Cycle Threshold Research Articles

Detection of porcine reproductive and respiratory syndrome virus type 2 in air emissions from grow-finish barns.

Diagnosis of porcine reproductive and respiratory syndrome virus (PRRSV) infections can be accomplished using various sample types and testing methods. The objective of this study was to evaluate the feasibility of using air emission samples to detect the onset of PRRSV type 2 infections in growing pigs. Air emissions and oral fluid samples were collected from three grow-finish barns, stocked with PRRSV-negative pigsevery 2 weeksfor 14-20 weeks. All samples were assayed for PRRSV using RT-PCR. In all barns, PRRSV was initially detected in both air emissions and oral fluid samples at the same sampling event. Compared with oral fluids, air emissions collected subsequently yielded higher cycle threshold values and showed a lower likelihood of being RT-PCR positive. Sampling was limited to three grow-finish barns. Air emission samples from pig barns are a biosecure and non-invasive sample type that is comparable to oral fluids fordetection of the onset of PRRSV infection.

Enhancing RT-PCR Throughput and Sensitivity through Large-Scale Sample Pooling Using a Nano-Hybrid Membrane.

During the pandemic surge, including SARS-CoV-2 and influenza, pooling samples emerged as an efficient strategy to identify infected individuals in large groups. While pooling enhances RT-PCR throughput, reducing costs and resources, it dilutes positive samples with negative ones, lowering sensitivity and increasing false negatives. This study proposes a new method to address the trade-off between pool sizes and RT-PCR accuracy. This method integrates large-scale pooling with sample enrichment using a nano-hybrid membrane, preventing the pooling-induced decrease in viral concentration and maintaining cycle threshold (Ct) values close to individual positive samples. The nano-hybrid membrane, named SIMPLE (streamlined, simple, and inexpensive method for preconcentration, lysis, and nucleic acid extraction), comprises layered red blood cell membranes, polyethersulfone, and silica membranes. Using SIMPLE, a Ct value reduction to≈2.6 is demonstrated in pooled COVID-19 samples with a pool size of 6 and found Ct values from larger pool sizes (8, 16, 32, 64, and 128) comparable to individual positive samples.

Effect and Safety of Fuzheng Huazhuo Decoction against Prolonged SARS-CoV-2 Clearance: A Retrospective Cohort Study.

To evaluate the effect and safety of Chinese medicine (CM) Fuzheng Huazhuo Decoction (FHD) in treating patients with coronavirus disease 2019 (COVID-19) who persistently tested positive for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This retrospective cohort study was conducted at Shanghai New International Expo Center shelter hospital in China between April 1 and May 30, 2022. Patients diagnosed as COVID-19 with persistently positive SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) test results for ⩾8 days after diagnosis were enrolled. Patients in the control group received conventional Western medicine (WM) treatment, while those in the FHD group received conventional WM plus FHD for at least 3 days. The primary outcome was viral clearance time. Secondary outcomes included negative conversion rate within 14 days, length of hospital stay, cycle threshold (Ct) values of the open reading frame 1ab (ORF1ab) and nucleocapsid protein (N) genes, and incidence of new-onset symptoms during hospitalization. Adverse events (AEs) that occurred during the study period were recorded. A total of 1,765 eligible patients were enrolled in this study (546 in the FHD group and 1,219 in the control group). Compared with the control group, patients receiving FHD treatment showed shorter viral clearance time for nucleic acids [hazard ratio (HR): 1.500, 95% confidence interval (CI): 1.353-1.664, P<0.001] and hospital stays (HR: 1.371, 95% CI: 1.238-1.519, P<0.001), and a higher negative conversion rate within 14 days (96.2% vs. 82.6%, P<0.001). The incidence of new-onset symptoms was 59.5% in the FHD group, similar to 57.8% in the control group (P>0.05). The Ct values of ORF1ab and N genes increased more rapidly over time in the FHD group than those in the control group post-randomization (ORF1ab gene: β =0.436±0.053, P<0.001; N gene: β =0.415 ±0.053, P<0.001). The incidence of AEs in the FHD group was lower than that in the control group (24.2% vs. 35.4%, P<0.001). No serious AEs were observed. FHD was effective and safe for patients with persistently positive SARS-CoV-2 PCR tests. (Registration No. ChiCTR2200063956).

Effect of Time and Temperature on the Detection of PRRSV RNA and Endogenous Internal Sample Control in Porcine Tongue Fluids.

Population-based sampling has improved pathogen monitoring in the US swine industry by increasing sensitivity while reducing costs. Postmortem tongue fluids (TF) have emerged as a practical option for monitoring porcine reproductive and respiratory syndrome virus (PRRSV) in breeding herds, but limited data exist on optimal storage conditions. This study evaluated PRRSV RNA detection via RT-qPCR in TF samples under various storage times, temperatures, and viral loads. Additionally, the porcine endogenous reference gene (internal sample control, ISC) was assessed for monitoring sample quality. Three studies were conducted: Study 1 compared fresh TF samples to freeze-thaw TF samples, with significantly lower cycle threshold (Ct) values in fresh samples. Studies 2 and 3 evaluated PRRSV RNA stability across six time points (0 to 336 h) and four temperatures (-20 °C, 4 °C, 22 °C, and 34 °C). At 4 °C, Ct value variation was minimal (≤1.1) over 14 days (336 h), with no significant changes within 48 h. In contrast, storage at 22 °C and 34 °C resulted in increased Ct values. The ISC RNA showed similar stability patterns, maintaining quality at 4 °C but degrading at higher temperatures. In conclusion, storing TF at ≤4 °C for up to 14 days for PRRSV and ISC RNA detection is the best scenario, ensuring optimal diagnostic quality.

Optimizing Tongue Fluid Sampling and Testing Protocols for Enhanced PRRSV Isolation from Perinatal Swine Mortalities.

Porcine reproductive and respiratory syndrome virus (PRRSV) remains a major concern for swine health. Isolating PRRSV is essential for identifying infectious viruses and for vaccine formulation. This study evaluated the potential of using tongue fluid (TF) from perinatal piglet mortalities for PRRSV isolation. Four collection protocols were tested: extracting TF from fresh tissues using phosphate-buffered saline (PBS group), extracting TF from fresh tissues using virus transportation medium (VTM group), extracting TF from freeze-thawed tissue (freeze-thaw group), and using tissue homogenates (homogenate group). Two cell lines (ZMAC and MARC-145) and primary alveolar macrophages (PAM) were evaluated for their effect on successful PRRSV isolation. An eligible PRRSV-positive unstable breeding herd in Midwestern USA was chosen for the study. Tongues were collected in 20 batches (~30 mortalities per batch). Within each batch, each tongue tissue was cut into four quarters, with each quarter randomly assigned to one of the four collection protocols and RT-qPCR tested. Virus isolation (VI) was attempted on 10 batches. The mean RT-qPCR cycle threshold (Ct) values for the PBS, VTM, freeze-thaw, and homogenate groups were 21.9, 21.8, 22.6, and 24.8, respectively. The VI success rate was 22.6%, 12.1%, 2.8%, and 2.8% in the PBS, VTM, freeze-thaw, and homogenate groups, respectively. The probability of successful VI was 3.1% and 21.0% in the MARC-145 and ZMAC cell lines, respectively, and 4.8% in the PAM cells. TF from perinatal mortalities is an option for PRRS VI, aiding in PRRSV monitoring and control programs.

Population-Level SARS-CoV-2 RT-PCR Cycle Threshold Values and Their Relationships with COVID-19 Transmission and Outcome Metrics: A Time Series Analysis Across Pandemic Years.

This study investigates the relationship between SARS-CoV-2 RT-PCR cycle threshold (Ct) values and key COVID-19 transmission and outcome metrics across five years of the pandemic in Jalisco, Mexico. Utilizing a comprehensive time-series analysis, we evaluated weekly median Ct values as proxies for viral load and their temporal associations with positivity rates, reproduction numbers (Rt), hospitalizations, and mortality. Cross-correlation and lagged regression analyses revealed significant lead-lag relationships, with declining Ct values consistently preceding surges in positivity rates and hospitalizations, particularly during the early phases of the pandemic. Granger causality tests and vector autoregressive modeling confirmed the predictive utility of Ct values, highlighting their potential as early warning indicators. The study further observed a weakening association in later pandemic stages, likely influenced by the emergence of new variants, hybrid immunity, changes in human behavior, and diagnostic shifts. These findings underscore the value of Ct values as scalable tools for public health surveillance and highlight the importance of contextualizing their analysis within specific epidemiological and temporal frameworks. Integrating Ct monitoring into surveillance systems could enhance pandemic preparedness, improve outbreak forecasting, and strengthen epidemiological modeling.

Multiplex lateral flow test sensitivity and specificity in detecting influenza A, B and SARS-CoV-2 in adult patients in a UK emergency department

BackgroundRapid identification of individuals with acute respiratory infections is crucial for preventing nosocomial infections. For rapid diagnosis, especially in EDs, lateral flow devices (LFDs) are a convenient, inexpensive option with...

Monitoring SARS-CoV-2 variants with complementary surveillance systems: risk evaluation of the Omicron JN.1 variant in France, August 2023 to January 2024.

BackgroundEarly detection and characterisation of SARS-CoV-2 variants have been and continue to be essential for assessing their public health impact. In August 2023, Santé publique France implemented enhanced surveillance for BA.2.86 and sub-lineage JN.1 because of their genetic divergence from other variants and increased prevalence.AimTo detail how combining epidemiological and laboratory data sources, targeted investigations and modelling enabled comprehensive characterisation of sub-lineage JN.1.MethodsData were collected from epidemiological investigations using a standardised questionnaire and from routine and novel (RELAB network) surveillance systems. JN.1 cases were compared with cases infected with previously circulating variants, such as EG.5, BA.4/BA.5 and other BA.2.86 sub-lineages. The growth rate and doubling time of JN.1 were estimated.ResultsJN.1 was first detected in September 2023 in the Île-de-France region, France, and spread widely across the country. By late November, doubling time was estimated to be 8.6 to 26.4 days depending on the region. For all data sources, cases infected by JN.1 showed similar demographics, rates of hospitalisation and RT-PCR cycle threshold values compared with those infected by previous variants. JN.1 cases also had older median age (54 years; 40-71 vs 47 years; 30-59), more frequent reports of feverish feeling and less frequent cough or nausea compared with BA.4/BA.5 cases. JN.1 cases had significantly higher frequency of anosmia compared with other BA.2.86 cases.ConclusionCombining different data sources played a key role in detecting emerging variant JN.1, for which no evidence of increased public health impact was found despite its genetic divergence.

Key Insights into Respiratory Virus Testing: Sensitivity and Clinical Implications.

Acute respiratory infections are a significant challenge in primary care and hospital settings. Viruses are the most common etiology and the overlapping symptomatology among major respiratory viruses, such as influenza, severe acute respiratory syndrome coronavirus 2, and respiratory syncytial virus, requires the use of diagnostic tests that deliver early and accurate results. With the increasing availability of rapid antigen tests (RATS), it is tempting to prefer them over polymerase chain reaction (PCR) tests. However, compelling arguments support the existing recommendations in some European countries to maintain PCR testing for patient management throughout the year. RATs show sensitivities below 30% with lower viral loads, which are common and can have significant clinical implications. RATs perform well at lower cycle threshold (Ct) values, with sensitivity reaching 97.9% for Ct values below 20, which drops significantly for values above 25. Factors affecting viral load include disease stage, vaccination status, and viral variants, all of which can compromise the accuracy of antigen tests. Multi-target PCR tests effectively overcome these issues, ensuring reliable diagnosis. Additionally, the early detection of paucisymptomatic cases is essential in primary care and hospital settings to facilitate isolation and prevent secondary infections. Economic analyses support the use of comprehensive PCR tests, such as triplex-type tests, detecting SARS-CoV-2, influenza viruses, and RSV, as a first-line approach, as they can reduce case numbers and healthcare resource utilization. Maintaining PCR testing year-round is therefore crucial for the effective management of respiratory infections.

Systematic Molecular Influenza A/B Screening Upon Hospital Admission in Belgium, January-April 2022: Positivity Ratios and Viral Loads According to Symptomatology, Age, and Vaccination Status.

Three hospitals implemented molecular point-of-care tests (POCTs) to screen patients for SARS-CoV-2 infection upon admission during the 2021/2022 influenza season, which in Belgium lasted from January to April 2022. The samples were simultaneously tested for influenza A/B. Influenza positivity at admission was examined in relation to patient characteristics and symptomatology. Influenza POCTs were performed on all patients requiring urgent hospitalization, regardless of the admission reason. A total of 9327 patients were included in the study, of which 411 (4.4%) tested positive for influenza A/B. Asymptomatic infection and mild illness accounted for respectively 11.2% (95% CI: 8.5%-14.6%), and 43.3% (95% CI: 38.6%-48.1%) of the cases. A total of 66% (95% CI: 60%-72%) of all patients in these symptom categories (asymptomatic and mild illness) showed a high viral load (cycle threshold [Ct] < 24). Only in 30 (7.3%, 95% CI: 5.2%-10.2%) of all cases and in two (4.4%, 95% CI: 1.2%-14.5%) of the asymptomatic cases, the symptomatology worsened during hospital stay. Coinfections with both influenza and SARS-CoV-2 occurred in 35 patients (8.5% of all influenza positive patients). There was no difference in symptomatology between patients with co-infections and those with an influenza mono-infection. Patients could not be reliably categorized into carriers with low versus high viral loads based on symptomatology, age, and vaccination status. More than half of the influenza-positive individuals were either asymptomatic or had mild symptoms upon admission, while often carrying high viral loads. Our results show that without screening of patients at hospital admission, a considerable number of patients with a high viral load may be incorrectly classified as being not infectious.

Correlation of polymerase chain reaction results with hematocrit levels and platelet counts in dengue patients in Batam City

Background Dengue hemorrhagic fever (DHF) is a viral disease transmitted by Aedes aegypti mosquitoes, posing global public health challenge. The Riau Islands Province has the highest incidence of DHF in Indonesia. Objective This study aimed to investigate the relationship between hematocrit and platelet levels with the cycle threshold (Ct) values of polymerase chain reaction (PCR) results in DHF cases in Batam City, Riau Islands Province. Methods A descriptive correlation study was conducted using data from 102 patients infected with the dengue virus. Hematocrit and platelet counts were measured using a hematology analyzer, while Ct values for DENV1, DENV2, DENV3, and DENV4 were obtained through real-time qRT-PCR. Pearson's correlation test was employed to analyze the relationship between these variables. Results The study found no significant gender difference in DHF incidence (males: 50%, females: 50%). The highest prevalence was observed in the 6–11 years age group (44.1%), followed by the 12–18 years group (25.5%), the &gt;18 years group (24.5%), and the 1–5 years group (11.8%). DENV3 was identified as the dominant serotype. No statistically significant correlation was found between Ct values and hematocrit (p = 0.607) or platelet counts (p = 0.323). Conclusion DHF cases in this study showed no gender disparity, with the most affected group being children aged 6–11 years, and DENV3 was the prevalent serotype. Ct values did not show a statistically significant correlation with hematocrit levels or platelet counts, suggesting that these hematological parameters may not predict viral load in DHF cases.

At what cycle threshold level are dogs able to detect SARS-CoV-2 in humans?

Dogs can discriminate between people infected with SARS-CoV-2 from those uninfected, although their results vary depending on the settings in which they are exposed to infected individuals or samples of urine, sweat or saliva. This variability likely depends on the viral load of infected people, which may be closely associated with physiological changes in infected patients. Determining this viral load is challenging, and a practical approach is to use the cycle threshold (Ct) value of a RT-qPCR test. The hypothesis was that dogs should have a specific Ct range at which they could detect people infected with SARS-CoV-2. Therefore, the objective was to determine this Ct range. Sweat samples and epidemiological data were collected from 89 infected and 289 non-infected individuals at real life settings (e.g. health centers, offices, football fields). To determine each person's infection status, the Norgen Biotek kit for RT-qPCR was used; targeting the N1 and N2 regions of the SARS-CoV-2 nucleocapsid N gene. The performance of 11 trained dogs was evaluated on sweat samples of 379 individuals to determine their sensitivity and specificity (± 95% Confidence Intervals; CI) in detecting SARS-CoV-2 infections. Additionally, the SARS-CoV-2 viral load was calculated from Ct values using a reference curve, and the Ct range at which dogs showed optimal performance was determined. Six dogs exhibited a marginal performance, as their sensitivity 95% CI overlapped with the region of randomness (50%). The remaining five dogs demonstrated sensitivity values between 67% and 87%, with none of their 95% CIs overlapping the randomness region. Regarding specificity, three dogs showed values between 87% and 92%, while all other dogs exhibited values of ≥ 90%. Dogs demonstrated higher detection accuracy in a range of Ct values between 18.49 and 29.17 for the N1 region and between 24.07 and 26.69 for the N2 region of the SARS-CoV-2 nucleocapsid gene. Detection significantly decreases for Ct values greater than 30 or less than 16, indicating an optimal range in which dogs are most effective. These performance values concur well with those reported for commercial rapid antigen tests for detecting SARS-CoV-2. Consequently, it is considered that using properly trained animals could offer a viable option to supplement existing diagnostic methods, allowing for rapid diagnosis while optimizing time and economic resources. Moreover, this approach is ecologically sustainable, as it generates less waste compared to the use of rapid tests, while continuing to confirm positive cases.

Correlation of the cycle threshold value of SARS-CoV-2 by RT-PCR with biomarker levels in the prognosis of patients hospitalized with COVID-19

ABSTRACT Background and Objective: The COVID-19 disease caused by SARS-CoV-2 was declared as pandemic by WHO soon after its emergence in 2019. This virus was known to cause serious clinical symptoms and severe illness. By using RT-PCR, which reports the cycle threshold value, the disease is diagnosed, whereas for the severity of the disease, biomarker levels, like IL-6, CRP, D-dimer, serum ferritin and serum procalcitonin, can be measured. We, thus, aimed to explore any potential correlation of the cycle threshold value and biomarker level with the outcome of COVID-19-positive hospitalized patients. Method: Patients with the cycle threshold (Ct) value &lt;35 were included in the study and their initial Ct values were noted. Different biochemical parameters, such as C-reactive protein (CRP), serum ferritin, D-dimer, Interleukin-6 and serum procalcitonin, were assessed for severity. They were classified according to Ct value into three groups: Group 1 &gt;30.0, Group 2 20.0–30.0 and Group 3 &lt;20.0. Results: The study included 370 hospitalized COVID-19 patients with a mean age (mean ± SD) of 51.08 (16.58%) years and 250 (67.5%) males and 120 (32.4%) females. Comparison of data with outcome shows that IL-6, CRP amongst the biomarker and Ct value (deduced by RT-PCR test) were significantly correlated with the mortality (P value &lt; 0.05). The ROC curve was also plotted for these parameters, which shows that IL-6, CRP, PCT and Ct value were better prognostic marker. Poor prognosis was found in Group 2 (Ct value 20.0–30.0) patients compared to Group 1 and Group 3. There was significant correlation (P value &lt; 0.05) between Ct value and outcome of the patient. Interpretation and Conclusion: This study depicts that low Ct value and elevated levels of IL-6 and CRP can be used as a screening tool to detect the mortality in COVID-19 patients as they are significantly correlated with the mortality.

Cerebrospinal fluid multiplex PCR cycle thresholds may predict ICU admission in community-acquired meningoencephalitis

Cerebrospinal fluid multiplex PCR cycle thresholds may predict ICU admission in community-acquired meningoencephalitis

Automated post-run analysis of arrayed quantitative PCR amplification curves using machine learning.

The TaqMan Array Card (TAC) is an arrayed, high-throughput qPCR platform that can simultaneously detect multiple targets in a single reaction. However, the manual post-run analysis of TAC data is time consuming and subject to interpretation. We sought to automate the post-run analysis of TAC data using machine learning models. We used 165,214 qPCR amplification curves from two studies to train and test two eXtreme Gradient Boosting (XGBoost) models. Previous manual analyses of the amplification curves by experts in qPCR analysis were used as the gold standard. First, a classification model predicted whether amplification occurred or not, and if so, a second model predicted the cycle threshold (Ct) value. We used 5-fold cross-validation to tune the models and assessed performance using accuracy, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and mean absolute error (MAE). For external validation, we used 1,472 reactions previously analyzed by 17 laboratory scientists as part of an external quality assessment for a multisite study. In internal validation, the classification model achieved an accuracy of 0.996, sensitivity of 0.997, specificity of 0.993, PPV of 0.998, and NPV of 0.991. The Ct prediction model achieved a MAE of 0.590. In external validation, the automated analysis achieved an accuracy of 0.997 and a MAE of 0.611, and the automated analysis was more accurate than manual analyses by 14 of the 17 laboratory scientists. We automated the post-run analysis of highly-arrayed qPCR data using machine learning models with high accuracy in comparison to a manual gold standard. This approach has the potential to save time and improve reproducibility in laboratories using the TAC platform and other high-throughput qPCR approaches.

Seaweeds: Nature's super therapeutics? Immunomodulatory and anti-viral properties of sulfated rhamnoxyloglucuronan isolated from Ulva fasciata Delile.

Green seaweeds, which make up a major population of total seaweed worldwide, possess various therapeutic properties. The aim of the study directed at isolating a (1 → 4) linked sulfated rhamno xyloglucuronan, designated as UFP-2, from the edible green seaweed Ulva fasciata Delile, and to evaluate its efficacy in modulating immune responses and inhibiting SARS-CoV-2 (Delta variant) infection. Anti-inflammatory potential of UFP-2 was demonstrated through the regulation of key cytokines involved in inflammatory responses triggered by viral infections, including interferons (IFN-α/γ), interleukin (IL-1β/12/33), and tumor necrosis factor (TNF-α). Confocal microscopy and flow cytometry analyses indicated downregulation of IFN-α and IL-1β, while TNF-α expression reduced from 29.28 % in lipopolysaccharide (LPS)-induced CALU-1 cells to 1.6-5.4 % upon UFP-2 treatment. Treatment with UFP-2 at a concentration of 125 μg/mL significantly downregulated the overexpression of IL-1β in SARS-CoV-2-infected CALU-1 cells. Administering UFP-2 to SARS-CoV-2 (delta variant) induced cells led to increased cell viability and higher cycle threshold (Ct) values (20.34), indicating reduced viral load, with viral copy numbers decreasing from over 6.5 × 107 to <2.5 × 107 per cell. Structure-activity relationship analysis suggested that sulfate groups and overall hydrophilicity of UFP-2 contribute to its binding affinity with target receptors, potentially disrupting viral entry and replication processes.

Agreement Between High-Risk Human Papillomavirus Testing in Paired Self-Collected and Clinician-Collected Samples from Cervical Cancer Screening in Spain.

Background: Implementing self-sampling (SS) in cervical cancer screening requires comparable results to clinician-collected samples (CCS). Agreement measures are essential for evaluating HPV test performance. Previous studies on non-paired samples have reported higher viral cycle threshold (Ct) values in SS compared to CCS, affecting sensitivity for detecting cervical intraepithelial neoplasia grade 2 or worse (CIN2+). Objectives: We aimed to evaluate the agreement of high-risk (hr)HPV testing results between SS and CCS using paired samples and to explore differences in Ct values. Methods: Women aged 30 to 65 years attending cervical cancer screening in two regions of Spain were invited to participated in this study. For each woman there was: CCS collected during the screening visit using liquid-based cytology and cytobrush, and a SS using a brush at home one month later. A PCR-based assay was used for hrHPV detection. Agreement in hrHPV results among both samples, Ct value differences, and their association with screening outcomes were analyzed. Results: This study included 981 women with paired samples. SS had a higher hrHPV prevalence than CCS (overall ratio of 1.3). Positive agreement for all hrHPV genotypes, HPV16, HPV18, and other hrHPV types were 85%, 91.3%, 66.7%, and 83.3%, respectively. Negative agreement was >95% for all results. Median Ct values was slightly higher in SS than in CSS (32.9 vs. 30.6, p = 0.02). Seven CIN2+ cases HPV positive were detected by both methods. One CIN3 case was missed by SS. Conclusions: This study showed a good agreement between SS and CCS for hrHPV testing in a routine screening in Spain. Despite the slightly higher Ct values for SS, no significant impact on sensitivity could be determined due to the low incidence of CIN2+ cases. Further research on larger paired samples is needed to assess the implications of Ct values on test sensitivity.

Evaluation of Plasma microRNA-222 as a Biomarker for Gastric Cancer.

Background: The dysregulation of microRNAs (miRNAs) has been detected in patients with gastric cancer (GC), which inspired the use of miRNAs as a novel biomarker for GC. In this study, we investigated the previously reported miRNA dysfunction in cancer tissues as a potential plasma biomarker for GC using quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Methods: The published miRNA abnormalities were searched in the microRNA Cancer Association Database. Plasma samples were collected from patients with GC (n = 26) and controls (n = 17). The sensitivity and specificity of polyadenylation RT-PCR (PA-RT) and stem-loop RT-PCR (SL-RT) were compared. Statistical comparisons between patients with GC and controls were performed to identify miRNA biomarkers, and correlation analyses between the threshold cycle (Ct) values of miRNAs and various blood biochemical parameters were performed to elucidate the confounding factors. Results: mir-17, mir-21, mir-31, mir-99b, mir-222, and U6 were selected. PA-RT showed greater sensitivity and lower specificity than SL-RT (PA-RT vs. SL-RT, mean Ct: 19.6 vs. 29.2; coefficient of variation: 0.42 vs. 0.10). Adopting SL-RT owing to its higher specificity, only mir-222 was significantly upregulated in patients with GC (GC vs. control, miRNA expression: 15.4 vs. 5.27, p = 0.0098). Regarding the correlation between blood biochemical parameters and cells with miRNA expression, mir-31 and mir-99b were correlated with blood urea nitrogen, mir-17, mir-21, and mir-99b were negatively correlated with platelets, and mir-21 was correlated with neutrophils. No obvious correlations were noted between mir-222 expression and blood parameters. Receiver operating characteristic (ROC) curve analysis indicated that mir-222 identified GC patients with a maximum area under the curve (0.73, 95% confidence interval 0.57-0.89). Conclusions: Plasma mir-222 was confirmed to be dysregulated in patients with GC, irrespective of blood biochemical parameters.

Detection and Phylogenetic Characterization of Influenza D in Swedish Cattle.

Increased evidence suggests that cattle are the primary host of Influenza D virus (IDV) and may contribute to respiratory disease in this species. The aim of this study was to detect and characterise IDV in the Swedish cattle population using archived respiratory samples. This retrospective study comprised a collection of a total 1763 samples collected between 1 January 2021 and 30 June 2024. The samples were screened for IDV and other respiratory pathogens using real-time reverse transcription quantitative PCR (rRT-qPCR). Fifty-one IDV-positive samples were identified, with a mean cycle threshold (Ct) value of 27 (range: 15-37). Individual samples with a Ct value of <30 for IDV RNA were further analysed by deep sequencing. Phylogenetic analysis was performed by the maximum likelihood estimation method on the whole IDV genome sequence from 16 samples. The IDV strains collected in 2021 (n = 7) belonged to the D/OK clade, whereas samples from 2023 (n = 4) and 2024 (n = 5) consisted of reassortants between the D/OK and D/660 clades, for the PB2 gene. This study reports the first detection of IDV in Swedish cattle and the circulation of D/OK and reassortant D/OK-D/660 in this population.

Diagnosis of West Nile virus infections: Evaluation of different laboratory methods.

The diagnosis of West Nile virus (WNV) is challenging due to short-term and low-level viremia, flavivirus cross-reactivity, and long immunoglobulin M (IgM) persistence. To evaluate different methods for WNV detection [reverse transcription-polymerase chain reaction (RT-PCR), IgM/IgG antibodies, IgG avidity] in serum, cerebrospinal fluid (CSF), and urine samples of patients with confirmed WNV infection. The study included patients with confirmed WNV neuroinvasive infection (n = 62), asymptomatic WNV seropositive individuals (n = 22), and individuals with false-positive WNV IgM antibodies (n = 30). WNV RNA was detected using RT-PCR. A commercial ELISA was used to detect WNV IgM/IgG antibodies with confirmation of cross-reactive samples using a virus neutralization test (VNT). IgG-positive samples were tested for IgG avidity. The WNV-RNA detection rates were significantly higher in the urine (54.5%)/serum (46.4%) than in CSF (32.2%). According to the sampling time, the WNV-RNA detection rates in urine collected within 7 days/8-14/≥ 15 days were 29.4/66.6/62.5% (P = 0.042). However, these differences were not observed in the CSF. The median RT-PCR cycle threshold values were significantly lower in urine (32.5, IQR = 28-34) than in CSF (34.5, IQR = 33-36). The frequency of positive WNV IgM and IgG significantly differed according to the sampling time in serum but not in CSF. Positive IgM/IgG antibodies were detected in 84.3/9.3% of serum samples collected within 7 days, 100/71.1% of samples collected 8-14, and 100% samples collected after ≥ 15 days. Recent WNV infection was confirmed by low/borderline avidity index (AI) in 13.6% of asymptomatic individuals. A correlation between ELISA and AI was strong negative for IgM and strong positive for IgG. No significant correlation between ELISA IgG and VNT was found. The frequency of WNV RNA and antibody detection depends on the sampling time and type of clinical samples. IgG avidity could differentiate recent WNV infections from long-persisting IgM antibodies.