risk factors for CDI. Patients with CDI over 60 years-old have a higher risk of mortality. Disclosures All Authors: No reported disclosures

P-2012. Predicting Persistent SARS-CoV-2 Shedding in Immunocompromised Patients: A Probability-Based Approach

Abstract Background Prolonged SARS-CoV-2 shedding frequently occurs in immunocompromised patients, making the determination of their isolation periods challenging. This study aims to estimate the probability of viral clearance in immunocompromised patients, stratified by elapsed days and other relevant variables. Figure 1 The estimated probability of viral clearance along days, stratified by SARS-CoV-2 PCR Ct value and immunocompromised conditions Methods This prospective study was conducted at Asan Medical Center, a 2,732-bed tertiary teaching hospital in the Republic of Korea, from January 2022 to May 2023 during the prevalence of the Omicron variant. We enrolled immunocompromised patients, defined as 'moderately or severely immunocompromised' according to CDC guidelines. All participants, diagnosed with COVID-19 via nasopharyngeal swab PCR, provided weekly respiratory specimens for viral load measurement using real-time RT-PCR. Positive samples underwent viral culture. The Cox time-varying proportional hazard model was applied to identify significant predictors of viral culture negative conversion, employing backward elimination for model refinement. Analyses were conducted using R software, version 4.3.2, and included the assessment of viral copy number dynamics and treatment effects on viral shedding probabilities. Baseline characteristics of the immunocompromised patients Results In a cohort of 70 immunocompromised patients, 48 (68.6%) had hematologic malignancies and 22 (31.4%) underwent solid organ transplants. Univariate and multivariate analyses revealed that the use of B-cell depleting agents, CAR T-cell therapy, and viral copy number significantly influenced viral culture negative conversion. These findings were incorporated into a refined model to predict viral shedding probabilities. Patients with a history of both B-cell depleting agents and CAR T-cell therapy exhibited the lowest negative conversion rates, with median durations of viral shedding from 76 days to undeterminable depending on SARS-CoV-2 Ct values. In contrast, patients receiving neither treatment showed markedly faster viral clearance, typically within one to two months after COVID-19 diagnosis, regardless of Ct values. Predictive factors for viral culture negative conversion in the immunocompromised patients Conclusion Ending the isolation of immunocompromised patients with COVID-19 should be determined individually, based on the viral copy number at specific time points and treatment conditions. Table 3 Probability of culture-negative conversion among immunocompromised patients based on treatments received, days elapsed after diagnosis, and SARS-CoV-2 PCR results Disclosures All Authors: No reported disclosures

Systemic infection of cowpea aphid-borne mosaic virus in Passiflora spp. occurs at the initial stage regardless of the species' resistance.

Passion fruit woodiness disease (PWD), caused by cowpea aphid-borne mosaic virus (CABMV), severely damages leaves and fruits, compromising passion fruit production. The dynamics of this infection in Passiflora spp. are still poorly understood. The objective of this study was to determine the systemic infection time of CABMV in Passiflora spp. and to quantify the viral titer throughout the infection. Plants of Passiflora edulis Sims. (BGP418, susceptible), P. cincinnata Mast. (BGP243, moderately resistant), P. setacea DC. (BRS Pérola do Cerrado, resistant), and P. suberosa L. (BGP152, resistant) were used. The study was conducted in a climate chamber, and mechanical inoculations were carried out on the first pair of basal leaves of the seedlings. Symptoms were assessed using a scale whose scores were converted into a disease index (DI%), and the viral titer was determined at different time points by real-time quantitative RT-PCR (RT-qPCR). The first symptoms of the virus were observed at seven days after inoculation (Dai) in P. edulis (DI = 5.15%) and at 10 Dai in P. cincinnata (DI = 8.86%). On the other hand, P. setacea and P. suberosa did not show typical symptoms of the disease (DI = 0.00%). Systemic CABMV infection was detected at 30 minutes after inoculation regardless of the level of resistance of the Passiflora species. There was an increase in viral titer with infection time with P. edulis and P. cincinnata, although in the case of P. edulis, the increase in CABMV titer occurred earlier, at 2 Dai, and in P. cincinnata at 8 Dai. In the asymptomatic species (P. setacea and P. suberosa), there was no variation in the viral titer over the time periods evaluated. This pioneering study provides information for the selection of time intervals for future molecular research into the interaction between Passiflora spp. and CABMV.

P-2324. Detections of Endemic Coronaviruses in Medically Attended Respiratory Illness: Clinical Characteristics and Seasonal Trends from Two Public Health Surveillance Studies in Minnesota

Abstract Background Human coronavirus (hCoV) strains OC43, NL63, HKU1, and 229E exist worldwide and have the potential to cause severe disease. Understanding hCoV seasonality, clinical symptoms, and risk factors for severe disease may help predict the impact of emerging respiratory viral pathogens. Methods Eighteen outpatient clinics submitted clinical data and respiratory specimens from patients with influenza-like illness between 2010-2023. Inpatient surveillance data was submitted from 3 hospitals (including one freestanding children’s hospital) between 2013-2018 from patients with acute respiratory illness. Respiratory specimens were tested using a multi-target real-time RT-PCR for 23 bacterial and viral respiratory pathogens. Results We tested 24,765 specimens (51.2% hospitalized, 48.8% outpatients) and identified 1,207 non-SARS-CoV-2 coronavirus-positive cases (42% hospitalized, 58% outpatient). 74% of inpatient cases were submitted by a standalone children’s hospital. For hCoV+ cases the median outpatient age in years was 36.5 and inpatient age was 0.6. All hCoV strains were detected at higher frequency during the winter months (Figure 1) and sequential winter seasons demonstrated different dominant strains of hCoV (Figure 2). hCoV was co-detected with other viral pathogens in 29% of cases but strains were not co-detected with each other (Figure 3). The median age for cough, diarrhea, fever, URI symptoms, seizure, vomiting and wheeze was ≤1.0 years, while headache, myalgia, and sore throat was 27-31 years (Figure 4). Among hospitalized children with only hCoV detected, comorbidities included neurologic disorder(22%), developmental delay(14%), seizure disorder(14%), cardiovascular disease(13%), prematurity(12%), and asthma(10%). 16% required mechanical ventilation, 27% required ICU admission, and 41% received antiviral therapy. Patients with hCoV+1 other pathogen compared to those with singular hCoV+ infection did not have an increased risk for ICU admission or mechanical ventilation. Conclusion hCoV circulation in Minnesota demonstrates temporal variation in dominant hCoV strain. hCoV infection may present with different symptoms according to the patient’s age. hCoV may contribute to severe respiratory disease in the absence of other pathogenic viruses. Disclosures Beth K. Thielen, MD, PhD, GSK: Honoraria|Merck: Grant/Research Support|Sanofi: Honoraria|Seegene: Honoraria

Comparison of AccuPower Diarrhea V1&amp;V2 RT-PCR to a Chromatographic Immunoassay for Detecting Viral Pathogens from Human Diarrheal Stool Specimens

Viruses are a frequent cause of self-limited diarrhea, with more severe outcomes in immunocompromised patients. This study aimed to compare the performance of Real-Time RT-PCR to chromatographic immunoassays (CIAs) for detecting the major gastrointestinal viruses in human stool. This study was conducted at the University Hospital of Split, Croatia, from October 2023 to May 2024. Stool samples were simultaneously analyzed with CIA (Acro Biotech Rotavirus and Adenovirus Combo Rapid Test Cassette, USA and JusChek Norovirus Rapid Test Cassette, China) and Real-Time RT-PCR (AccuPower Diarrhea V1&amp;V2 Real-Time RT-PCR, Bioneer, Republic of Korea), according to the manufacturers’ instructions. Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) were calculated. For norovirus, CIA had a low PPA (25%), indicating that it missed 75% of norovirus-positive cases identified by RT-PCR. Adenovirus detection by CIA showed poor agreement with RT-PCR (PPA 0%; NPA 100%). Rotavirus detection presented a relatively better performance with CIA (PPA 90.9% and OPA 84.13%). However, the presence of false positives (15.8%) highlights the need for confirmatory RT-PCR testing. One specimen was sapovirus-RT-PCR-positive, marking the first documented case from human specimens in Croatia. Although CIA provided rapid results, limitations regarding reliability highlight the value of RT-PCR, particularly in the case of ambiguous clinical cases with negative antigenic test results and newly emerged viruses. A two-step diagnostic approach, with initial CIA screening followed by confirmatory RT-PCR, could balance cost-effectiveness with diagnostic accuracy.

Detection and genomic characterisation of foot-and-mouth disease virus serotypes circulating in Cameroon using environmental sampling

Foot-and-mouth disease virus (FMDV) is a highly contagious, economically important disease of livestock and wildlife species. Active monitoring and understanding the epidemiology of FMDV underpin the foundations of control programmes. In many endemic areas, however, veterinary resources are limited, resulting in a requirement for simple sampling techniques to increase and supplement surveillance efforts. In this study, environmental sampling was used for the first time at livestock markets and abattoirs across Cameroon to assess the opportunities for broad scale, non-invasive disease surveillance at such sites. Environmental samples (n = 1994) were collected from six locations across Cameroon between May and July 2019. Concurrent with environmental sampling, a questionnaire was used to gather descriptive information on the use and practices of market and abattoir sites. Samples were screened for the presence of FMDV RNA using a pan-serotype FMDV specific real-time RT-PCR assay. Positive samples were characterised at the genomic level using next generation sequencing in combination with a novel probe-based enrichment strategy. A total of 173/1994 (8.68%) environmental samples were found to be positive for FMDV RNA. Genome length sequences were obtained from environmental samples, with phylogenetically relevant capsid sequences obtained from 14 samples, with representatives of serotypes O (n = 6), A (n = 7) and SAT 2 (n = 3). The questionnaire results revealed that animals in Cameroon can be transported long distances to markets and abattoirs, with varying levels of control and biosecurity practices in place. The approaches used in this study have highlighted that environmental sampling is an effective and non-invasive approach to assessing FMDV presence. Furthermore, the study has demonstrated that livestock markets, abattoirs and trucks could be targeted for the introduction of biosecurity interventions as well as providing opportunities for carrying out disease surveillance. Information resulting from such surveillance could provide valuable knowledge of circulating viruses within a region of interest, aiding strategic approaches for surveillance and control of FMDV.

A nucleocapsid monoclonal antibody based sandwich ELISA for the general detection of both PRRSV-2 and PRRSV-1.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes reproductive failure in sows and respiratory disease in growing pigs, leading to significant economic losses worldwide. Due to the constant mutation and recombination, PRRSV exhibits significant genetic diversity, the general detection of all PRRSV-2 and PRRSV-1 strains is thus needed. In our study, four monoclonal antibodies (mAbs) against PRRSV nucleocapsid (N) protein were generated and the precise and novel B cell epitopes (52KPHF55 and 109HHTVR113) were identified. The epitope 52KPHF55 is highly conserved across all strains of PRRSV-2 lineages and PRRSV-1 subtypes, and the corresponding two mAbs (6D7, 4D12) were selected to develop a sandwich ELISA that was able to detect all tested PRRSV-2 and PRRSV-1 strains. The developed sandwich ELISA demonstrated high specificity, sensitivity and repeatability. In detection of 133 clinical samples, the sandwich ELISA achieved 84.21 % coincidence with the real-time RT-PCR. In conclusion, the mAb based sandwich ELISA can be suitable for detection of potential all PRRSV-2 lineages and PRRSV-1 subtypes, providing a simple, quick and high content method for diagnosis of PRRS.

Development of a TaqMan real-time RT-PCR protocol for rice stripe necrosis virus detection in plant material and soil

Development of a TaqMan real-time RT-PCR protocol for rice stripe necrosis virus detection in plant material and soil

SNORA37/CMTR1/ELAVL1 feedback loop drives gastric cancer progression via facilitating CD44 alternative splicing

BackgroundEmerging evidence shows that small nucleolar RNA (snoRNA), a type of highly conserved non-coding RNA, is involved in tumorigenesis and aggressiveness. However, the roles of snoRNAs in regulating alternative splicing crucial for cancer progression remain elusive.MethodsHigh-throughput RNA sequencing and comprehensive analysis were performed to identify crucial snoRNAs and downstream alternative splicing events. Biotin-labeled RNA pull-down, mass spectrometry, cross-linking RNA immunoprecipitation, and in vitro binding assays were applied to explore interaction of snoRNAs with protein partners. Alternative splicing and gene expression was observed by real-time quantitative RT-PCR and western blot assays. In vitro and in vivo studies were performed to investigate biological effects of snoRNAs and their protein partners in gastric cancer. Survival analysis was undertaken by using Kaplan-Meier method and log-rank test.ResultsSNORA37 was identified as an up-regulated snoRNA essential for tumorigenesis and aggressiveness of gastric cancer. Gain- and loss-of-function studies indicated that SNORA37 promoted the growth, invasion, and metastasis of gastric cancer cells in vitro and in vivo. Mechanistically, as an ELAV like RNA binding protein 1 (ELAVL1)-generated snoRNA, SNORA37 directly bound to cap methyltransferase 1 (CMTR1) to facilitate its interaction with ELAVL1, resulting in nuclear retention and activity of ELAVL1 in regulating alternative splicing of CD44. Rescue studies revealed that SNORA37 exerted oncogenic roles in gastric cancer progression via facilitating CMTR1-ELAVL1 interaction. In clinical gastric cancer cases, high levels of SNORA37, CMTR1, ELAVL1, or CD44 were associated with shorter survival and poor outcomes of patients.ConclusionsThese results indicated that SNORA37/CMTR1/ELAVL1 feedback loop drives gastric cancer progression via facilitating CD44 alternative splicing.

Histone Demethylase KDM6B Promotes Chondrogenic Differentiation Potential of Stem Cells from the Apical Papilla Via HES1.

Mesenchymal stem cell (MSC)-based therapies have emerged as a promising approach for treating articular cartilage injuries. However, enhancing the chondrogenic differentiation potential of MSCs remains a significant challenge. KDM6B, a histone demethylase that specifically removes H3K27me3 marks, is essential in controlling the maturation of chondrocytes. In this study, we examined how KDM6B influences chondrogenic differentiation in SCAPs and investigated the underlying mechanisms involved. SCAPs were utilized. Alcian Blue staining, pellet culture, and cell transplantation in rabbit knee cartilage defect models assessed MSC chondrogenic differentiation. Western blot, Real-time RT-PCR, and Microarray analysis examined the underlying molecular mechanisms. KDM6B promotes the expression of Aggrecan, COL2A1, COL5, glycosaminoglycans, and collagen fibers, while also increasing the COL2/COL1 ratio in SCAPs. In vivo, SCAPs overexpressing KDM6B significantly enhanced the repair and regeneration of knee cartilage and subchondral bone, with higher levels of glycosaminoglycan and COL5 expression observed within the tissue. KDM6B promotes the chondrogenic differentiation potential of SCAPs by repressing HES1. In addition, knock-down of HES1 enhanced the chondrogenic differentiation of SCAPs. KDM6B enhances the differentiation of SCAPs into chondrocytes and demonstrated its effectiveness in the repair and regeneration of cartilage tissue and subchondral bone in vivo experiments. These findings provide an important foundation for future research on the use of dental tissue-derived stem cells to treat cartilage injuries.

Evidence of avian and human influenza A virus infection in farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

Crocodilians are susceptible to a range of virus infection including influenza A virus (IAV). However, little is known about the ecology and epidemiology of IAV in crocodile species. This study aimed to investigate IAV infection in farmed Siamese crocodiles in central Thailand. We collected plasma samples and pharyngeal swab samples from Siamese crocodiles residing in 13 crocodile farms in 9 provinces of central Thailand during 2019. Additional archival plasma samples of Siamese crocodiles collected in 2012 and 2018 were also included in the study. Plasma samples were screened for influenza A antibodies by a hemagglutination inhibition (HI) assay and positive were evaluated by a cytopathic effect/hemagglutination based-microneutralization (MN) assay. Swab samples were tested for influenza viral RNA by a real-time RT-PCR assay targeting the influenza matrix (M) gene. Among 246 tested plasma samples, the overall seroprevalence of antibodies against IAV in farmed Siamese crocodiles was 17.5% (43/246). The most common hemagglutinin (HA) subtype was H2 (46.5%, 20/43) followed by H9 (39.5%, 17/43), human H1 (14%, 6/43) and H1 (7%, 3/43). Multiple HA subtypes were also detected in 7% (3/43) of infected crocodiles with combination of H1 and H2 subtypes. All 126 tested swab samples were negative for influenza viral RNA. In addition, we demonstrated the ability of wild-type IAV subtypes (H1, H2, H9 and human H1) to infect primary Siamese crocodile fibroblast cells. To our knowledge, this is the first report of serological evidences of avian and human IAV infection in Siamese crocodiles. Our findings highlighted the role of crocodile species in the ecology of IAV particularly the potential to serve as the reservoir or mixing vessel for the viruses that significantly threaten both human and animal health.

The Prevalence Of Covid-19 In High-Risk District Of Kpk, Mardan, Pakistan

Objectives: to investigate the prevalence of COVID-19 and to find out the prevalence of COVID-19 positive individuals with full-vaccination, partial-vaccination and those who were non-vaccinated individuals in Mardan, Khyber Pakhtunkhwa, Pakistan. Study Design: This Multi-Center and retrospective study was carried out to determine the prevalence of COVID-19 in children, adults and aged old individuals using RT-PCR as a method of detection. Place and Duration of the Study: Department of Pathology at Mardan Medical Complex from August 1st, 2021 to August 31st 2021. Methodology: A random sample was taken from the population as well as all suspected persons who were admitted to various isolation wards and quarantine centres throughout District Mardan to be tested. The real-time RT-PCR tests were performed for testing the COVID-19 samples. The statistical analysis was performed with a 5% significance level. Results: A total of 2960 samples were collected. Among all the samples, 1536 individuals were male while females were 1424. The ages of the participants ranged from 0 to 80 years, with the average age being 40 years. The patients' median age was discovered to be 40.66 years. The youngest patient was only 1.5 months old, and the oldest patient was 80 years old. Males were particularly affected when compared to females. The RT-PCR tests were performed for testing the COVID-19 samples. The overall prevalence in Mardan was found 10.03 % (297 out of 2960), in which 161 out of 1536 (10.48%) were males positive cases and 136 out of 1424 (9.55%) were female positive COVID-19 cases whereas 2663 out of 2960 (89.96%) were reported as negative COVID-19 cases. Conclusions: The prevalence of COVID-19 in the Mardan district is extremely high, necessitating the implementation of WHO-recommended SOPs to control the spread of COVID-19. Keywords: Sars-Corona virus-2, COVID-19, Prevalence, RT-PCR, Statistical Package for Social Sciences version 28 (SPSSv28), Mardan Khyber Pakhtunkhwa, Pakistan

P-Glycoprotein Drives Glioblastoma Survival and Chemotherapy Resistance: Potential as a Promising Liquid Biopsy Biomarker.

Drug resistance is a major challenge in cancer therapy, and the expression of efflux pumps such as P-glycoprotein (P-gp, ABCB1) often correlates with poor prognosis in various tumors, including glioblastoma (GB). Considering that different roles for these proteins have been established in the biology of various tumors, this study aimed to investigate the functions of P-gp in GB-derived cells by evaluating its survival, migratory, and apoptosis-regulating capabilities, as well as its potential as a liquid biopsy biomarker. P-gp expression was diminished via siRNA to determine its exact role in GB biology. The P-gp mRNA levels were evaluated by using quantitative real-time RT-PCR. With respect to liquid biopsy, circulating cell-free RNA was extracted from plasma belonging to patients diagnosed with GB, and P-gp levels were compared with matching tumor tissues using digital PCR. The results showed that P-gp silencing significantly decreased viability, increased apoptosis, and enhanced chemotherapy sensitivity in GB cells, although it did not affect migratory patterns. Finally, P-gp expression levels in circulating cell-free RNA from patients with GB matched tumor tissue, whereas healthy volunteers seemed to bear no circulating P-gp. Taken together, the results indicate that P-gp affects GB tumor biology beyond its known role in drug resistance and could integrate a broader molecular signature for future diagnosis via liquid biopsy.

Molecular Diagnosis of Phlebovirus riftense Infection Using an L-Segment-Based Real-Time RT-PCR Method.

Rift Valley fever (RVF) is one of the main vector-borne zoonotic diseases that affects a wide range of ruminants and humans in Africa and the Arabian Peninsula.Several techniques involving cell culture and molecular biology methods have been developed to diagnose RVF infection. Success partly relies on sending samples to a national or reference laboratory in good conditions and having the capacity to perform the appropriate diagnostic test in the matrix of interest during the period of viremia where high loads of viral particles are present. Conventional and duplex/multiplex real-time reverse transcriptase polymerase chain reaction (RT-PCR) assays are currently the most rapid and sensitive molecular tests for the detection and quantification of Rift Valley fever virus (RVFV) during outbreaks. The one-step real-time duplex RT-PCR technique that is detailed in this chapter is based on the L segment of RVFV. Results must be carefully validated and interpreted, taking into account the results of both positive and negative controls.

Epidemiology and Genotype Dynamics of Dengue in Hospitalized Patients in Northern Vietnam Between 2020 and 2022.

Arboviruses, including Dengue (DENV), Zika, and chikungunya, cause recurrent outbreaks of varying intensity in tropical countries. This study aimed to investigate other arboviruses, including Zika and chikungunya, in patients clinically suspected of Dengue and to characterize the circulating Dengue serotypes and genotypes in Northern Vietnam from 2020 to 2022. To date, information on this topic in the region has been limited. Multiplex real-time polymerase chain reaction (PCR) was used to detect Dengue, Zika, and chikungunya RNA, and DENV serotypes were identified via real-time reverse transcriptase PCR from 426 clinically Dengue suspected patients. Patients were screened for NS1 antigen and anti-DENV immunoglobulin (Ig) G/IgM antibodies. Phylogenetic analysis of DENV Capsid-premembrane gene sequences was performed to investigate genotype distribution. Dengue was confirmed in 95% of cases, with no Zika or chikungunya RNA detected. DENV-2 was the predominant serotype (61%), followed by DENV-1 (31%) and DENV-4 (7%). Coinfections were observed, with DENV-1 and DENV-2 being the most common. In 2022, a high incidence of Dengue cases with warning signs and severe Dengue was observed, accompanied by elevated liver enzyme levels and reduced platelet counts. Phylogenetic analysis revealed that the DENV-1 and DENV-4 serotypes clustered with previously reported regional virus, while DENV-2 showed a shift from genotype Asian I to Cosmopolitan over the study period. This study underscores a significant rise in Dengue severity and shifts in DENV genotypes in recent years in Northern Vietnam, emphasizing the importance of understanding genotype dynamics and clinical dynamics for improving outbreak preparedness and response strategies.