Performance comparison of three commercial multiplex molecular panels for respiratory viruses at a South African academic hospital.

Background: Respiratory infections are a major contributor to hospital admissions. Identification of respiratory pathogens by means of conventional culture and serology methods remains challenging. Multiplex molecular assays are an appealing alternative that endeavours to be rapid, more accurate and less arduous. Objective: The study aimed to compare the clinical performance of three commercial multiplex molecular assays for respiratory viruses. Methods: Forty-eight respiratory specimens obtained from patients at Tygerberg Hospital in the Western Cape province of South Africa were studied. These specimens were collected between May 2020 and August 2020. The results of the Seegene Anyplex™ II RV16, FilmArray® Respiratory 2.1 plus Panel (FARP), and QIAstat-Dx® Respiratory SARS-CoV-2 Panel (QRP) were analysed based on the overlapping targets. A composite reference standard was applied to provide a standard reference for comparison. Results: The overall sensitivity of the Seegene Anyplex™ II RV16 was 96.6% (57/59), the FARP 98.2% (56/57) and the QRP 80.7% (46/57). The overall specificities were 99.8% (660/661), 99.0% (704/711) and 99.7% (709/711), respectively. The QRP failed to detect coronaviruses and parainfluenza viruses in 41.7% (5/12) and 28.6% (4/14) of positive specimens, respectively, while the FARP produced the lowest target specificity of 88.4% (38/43) for rhinovirus/enterovirus. Conclusion: The overall specificity of all three platforms was comparable; however, the sensitivity of the QRP was inferior to that of the ARV and FARP. What this study adds: This study adds to the body of performance characteristics described for respiratory multiplex panels, especially in the African context where molecular diagnostics for infectious diseases are gaining momentum.

Molecular detection and genotyping of bovine viral diarrhea virus in Selangor, Malaysia.

Objective: Bovine viral diarrhea (BVD) disease is a viral infection in cows caused by a single-stranded plus-sense RNA virus of the Pestivirus genus under the Flaviviridae family. The clinical manifestation of BVD mainly includes diarrhea and immunosuppression, thereby exacerbating various respiratory diseases. This study was conducted to detect and molecularly characterize the bovine viral diarrhea disease virus (BVDV) in cattle on selected farms in Selangor, Malaysia. Materials and Methods: A reverse transcription polymerase chain reaction (RT-PCR) was performed for antigen detection in 253 plasma samples collected from cows using a cross-sectional study design. We selected the 5 untranslated regions (5'-UTR) region and the E2 region to compare the genetic differences between the isolates. Results: One sample was found to be positive (1/253) following RT-PCR targeting the conserved 5'-UTR region of BVDV. Thus, BVDV antigen prevalence was 0.40% (95% confidence interval: 0.0%-2.2%). By targeting the hypervariable E2 region of the isolated virus, UPM/MAL/BVDV/D17, the virus was classified under the subgenotype BVDV-1a. Conclusion: BVDV is present and circulating on selected cattle farms in Selangor, Malaysia. Given the presence of BVDV in several subgenotypes, the screening of all incoming cattle at Malaysia's border is pertinent to prevent the entry of other BVDV subgenotypes into the country.

Long-term trends in cycle threshold values: a comprehensive analysis of COVID-19 dynamics, viral load, and reproduction number in South Korea.

Background: With the emergence of COVID-19 cases, governments quickly responded with aggressive testing, contact tracing, isolation and quarantine measures. South Korea's testing strategy primarily relied on real-time reverse-transcriptase polymerase chain reaction (real-time RT-PCR), focusing on cycle threshold (Ct) values, indicative of viral load, to determine COVID-19 positivity. This study examined the long-term time series distribution of Ct values measured in the same laboratory using a nationally standardized testing type and sampling method in South Korea. It aimed to link Ct values, new COVID-19 cases, and the reproduction number (Rt), setting the stage for using Ct values effectively. Methods: This study analyzed nationally collected 296,347 samples Ct values from February 2020 to January 2022 and examined their associations with the number of new cases and Rt trends. The data were categorized into four COVID-19 periods for in-depth analysis. Statistical methods included time series trend analysis, local regression for smoothing, linear regression for association analysis, and calculation of correlation coefficients. Results: The median Ct values across four COVID-19 periods decreased gradually from 31.71 in the initial period to 21.27 in the fourth period, indicating higher viral load. The comparison of trends between Ct values and the number of new cases revealed that the decline in Ct values preceded the surge in new cases, particularly evident during the initial stages when new cases did not undergo a significant increase. Also, during variant emergence and vaccination rollout, marked shifts in Ct values were observed. Results from linear regression analysis revealed a significant negative relationship between Ct values and new cases (ß = -0.33, p < 0.001, R 2 = 0.67). This implies that as Ct values decrease, new case numbers increase. Conclusion: This study demonstrates the potential of Ct values as early indicators for predicting confirmed COVID-19 cases during the initial stages of the epidemic and suggests their relevance in large-scale epidemic monitoring, even when case numbers are similar.

Comparison of Different Reverse Transcriptase-Polymerase Chain Reaction-Based Methods for Wastewater Surveillance of SARS-CoV-2: Exploratory Study.

BACKGROUND: Many countries have applied the wastewater surveillance of the COVID-19 pandemic to their national public health monitoring measures. The most used methods for detecting SARS-CoV-2 in wastewater are quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR) and reverse transcriptase-droplet digital polymerase chain reaction (RT-ddPCR). Previous comparison studies have produced conflicting results, thus more research on the subject is required. OBJECTIVE: This study aims to compare RT-qPCR and RT-ddPCR for detecting SARS-CoV-2 in wastewater. It also aimed to investigate the effect of changes in the analytical pipeline, including the RNA extraction kit, RT-PCR kit, and target gene assay, on the results. Another aim was to find a detection method for low-resource settings. METHODS: We compared 2 RT-qPCR kits, TaqMan RT-qPCR and QuantiTect RT-qPCR, and RT-ddPCR based on sensitivity, positivity rates, variability, and correlation of SARS-CoV-2 gene copy numbers in wastewater to the incidence of COVID-19. Furthermore, we compared 2 RNA extraction methods, column- and magnetic-bead-based. In addition, we assessed 2 target gene assays for RT-qPCR, N1 and N2, and 2 target gene assays for ddPCR N1 and E. Reverse transcription strand invasion-based amplification (RT-SIBA) was used to detect SARS-CoV-2 from wastewater qualitatively. RESULTS: Our results indicated that the most sensitive method to detect SARS-CoV-2 in wastewater was RT-ddPCR. It had the highest positivity rate (26/30), and its limit of detection was the lowest (0.06 gene copies/µL). However, we obtained the best correlation between COVID-19 incidence and SARS-CoV-2 gene copy number in wastewater using TaqMan RT-qPCR (correlation coefficient [CC]=0.697, P<.001). We found a significant difference in sensitivity between the TaqMan RT-qPCR kit and the QuantiTect RT-qPCR kit, the first having a significantly lower limit of detection and a higher positivity rate than the latter. Furthermore, the N1 target gene assay was the most sensitive for both RT-qPCR kits, while no significant difference was found between the gene targets using RT-ddPCR. In addition, the use of different RNA extraction kits affected the result when the TaqMan RT-qPCR kit was used. RT-SIBA was able to detect SARS-CoV-2 RNA in wastewater. CONCLUSIONS: As our study, as well as most of the previous studies, has shown RT-ddPCR to be more sensitive than RT-qPCR, its use in the wastewater surveillance of SARS-CoV-2 should be considered, especially if the amount of SARS-CoV-2 circulating in the population was low. All the analysis steps must be optimized for wastewater surveillance as our study showed that all the analysis steps including the compatibility of the RNA extraction, the RT-PCR kit, and the target gene assay influence the results. In addition, our study showed that RT-SIBA could be used to detect SARS-CoV-2 in wastewater if a qualitative result is sufficient.

Comparable Efficacy of Lopinavir/Ritonavir and Remdesivir in Reducing Viral Load and Shedding Duration in Patients with COVID-19.

The spread of COVID-19 has significantly increased research on antiviral drugs and measures such as case isolation and contact tracing. This study compared the effects of lopinavir/ritonavir and remdesivir on COVID-19 patients with a control group receiving no antiviral drugs. Patients confirmed to have a SARS-CoV-2 infection via real-time RT-PCR were divided into three groups: lopinavir/ritonavir, remdesivir, and control. We assessed the efficacy of these drugs in reducing viral load and viral shedding duration using real-time RT-PCR and Vero E6 cell cultures. Lopinavir/ritonavir led to no detectable infectious SARS-CoV-2, with a median viral clearance time of one day, whereas one remdesivir-treated case remained culture-positive until day 12. Lopinavir/ritonavir significantly reduced viral load compared to remdesivir and control groups (p = 0.0117 and p = 0.0478). No infectious virus was detected in the lopinavir/ritonavir group, and the non-infectious SARS-CoV-2 proportion remained constant at 90%, higher than in the remdesivir and control groups (p = 0.0097). There was a significant difference in culture positivity among the groups (p = 0.0234), particularly between the lopinavir/ritonavir and remdesivir groups (p = 0.0267). These findings suggest that lopinavir/ritonavir reduces viral load and shortens the viral shedding duration compared to remdesivir, despite not being an effective treatment option.

Development and evaluation of a high-sensitivity RT-PCR lateral flow assay for early detection of HIV-1 infection.

Early diagnosis of HIV-1 is crucial to minimize transmission, morbidity, and mortality, particularly for neonates with developing immune systems. This study aimed to develop and evaluate a simplified, high-sensitivity assay for early HIV-1 detection before seroconversion. The assay utilizes reverse-transcription-polymerase chain reaction (RT-PCR) to amplify the HIV-1 RNA protease gene. Digoxigenin (dig)-labeled forward, and biotin-labeled universal reverse primers are used, generating digoxigenin-amplicon-biotin (DAB) products. These products are detected using a lateral flow assay (LFA) containing a conjugated pad with colloidal gold-labeled 6-histidine tag-fused maltose-binding protein-monomeric streptavidin (6HISMBP-mSA-CGC). Anti-dig monoclonal antibody (mAb) and biotinylated-BSA are immobilized in the test and control line zones, respectively. Five plasma samples with known viral load (VL) were used to simulate the efficacy of early HIV-1 detection. RNA extracted from these samples was amplified by RT-PCR using the labeled primers, and DAB products were examined on agarose gel electrophoresis and LFA. RT-PCR from diluted clinical samples yielded visible DNA bands in agarose gel electrophoresis, consistent with positive LFA results. Conversely, negative samples only displayed the control line on LFA. This assay exhibited a limit of detection (LOD) of 82.29 RNA copies/mL, comparable to other nucleic acid amplification tests (NAATs). This novel technique provides a highly sensitive assay for early HIV-1 diagnosis, even with low VL, making it suitable for resource-limited settings.

Clinical Features and Characteristics of Hand, Foot, and Mouth Disease Caused by Recent Coxsackievirus A6: Five Cases in Japan from 2019 to 2022.

Hand, foot, and mouth disease (HFMD) is a common infectious disease caused by enteroviruses. Coxsackievirus A6 (CV-A6)-associated HFMD has recently emerged as a predominant disease worldwide. Here, we describe five HFMD cases caused by CV-A6 in Japan from 2019 to 2022. All clinical courses were not severe and were self-limited, and the skin exanthema with vesicles differed from that in classical HFMD. Phylogenetic analysis showed that the major epidemic strain cluster of CV-A6 was formed independently in 2011, and our latest CV-A6 strains in Japan were detected within this cluster. The five cases described in this report indicate the recent shift in the predominant and continuous disease manifestation of CV-A6-associated HFMD.

First detection of tick-borne encephalitis virus (TBEV) in raw milk samples in North-Western Iran.

Tick-borne encephalitis virus (TBEV) is a significant cause of flaviviral infections affecting the human central nervous system, primarily transmitted through tick bites and the consumption of unpasteurized milk. This study aimed to assess the prevalence of TBEV and identify new natural foci of TBEV in livestock milk. In this cross-sectional study, unpasteurized milk samples were collected from livestock reared on farms and analysed for the presence and subtyping of TBEV using nested reverse transcription-polymerase chain reaction , alongside the detection of anti-TBEV total IgG antibodies using ELISA. The findings revealed that the highest prevalence of TBEV was observed in goat and sheep milk combined, whereas no TBEV was detected in cow milk samples. All identified strains were of the Siberian subtype. Moreover, the highest prevalence of anti-TBEV antibodies was detected in sheep milk. These results uncover new foci of TBEV in Iran, underscoring the importance of thermal processing (pasteurization) of milk prior to consumption to mitigate the risk of TBEV infection.

Time- and Region-Specific Selection of Reference Genes in the Rat Brain in the Lithium-Pilocarpine Model of Acquired Temporal Lobe Epilepsy.

Reverse transcription followed by quantitative polymerase chain reaction (RT-qPCR) is a commonly used tool for gene expression analysis. The selection of stably expressed reference genes is required for accurate normalization. The aim of this study was to identify the optimal reference genes for RT-qPCR normalization in various brain regions of rats at different stages of the lithium-pilocarpine model of acquired epilepsy. We tested the expression stability of nine housekeeping genes commonly used as reference genes in brain research: Actb, Gapdh, B2m, Rpl13a, Sdha, Ppia, Hprt1, Pgk1, and Ywhaz. Based on four standard algorithms (geNorm, NormFinder, BestKeeper, and comparative delta-Ct), we found that after pilocarpine-induced status epilepticus, the stability of the tested reference genes varied significantly between brain regions and depended on time after epileptogenesis induction (3 and 7 days in the latent phase, and 2 months in the chronic phase of the model). Pgk1 and Ywhaz were the most stable, while Actb, Sdha, and B2m demonstrated the lowest stability in the analyzed brain areas. We revealed time- and region-specific changes in the mRNA expression of the housekeeping genes B2m, Actb, Sdha, Rpl13a, Gapdh, Hprt1, and Sdha. These changes were more pronounced in the hippocampal region during the latent phase of the model and are thought to be related to epileptogenesis. Thus, RT-qPCR analysis of mRNA expression in acquired epilepsy models requires careful selection of reference genes depending on the brain region and time of analysis. For the time course study of epileptogenesis in the rat lithium-pilocarpine model, we recommend the use of the Pgk1 and Ywhaz genes.

Identifying the stability of housekeeping genes to be used for the quantitative real-time PCR normalization in retinal tissue of streptozotocin-induced diabetic rats.

AIM: To investigate the stability of the seven housekeeping genes: beta-actin (ActB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18s ribosomal unit 5 (18s), cyclophilin A (CycA), hypoxanthine-guanine phosphoribosyl transferase (HPRT), ribosomal protein large P0 (36B4) and terminal uridylyl transferase 1 (U6) in the diabetic retinal tissue of rat model. METHODS: The expression of these seven genes in rat retinal tissues was determined using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) in two groups; normal control rats and streptozotocin-induced diabetic rats. The stability analysis of gene expression was investigated using geNorm, NormFinder, BestKeeper, and comparative delta-Ct (ΔCt) algorithms. RESULTS: The 36B4 gene was stably expressed in the retinal tissues of normal control animals; however, it was less stable in diabetic retinas. The 18s gene was expressed consistently in both normal control and diabetic rats' retinal tissue. That this gene was the best reference for data normalisation in RT-qPCR studies that used the retinal tissue of streptozotocin-induced diabetic rats. Furthermore, there was no ideal gene stably expressed for use in all experimental settings. CONCLUSION: Identifying relevant genes is a need for achieving RT-qPCR validity and reliability and must be appropriately achieved based on a specific experimental setting.

Cost-effective in-house COVID-19 reverse transcription-polymerase chain reaction testing with yeast-derived Taq polymerase.

BACKGROUND: Despite the decline of the COVID-19 pandemic, there continues to be a persistent requirement for reliable testing methods that can be adapted to future outbreaks and areas with limited resources. While the standard approach of using reverse transcription-polymerase chain reaction (RT-PCR) with Taq polymerase is effective, it faces challenges such as limited access to high-quality enzymes and the presence of bacterial DNA contamination in commercial kits, which can impact the accuracy of test results. METHODS: This study investigates the production of recombinant Taq polymerase in yeast cells and assesses its crude lysate in a multiplex RT-PCR assay for detecting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRP) and N genes, with human Ribonuclease P serving as an internal control. RESULTS: The unpurified yeast Taq polymerase demonstrates sensitivity comparable to commercially purified bacterial Taq polymerase and unpurified bacterial counterparts in detecting the RdRP and N genes. It exhibits the highest specificity, with 100% accuracy, for the N gene. The specificity for the RdRP gene closely aligns with that of commercially purified bacterial Taq polymerase and unpurified bacterial Taq polymerase. CONCLUSIONS: The use of unpurified recombinant yeast Taq polymerase shows promise as a cost-effective approach for conducting in-house COVID-19 RT-PCR testing. By eliminating the need for chromatography purification steps, the production of RT-PCR kits can be streamlined, potentially improving accessibility and scalability, especially in resource-limited settings and future pandemics.

LncRNA PCAT6 is a predictor of poor prognosis of colorectal cancer.

Background: The long non-coding RNA (lncRNA) prostate cancer-associated transcript 6 (PCAT6) has been studied in many cancers, yet its relationship with colorectal cancer (CRC) remains poorly defined. Here, we conducted an analysis of The Cancer Genome Atlas (TCGA) database to better clarify the role of PCAT6 in this cancer type. Methods: Wilcoxon rank-sum tests were utilized to assess relative levels of PCAT6 in CRC tumors and normal tissues, while logistic regression analyses were utilized to compare the relationships between PCAT6 levels and clinicopathological findings. Kaplan-Meier curves and Cox regression analyses were used to gauge correlations between PCAT6 and patient survival outcomes, while the biological roles of this lncRNA were investigated via a gene set enrichment analysis (GSEA) approach. The expression level of PCAT6 in CRC cell lines was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results: PCAT6 levels were significantly correlated with CRC patient lymph node metastasis (N) stage [odds ratio (OR) =1.8 for N1 & N2 vs. N0], lymphatic invasion [OR =1.9 for yes vs. no), distant metastasis (M stage) (OR =2.1 for M1 vs. M0), carcinoembryonic antigen (CEA) level (OR =1.9 for >5 vs. ≤5), perineural invasion (OR =1.9 for yes vs. no), pathologic stage (OR =1.9 for stage III/IV vs. stage I/II), and neoplasm type (OR =2.1 for rectal adenocarcinoma vs. colon adenocarcinoma) (all P<0.05). CRC patients expressing higher PCAT6 levels exhibited poorer survival outcomes than those expressing low levels of this lncRNA (P=0.017), and in univariate analyses, higher PCAT6 levels were linked to worse overall survival [hazard ratio (HR) =1.540; 95% confidence interval (CI): 1.079-2.199; P=0.017], with this relationship also being preserved in a multivariate analysis (HR =6.892; 95% CI: 1.713-27.727, P=0.007). GSEA revealed high PCAT6 expression to be linked to differential DNA methylation enrichment, with high PCAT6 levels being associated with changes in base excision repair, cellular senescence, G2/M DNA damage checkpoint, chromatin-modifying enzyme, and gene silencing by RNA activity. The high expression of lncRNA PCAT6 in CRC cell lines was demonstrated by PCR experiments. Conclusions: PCAT6 represents a promising prognostic biomarker of poor CRC patient survival outcomes, with DNA methylation and RNA-mediated gene silencing being potentially promising mechanistic pathways whereby this lncRNA may shape patient outcomes.

Pneumonia Caused by Chlamydia psittaci and SARS-CoV-2 Coinfection Diagnosed Using Metagenomic Next-Generation Sequencing: A Case Report.

We report a case of pneumonia caused by coinfection with Chlamydia psittaci and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB.1 variant, confirmed using metagenomic next-generation sequencing (mNGS) and quantitative polymerase chain reaction (qPCR). C. psittaci and SARS-CoV-2 were detected in bronchoalveolar lavage fluid using mNGS. Additionally, mNGS detected C. psittaci in blood and nasopharyngeal specimens and was more sensitive than qPCR. The patient recovered after treatment with moxifloxacin. This report highlights the use of coinfections of C. psittaci and SARS-CoV-2, as mNGS has already been recognized to be a diagnostic tool for identifying coinfections.

Suppression of Streptococcus mutans Biofilm Formation and Gene Expression by PRG Barrier Coat: A Molecular and Microscopic Study for Preventing Dental Caries.

PURPOSE: This study aimed to investigate the inhibitory effect of a PRG Barrier Coat on biofilm formation and structure by Streptococcus mutans and propose an effective method for preventing dental caries. MATERIALS AND METHODS: Streptococcus mutans MT8148 biofilms were obtained from hydroxyapatite disks with and with- out a PRG Barrier Coat. Scanning electron microscopy (SEM) was used to observe the 12- and 24-h-cultured biofilms, while reverse-transcription polymerase chain reaction (qRT-PCR) was used to quantify caries-related genes. Biofilm adhe- sion assessments were performed on glass. Statistical analysis was performed using a two-sample t-test. RESULTS: A statistically significant difference in Streptococcus mutans biofilm adhesion rate was observed between the con- trol and PRG Barrier Coat-coated samples (p < 0.01). However, there was no statistically significant difference in total bacter- ial count or biofilm volume (p > 0.05). SEM revealed that the PRG Barrier Coat inhibited biofilm formation by Streptococcus mutans. Real-time RT-PCR revealed that the material restricted the expression of genes associated with caries-related bio- film formation. However, the suppression of gtfD and dexB differed from that of other genes. CONCLUSION: PRG Barrier Coat suppressed biofilm formation by Streptococcus mutans by inhibiting the expression of in- soluble glucan synthase, which is associated with primary biofilm formation. The material also affected gene expression and altered the biofilm structure. Tooth surface-coating materials, such as PRG Barrier Coat, may improve caries preven- tion in dental practice.

Laboratory diagnosis of measles infection using molecular and serology during 2019-2020 outbreak in Brazil.

INTRODUCTION: Laboratory diagnosis of measles can be challenging, and the reintroduction of the measles virus in Brazil has brought about new issues. The aim of this study was to analyze the qPCR results of swab and urine samples and compare them with those of immunological methods for the diagnosis of measles. METHODS: This was a cross-sectional study based on a retrospective analysis of 3,451 suspected cases using laboratory test surveillance databases for qPCR (respiratory swabs and urine) and serologic tests for IgM and paired IgG. Sensitivity, specificity, positive predictive value, negative predictive value, accuracy, and agreement through kappa and adjusted kappa coefficients (PABAK) were calculated using different diagnostic strategies. RESULTS: The swab and urine samples obtained using real-time qPCR were equivalent. Samples collected simultaneously and the combined samples showed moderate agreement between IgM ELISA and real-time qPCR; however, 48.9 % of the IgM ELISA analyses did not demonstrate detectable qPCR concentrations during simultaneous collections and 43.9 % of combined collections. The paired analysis of IgG showed an accuracy of 67.5 % for IgM and 90.7 % for real-time qPCR. CONCLUSIONS: Diagnosis based on IgM presents detection delimitation in samples collected early (1-5 days), suggesting that these individuals satisfy at least two criteria. In addition to qPCR, paired analysis of IgG using ELISA can be used to increase the sensitivity and specificity of laboratory diagnoses.

Trimethylamine increases intestinal fatty acid absorption: in vitro studies in a Caco-2 cell culture system.

Although elevated blood levels of trimethylamine N-oxide (TMAO) have been associated with atherosclerosis development in humans, the role of its gut microbiota-derived precursor, TMA, in this process has not been yet deciphered. Taking this into account, and the fact that increased intestinal fatty acid absorption contributes to atherosclerosis onset and progression, this study aimed to evaluate the effect of TMA on fatty acid absorption in a cell line that mimics human enterocytes. Caco-2 cells were treated with TMA 250 µM for 24 h. Fatty acid absorption was assessed by measuring the apical-to-basolateral transport and the intracellular levels of BODIPY-C12, a fluorescently labelled fatty acid analogue. Gene expression of the main intestinal fatty acid transporters was evaluated by real-time quantitative reverse transcription PCR. Compared to control conditions, TMA increased, in a time-dependent manner and by 20-50 %, the apical-to-basolateral transport and intracellular levels of BODIPY-C12 fatty acid in Caco-2 cells. Fatty acid transport protein 4 (FATP4) and fatty acid translocase (FAT)/CD36 gene expression were not stimulated by TMA, suggesting that TMA-induced increase in fatty acid transport may be mediated by an increase in FAT/CD36 and/or FATP4 activity and/or fatty acid passive transport. This study demonstrated that TMA increases the intestinal absorption of fatty acids. Future studies are necessary to confirm if this may constitute a novel mechanism that partially explains the existing positive association between the consumption of a diet rich in TMA sources (e.g. red meat) and the increased risk of atherosclerotic diseases.

Monitoring SARS-CoV-2 Using Infoveillance, National Reporting Data, and Wastewater in Wales, United Kingdom: Mixed Methods Study.

BACKGROUND: The COVID-19 pandemic necessitated rapid real-time surveillance of epidemiological data to advise governments and the public, but the accuracy of these data depends on myriad auxiliary assumptions, not least accurate reporting of cases by the public. Wastewater monitoring has emerged internationally as an accurate and objective means for assessing disease prevalence with reduced latency and less dependence on public vigilance, reliability, and engagement. How public interest aligns with COVID-19 personal testing data and wastewater monitoring is, however, very poorly characterized. OBJECTIVE: This study aims to assess the associations between internet search volume data relevant to COVID-19, public health care statistics, and national-scale wastewater monitoring of SARS-CoV-2 across South Wales, United Kingdom, over time to investigate how interest in the pandemic may reflect the prevalence of SARS-CoV-2, as detected by national testing and wastewater monitoring, and how these data could be used to predict case numbers. METHODS: Relative search volume data from Google Trends for search terms linked to the COVID-19 pandemic were extracted and compared against government-reported COVID-19 statistics and quantitative reverse transcription polymerase chain reaction (RT-qPCR) SARS-CoV-2 data generated from wastewater in South Wales, United Kingdom, using multivariate linear models, correlation analysis, and predictions from linear models. RESULTS: Wastewater monitoring, most infoveillance terms, and nationally reported cases significantly correlated, but these relationships changed over time. Wastewater surveillance data and some infoveillance search terms generated predictions of case numbers that correlated with reported case numbers, but the accuracy of these predictions was inconsistent and many of the relationships changed over time. CONCLUSIONS: Wastewater monitoring presents a valuable means for assessing population-level prevalence of SARS-CoV-2 and could be integrated with other data types such as infoveillance for increasingly accurate inference of virus prevalence. The importance of such monitoring is increasingly clear as a means of objectively assessing the prevalence of SARS-CoV-2 to circumvent the dynamic interest and participation of the public. Increased accessibility of wastewater monitoring data to the public, as is the case for other national data, may enhance public engagement with these forms of monitoring.

Detection of Alpha- and Betacoronaviruses in Small Mammals in Western Yunnan Province, China.

The genetic diversity of coronaviruses (CoVs) is high, and their infection in animals has not yet been fully revealed. By RT-PCR detection of the partial RNA-dependent RNA polymerase (RdRp) gene of CoVs, we screened a total of 502 small mammals in the Dali and Nujiang prefectures of Western Yunnan Province, China. The number of overall CoV positives was 20, including ß-CoV (n = 13) and α-CoV (n = 7), with a 3.98% prevalence in rectal tissue samples. The identity of the partial RdRp genes obtained for 13 strains of ß-CoV was 83.42-99.23% at the nucleotide level, and it is worth noting that the two strains from Kachin red-backed voles showed high identity to BOV-36/IND/2015 from Indian bovines and DcCoV-HKU23 from dromedary camels (Camelus dromedarius) in Morocco; the nucleotide identity was between 97.86 and 98.33%. Similarly, the identity of the seven strains of α-CoV among the partial RdRp sequences was 94.00-99.18% at nucleotide levels. The viral load in different tissues was measured by quantitative RT-PCR (qRT-PCR). The average CoV viral load in small mammalian rectal tissue was 1.35 × 106 copies/g; differently, the mean CoV viral load in liver, heart, lung, spleen, and kidney tissue was from 0.97 × 103 to 3.95 × 103 copies/g, which revealed that CoV has extensive tropism in rectal tissue in small mammals (p < 0.0001). These results revealed the genetic diversity, epidemiology, and infective tropism of α-CoV and ß-CoV in small mammals from Dali and Nujiang, which deepens the comprehension of the retention and infection of coronavirus in natural hosts.

NAP (Davunetide): The Neuroprotective ADNP Drug Candidate Penetrates Cell Nuclei Explaining Pleiotropic Mechanisms.

(1) Background: Recently, we showed aberrant nuclear/cytoplasmic boundaries/activity-dependent neuroprotective protein (ADNP) distribution in ADNP-mutated cells. This malformation was corrected upon neuronal differentiation by the ADNP-derived fragment drug candidate NAP (davunetide). Here, we investigated the mechanism of NAP nuclear protection. (2) Methods: CRISPR/Cas9 DNA-editing established N1E-115 neuroblastoma cell lines that express two different green fluorescent proteins (GFPs)-labeled mutated ADNP variants (p.Tyr718* and p.Ser403*). Cells were exposed to NAP conjugated to Cy5, followed by live imaging. Cells were further characterized using quantitative morphology/immunocytochemistry/RNA and protein quantifications. (3) Results: NAP rapidly distributed in the cytoplasm and was also seen in the nucleus. Furthermore, reduced microtubule content was observed in the ADNP-mutated cell lines. In parallel, disrupting microtubules by zinc or nocodazole intoxication mimicked ADNP mutation phenotypes and resulted in aberrant nuclear-cytoplasmic boundaries, which were rapidly corrected by NAP treatment. No NAP effects were noted on ADNP levels. Ketamine, used as a control, was ineffective, but both NAP and ketamine exhibited direct interactions with ADNP, as observed via in silico docking. (4) Conclusions: Through a microtubule-linked mechanism, NAP rapidly localized to the cytoplasmic and nuclear compartments, ameliorating mutated ADNP-related deficiencies. These novel findings explain previously published gene expression results and broaden NAP (davunetide) utilization in research and clinical development.