RT-qPCR Test Research Articles

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.

IL-20RA is Associated with the Risk of Diabetic Microangiopathy: A Bidirectional Mendelian Randomization Analysis and Clinical Validation.

Studies have demonstrated a link between chronic inflammatory responses and diabetic microangiopathy, which include diabetic nephropathy, diabetic retinopathy, and diabetic neuropathy. However, it remains unclear whether there is a causal association between circulating inflammatory cytokines and the development of diabetic microvascular complications. This study aimed to investigate whether altered genetically predicted concentrations of circulating inflammatory cytokines were associated with the development of diabetic microvascular complications using two-sample Mendelian randomization (MR) analysis and clinical validation. Pooled data on diabetic nephropathy, diabetic retinopathy, diabetic neuropathy, and 91 circulating inflammatory cytokines were obtained from publicly available databases. The analysis was conducted mainly using the inverse variance weighting (IVW) method and the results were assessed based on the odds ratio (OR) and 95% confidence interval (CI). In addition, the stability and reliability of the results were verified using the leave-one-out method, heterogeneity tests, and horizontal pleiotropy. Finally, ELISA and RT-qPCR were utilized to assess the expression of relevant inflammatory cytokines associated with diabetic microvascular complications. Mendelian randomization analysis identified a total of 9 circulating inflammatory cytokines that exhibit causal associations with the diabetic microangiopathy, with IL-20RA being a common risk factor for all three conditions. Clinical studies have found elevated plasma IL-20RA concentrations in patients with diabetic peripheral neuropathy, and RT-qPCR testing of peripheral blood mononuclear cells revealed significantly higher IL-20RA mRNA expression in patients with diabetic peripheral neuropathy as compared to normal individuals. This study highlights the potential role of specific inflammatory cytokines in the development of diabetic microangiopathy (diabetic nephropathy, diabetic retinopathy and diabetic neuropathy). Additionally, IL-20RA emerges as a potential common risk factor for three diabetic microvascular complications. These findings may provide novel insights into early prevention and new therapeutic strategies for diabetic microvascular complications.

Development and Validation of One-Step Reverse Transcription-Droplet Digital PCR for Plum Pox Virus Detection and Quantification from Plant Purified RNA and Crude Extract.

Plum pox virus (PPV) is the etiological agent of sharka, the most important viral disease of stone fruit worldwide. In this study, a one-step reverse transcription real-time PCR test (RT-qPCR) was modified and translated as a one-step RT-droplet digital PCR (RT-ddPCR) for sensitive, direct, and accurate detection and quantification of PPV. The modified RT-qPCR and RT-ddPCR PPV detection tests were validated using both plant purified total RNA (TRNA) and crude extract as templates. The proposed tests were sensitive, specific, selective, repeatable, and reproducible in detecting PPV from fresh, lyophilized, and in vitro plant samples. RT-ddPCR was more sensitive than RT-qPCR in detecting PPV using purified TRNA while showing the same sensitivity using crude extract. This work highlights the robustness, time-saving, and cost-effective nature of the proposed one-step RT-ddPCR test, offering a potential reduction in resources for PPV detection and quantification even with raw extracts.

Exploration of the Active Components and Mechanism of Jiegeng (Platycodonis Radix) in the Treatment of Influenza Virus Pneumonia Through Network Pharmacology Analysis and Experimental Verification.

This study aimed to explore the pathogenesis of platycodin D and luteolin, which are both active components in Jiegeng (Platycodonis Radix), in the treatment of influenza virus pneumonia through network pharmacology analysis combined with experimental verification. The bioactive components of Jiegeng (Platycodonis Radix) were screened by TCMSP and literature mining, and the results were standardized via the UniProt database. The action targets for the disease were identified from databases including OMIM, GeneCards, TTD, DisGeNET, and PharmGKB. Then, the visualized key target regulatory network and protein-protein interaction (PPI) network for the active components were established using Cytoscape3.7.1 software. The findings were illustrated through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The intervention concentrations of platycodin D and luteolin were screened by the CCK8 method, and the important signaling pathways of platycodin D and luteolin for treating influenza virus pneumonia were verified by RT-qPCR and Western blot tests. From data mining, 89 common drug-disease targets were screened out, and five major active components of Jiegeng (Platycodonis Radix), including platycodin D and luteolin, were obtained. Besides, 11 therapeutic targets including IL-17, IL-6, TNF-α, JUN, and MAKP1 were identified by PPI network analysis. GO and KEGG enrichment analyses showed that the pathways most related to the mechanisms of Jiegeng (Platycodonis Radix) against influenza virus pneumonia included the TNF and IL-17 signaling pathways and apoptosis. Invitro experiments demonstrated that the model group exhibited a notable elevation in mRNA levels of IL-6, IL-17, TNF-α, JUN, MAPK1, and the IL-17/-acting protein ratio, as compared to the control group (p < 0.05). In contrast to the model group, the IL-6, IL-17, TNF-α, JUN, MAPK1 mRNA expression levels, and the IL-17 protein ratio in both the platycodin D group and luteolin group were considerably decreased (p < 0.05). Combined with network pharmacology and experimental verification, this study revealed that platycodin D and luteolin in Jiegeng (Platycodonis Radix) may treat influenza virus pneumonia by regulating inflammation through the IL-17 signaling pathway.

Transcriptome analysis revealed the mechanism of skeletal muscle growth and development in different hybrid sheep.

To analyze the molecular mechanism of heterosis in East Friesian sheep × Hu sheep (EH) hybrid sheep and Suffolk × EH (SHE) hybrid sheep (Ovis aries). In this research, the growth performance data of Hu sheep (H), EH and SHE from birth to 8 months of age were analyzed. Three 8-month-old sheep of each of the three strains (9 sheep in total) were chosen and their longissimus dorsi muscles were collected for transcriptome sequencing. We verified the expression of seven differentially expressed genes (DEGs) by RT-qPCR. The results showed: (1) body weight and chest circumference of EH were significantly greater than H (p<0.05), except at 4 months of age. Body weight and chest circumference of SHE was significantly higher than EH (p<0.05), except at 6 months of age. (2) 310 DEGs were screened in the EH and H, GO and KEGG showed DEGs were mainly concentrate on the categories of actin cytoskeleton, calcium binding, cGMP-PKG and MAPK signaling pathway, which correlating the development of skeletal muscle and energy metabolism. 329 DEGs were screened in the SHE and EH. DEGs were mainly enriched in ECM-receptor interactions and cell adhesion molecules. (3) PPI screening yielded five (MYL2, TNNI1, TNNI3, MYH11, TNNC1) and three (SOX10, COL2A1, MPZ) pivotal DEGs regulating muscle development in EH and SHE. (4) RT-qPCR test results were consistent with transcriptome sequencing. This study provides candidate genes for improving sheep growth traits. It provides a theoretical basis for analyzing the mechanism of muscle development in crossbred sheep.

Evaluation of a Commercial Rapid Molecular Point-of-Care Assay for Differential Diagnosis Between SARS-CoV-2 and Flu A/B Infections in a Pediatric Setting.

Given the ongoing COVID-19 pandemic, there is a need to identify SARS-CoV-2 and to differentiate it from other respiratory viral infections, especially influenza A and B, in various critical settings. Since their introduction, the use of rapid antigen tests has spread worldwide, but there is variability in their diagnostic accuracy. In the present study, we evaluated the clinical performance of the ID NOW™ COVID-19 2.0, a molecular point-of-care test (POCT) based on enzymatic isothermal amplification for the differential diagnosis of SARS-CoV-2 and influenza A/B in a pediatric emergency setting. A cohort of pediatric patients admitted between December 2022 and February 2023 were simultaneously tested with the POCT and standard laboratory molecular assay. Our findings showed high negative agreement of the POCT assay across the different age groups for SARS-CoV-2, influenza A, and influenza B (more than 98.0%), while its positive agreement varied significantly for the abovementioned viral species from 50.0% to 100%. These results highlight the potential of the ID NOW™ COVID-19 2.0 POCT assay as a reliable and rapid tool for excluding SARS-CoV-2 and influenza A/B infections in symptomatic pediatric patients, although its variable positive agreement suggests a need for confirmatory RT-qPCR testing in certain clinical and epidemiological settings in order to ensure accurate diagnosis and appropriate patient management.

How control and eradication of BVDV at farm level influences the occurrence of calf diseases and antimicrobial usage during the first six months of calf rearing

BackgroundBovine viral diarrhoea (BVD) is one of the major cattle diseases causing economic losses worldwide. Nowadays the disease manifests mainly as virus-induced immunosuppression and early embryonic death, impacting overall herd performance and contributing to increased antibiotic usage in calf rearing.MethodsIn our study we investigated the effect of rapid BVDV control measures on calf diseases and antimicrobial usage after weaning on a large industrial dairy farm. Persistently infected (PI) animals were identified and removed from the herd within a short period of time, and all susceptible animals were vaccinated against BVDV. Recorded herd parameters and AB usage were monitored retrospectively and compared with data collected after starting the BVD control program.Results and discussionThe programme began in January 2023 with identifying and eliminating PI animals from the farm. Twenty-one PI animals were found by using RT-qPCR testing of blood sera out of the 1571 animals tested (1.33%). Subsequent testing (January and December 2023) identified further 28 PI animals amongst the 542 calves tested shortly after birth, and all were instantly removed from the farm. In parallel with the BVDV eradication measures, AB usage dropped by more than 50% compared to previous years. Calf mortality also decreased from 7.45 to 4.38% as the control program progressed. Correspondingly, both the number of respiratory and diarrhoea cases decreased dramatically on the farm while the eradication measures were in place.ConclusionOur study clearly demonstrated the positive effects of BVDV eradication on the improvement of calf health and importantly, a reduction of AB usage, contributing to the One Health perspective of farm animal production.

The Immune-Related 27-Gene Signature DetermaIO Predicts Response to Neoadjuvant Atezolizumab plus Chemotherapy in Triple-Negative Breast Cancer.

We assessed the 27-gene RT-qPCR-based DetermaIO assay and the same score calculated from RNA sequencing (RNA-seq) data as predictors of sensitivity to immune checkpoint therapy in the neoTRIPaPDL1 randomized trial that compared neoadjuvant carboplatin/nab-paclitaxel chemotherapy (CT) plus atezolizumab with CT alone in stage II/III triple-negative breast cancer. We also assessed the predictive function of the immuno-oncology (IO) score in expression data of patients treated with pembrolizumab plus paclitaxel (N = 29) or CT alone (N = 56) in the I-SPY2 trial. RNA-seq data were obtained from pretreatment core biopsies from 242 (93.8%) of the 258 patients in the per-protocol-population. The DetermaIO RT-qPCR test, performed in the CAP/CLIA-accredited laboratory of Oncocyte Corp., was available for 220 patients (85.3%). A previously established threshold was used to assign DetermaIO-positive versus DetermaIO-negative status. Publicly available microarray data were used from I-SPY2. IO scores calculated from RNA-seq and RT-qPCR data were highly concordant. In neoTRIPaPDL1, DetermaIO-positive cancers (N = 92, 41.8%) had pathologic complete response (pCR) rates of 69.8% and 46.9% in the CT + atezolizumab and CT arms, respectively. In DetermaIO-negative cases, pCR rates were similar in both arms (44.6% vs. 49.2%; interaction test P = 0.04). PDL1 protein expression and stromal tumor-infiltrating lymphocyte count were not predictive of differential benefit from atezolizumab. In I-SPY2, IO-positive cancers (45.9%) had pCR rates of 85.7% and 16%, with and without immunotherapy, respectively. In IO-negative cancers, pCR rates were 46.7% versus 16.1%. DetermaIO identified patients who benefited from neoadjuvant immunotherapy resulting in improved pCR rate, independently of PDL1.

Managing the largest school-based COVID-19 outbreak: insights and mitigation strategies

The COVID-19 pandemic, caused by SARS-CoV-2, has significantly impacted educational environments due to their role in community transmission. Early reports indicated low transmission rates in schools, but subsequent studies showed variability, including significant outbreaks like the one in Israel. This study investigates a large COVID-19 outbreak in a boarding school in Brazil, analyzing transmission dynamics and the effectiveness of control measures such as mass testing and isolation. A total of 291 individuals from the school community were tested, revealing an overall infection rate of 64.6%. Initial cases were identified through antigen testing, followed by extensive RT-qPCR testing. Mass testing and timely isolation of infected individuals significantly reduced transmission rates. Whole-genome sequencing was performed on seven positive samples, and environmental contamination was confirmed, with 11 of 35 surface samples testing positive for SARS-CoV-2. The outbreak was controlled within 30 days through rigorous testing, isolation, and environmental disinfection. The study emphasizes the importance of mass testing, including asymptomatic individuals, and highlights the role of environmental contamination in transmission. The findings contribute to understanding COVID-19 outbreaks in educational settings and provide practical insights into managing future outbreaks through comprehensive testing and control strategies. These results may guide policymakers and public health officials in improving pandemic response measures in similar environments.

The impact of photobiomodulation on angiogenic differentiation of two different dental derived stem cells using two irradiation protocols: an in vitro investigation

The present study aimed to compare the effect of photobiomodulation with different energy densities on the angiogenic differentiation of human periodontal ligament stem cells (hPDLSCs) and stem cells from human exfoliated deciduous teeth (SHED). Photobiomodulation therapy with a 660 nm diode laser (2.4 J/cm2 and 3.9 J/cm2) on two consecutive days post-culture was applied to two types of stem cells (hPDLSCs and SHED). The Quantitative Real-time Polymerase Chain Reaction (RT-qPCR) test was undertaken to investigate Vascular Endothelial Growth Factor-A (VEGF-A) and Angiopoietin I (ANG-I) genes on days 1, 3, 5, 7, and 10 after the first session of laser application. The 4′,6-diamidino-2-phenylindole (DAPI) staining and Methyl Thiazolyl Tetrazolium (MTT) test were conducted on days 1, 3, and 5 after the first session of laser application, to assess the cell viability. The Two-way ANOVA with Tukey post hoc test was used to analyze the outcomes of the MTT and RT-qPCR tests. The results of the MTT and DAPI convergently illustrated that the groups receiving photobiomodulation with 2.4 J/cm2 had higher cell viability compared to 3.9 J/cm2. All experimental groups showed an upregulation of VEGF-A and ANG-I gene expression from day 1 to 5, followed by a downregulation from day 5 to 10. The groups with cultured hPDLSCs and SHED receiving photobiomodulation using 2.4 J/cm2 had the most amounts of VEGF-A and ANG-I gene expression on day 5, respectively. In conclusion, the 660 nm mediated photobiomodulation therapy of cultured SHED and hPDLSCs with 2.4 J/cm2 energy density may be associated with higher angiogenic differentiation (the expression of VEGF-A and ANG-I) as well as higher cell viability compared to the photobiomodulation therapy with 3.9 J/cm2.

Personal characteristics and transmission dynamics associated with SARS-CoV-2 semi-quantitative PCR test results: an observational study from Belgium, 2021-2022.

Following harmonization efforts by the Belgian National Reference Center for SARS-CoV-2, semi-quantitative PCR test (SQ-PCR) results, used as a proxy for viral load, were routinely collected after performing RT-qPCR tests. We investigated both the personal characteristics associated with SQ-PCR results and the transmission dynamics involving these results. We used person-level laboratory test data and contact tracing data collected in Belgium from March 2021 to February 2022. Personal characteristics (age, sex, vaccination, and laboratory-confirmed prior infection) and disease stage by date of symptom onset were analyzed in relation to SQ-PCR results using logistic regression. Vaccine effectiveness (VE) against a high viral load (≥107 copies/mL) was estimated from the adjusted probabilities. Contact tracing involves the mandatory testing of high-risk exposure contacts (HREC) after contact with an index case. Odds ratios for test positivity and high viral load in HREC were calculated based on the SQ-PCR result of the index case using logistic regression models adjusted for age, sex, immunity status (vaccination, laboratory-confirmed prior infection), variant (Alpha, Delta, Omicron), calendar time, and contact tracing covariates. We included 909,157 SQ-PCR results of COVID-19 cases, 379,640 PCR results from index cases, and 72,052 SQ-PCR results of HREC. High viral load was observed more frequently among recent cases, symptomatic cases, cases over 25 years of age, and those not recently vaccinated (>90 days). The vaccine effectiveness (VE) of the primary schedule in the first 30 days after vaccination was estimated at 47.3% (95%CI 40.8-53.2) during the Delta variant period. A high viral load in index cases was associated with an increased test positivity in HREC (OR 2.7, 95%CI 2.62-2.79) and, among those testing positive, an increased likelihood of a high viral load (OR 2.84, 95%CI 2.53-3.19).

The 2023 South Sudanese outbreak of Hepatitis E emphasizes ongoing circulation of genotype 1 in North, Central, and East Africa

In April 2023, an outbreak of acute hepatitis was reported amongst internally displaced persons in the Nazareth community of South Sudan. IgM serology-based screening suggested the likely etiologic agent to be Hepatitis E virus (HEV). In this study, plasma specimens collected from anti-HEV IgM-positive cases were subjected to additional RT-qPCR testing and sequencing of extracted nucleic acids, resulting in the recovery of five full and eight partial HEV genomes. Maximum likelihood phylogenetic reconstruction confirmed the genomes belong to HEV genotype 1. Using distance-based methods, we show that genotype 1 is best split into three sub-genotypes instead of the previously proposed seven, and that these sub-genotypes are geographically restricted. The South Sudanese sequences confidently cluster within sub-genotype 1e, endemic to northeast, central, and east Africa. Bayesian Inference of phylogeny incorporating sampling dates shows that this new outbreak is not directly descended from other recent local outbreaks for which sequence data is available. However, the analysis suggests that sub-genotype 1e has been consistently and cryptically circulating locally for at least the past half century and that the known outbreaks are often not directly descended from one another. The ongoing presence of HEV, combined with poor sanitation and hygiene in the conflict-affected areas in the region, place vulnerable populations at risk for infection and its more serious effects, including progression to fulminant hepatitis.

Transcriptomic Analysis Provides Insights into the Energetic Metabolism and Immune Responses in Litopenaeus vannamei Challenged by Photobacterium damselae subsp. damselae

To explore the molecular mechanisms of the Litopenaeus vannamei response to infection by Photobacterium damselae, reveal its immune response and energetic metabolic effect, and provide a valuable genetic data source for the scientific prevention and control of Vibrio infection, transcriptomic analysis, RT-qPCR, and physiological and biochemical tests were conducted. The results showed that the expression of key genes involved in lipid and carbohydrate transport, such as apolipoprotein and TPS, was upregulated after pathogenic infection, which brought the accumulation of triacylglycerol and trehalose into the hemolymph. Additionally, the pathogenic infection selectively triggered an immune response in infected L. vannamei, activating certain immune pathways, such as the serpins and MAPK pathways. The pathogenic infection suppressed the activity of phenoloxidase (PO), and the prophenoloxidase (PPO) cascade responses were suppressed by the invasive bacteria. This paper will help us understand the energetic metabolism, immune response, and activation of the immune recognition response after pathogenic infection by P. damselae, and it lays a theoretical foundation for the biological prevention and control of P. damselae infection.

Assessment of DNA/RNA Defend Pro: An Inactivating Sample Collection Buffer for Enhanced Stability, Extraction-Free PCR, and Rapid Antigen Testing of Nasopharyngeal Swab Samples.

This study comprehensively evaluated the DNA/RNA Defend Pro (DRDP) sample collection buffer, designed to inactivate and stabilize patient samples. The primary objectives were to assess DRDP's efficacy in ensuring sample stability, facilitating extraction-free polymerase chain reaction (PCR), and ensuring compatibility with rapid antigen testing (RAT). Ninety-five diagnostic nasopharyngeal swab samples tested for influenza virus (influenza A), respiratory syncytial virus (RSV A), and/or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were 10-fold diluted with DRDP and anonymized. Initial characterization and retesting of these samples using cobas Liat confirmed 88 samples as positive, validating the presence of viral targets. Results from rapid antigen testing showed lower sensitivity compared to nucleic acid amplification testing (NAAT) but maintained perfect specificity, with 40 out of 88 positive samples by cobas Liat also testing positive for RAT. Direct RT-qPCR of DRDP-diluted samples demonstrated robust compatibility, with 72 out of 88 samples positive for cobas Liat also testing positive by direct RT-qPCR. Non-concordant results could be explained by the 200-fold lower input of extraction-free NAAT. Stability testing involved incubating 31 positive samples at 4 °C, 20 °C, and 37 °C for 7 days, with extraction-free NAAT. DRDP guaranteed viral RNA stability at all temperatures for influenza A, SARS-CoV-2, and RSV A, showing stability up to 7 days at 4 °C. In conclusion, DRDP is an effective stabilizing medium compatible with direct RT-qPCR and rapid antigen testing and shows great potential for optimizing diagnostic processes, particularly in resource-limited or time-sensitive scenarios.

Influenza virus shedding and symptoms: Dynamics and implications from a multiseason household transmission study.

Isolation of symptomatic infectious persons can reduce influenza transmission. However, virus shedding that occurs without symptoms will be unaffected by such measures. Identifying effective isolation strategies for influenza requires understanding the interplay between individual virus shedding and symptom presentation. From 2017 to 2020, we conducted a case-ascertained household transmission study using influenza real-time RT-qPCR testing of nasal swabs and daily symptom diary reporting for up to 7 days after enrolment (≤14 days after index onset). We assumed real-time RT-qPCR cycle threshold (Ct) values were indicators of quantitative virus shedding and used symptom diaries to create a score that tracked influenza-like illness (ILI) symptoms (fever, cough, or sore throat). We fit phenomenological nonlinear mixed-effects models stratified by age and vaccination status and estimated two quantities influencing isolation effectiveness: shedding before symptom onset and shedding that might occur once isolation ends. We considered different isolation end points (including 24 h after fever resolution or 5 days after symptom onset) and assumptions about the infectiousness of Ct shedding trajectories. Of the 116 household contacts with ≥2 positive tests for longitudinal analyses, 105 (91%) experienced ≥1 ILI symptom. On average, children <5 years experienced greater peak shedding, longer durations of shedding, and elevated ILI symptom scores compared with other age groups. Most individuals (63/105) shed <10% of their total shed virus before symptom onset, and shedding after isolation varied substantially across individuals, isolation end points, and infectiousness assumptions. Our results can inform strategies to reduce transmission from symptomatic individuals infected with influenza.

Trends in SARS-CoV-2 Cycle Threshold Values in Bosnia and Herzegovina-A Retrospective Study.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which led to the COVID-19 pandemic, has significantly impacted global public health. The proper diagnosis of SARS-CoV-2 infection is essential for the effective control and management of the disease. This study investigated the SARS-CoV-2 infection using RT-qPCR tests from laboratories in Bosnia and Herzegovina. We performed a retrospective study of demographic data and Ct values from 170,828 RT-qPCR tests from April 2020 to April 2023, representing 9.3% of total national testing. Samples were collected from 83,413 individuals across different age groups. Of all tests, 33.4% were positive for SARS-CoV-2, with Ct values and positivity rates varying across demographics and epidemic waves. The distribution was skewed towards older age groups, although lower positivity rates were observed in younger age groups. Ct values, indicative of viral load, ranged from 12.5 to 38. Lower Ct values correlated with higher positive case numbers, while higher Ct values signaled outbreak resolution. Additionally, Ct values decreased during epidemic waves but increased with the dominance of certain variants. Ct value-distribution has changed over time, particularly after the introduction of SARS-CoV-2 variants of interest/concern. Established Ct value trends might, therefore, be used as an early indicator and additional tool for informed decisions by public health authorities in SARS-CoV-2 and future prospective pandemics. Moreover, they should not be overlooked in future epidemiological events.

Host Jump of an Exotic Fish Rhabdovirus into a New Class of Animals Poses a Disease Threat to Amphibians.

Spring viremia of carp virus (SVCV) is a rhabdovirus that primarily infects cyprinid finfishes and causes a disease notifiable to the World Organization for Animal Health. Amphibians, which are sympatric with cyprinids in freshwater ecosystems, are considered non-permissive hosts of rhabdoviruses. The potential host range expansion of SVCV in an atypical host species was evaluated by testing the susceptibility of amphibians native to the Pacific Northwest. Larval long-toed salamanders Ambystoma macrodactylum and Pacific tree frog Pseudacris regilla tadpoles were exposed to SVCV strains from genotypes Ia, Ib, Ic, or Id by either intraperitoneal injection, immersion, or cohabitation with virus-infected koi Cyprinus rubrofuscus. Cumulative mortality was 100% for salamanders injected with SVCV, 98-100% for tadpoles exposed to virus via immersion, and 0-100% for tadpoles cohabited with SVCV-infected koi. Many of the animals that died exhibited clinical signs of disease and SVCV RNA was found by in situ hybridization in tissue sections of immersion-exposed tadpoles, particularly in the cells of the gastrointestinal tract and liver. SVCV was also detected by plaque assay and RT-qPCR testing in both amphibian species regardless of the virus exposure method, and viable virus was detected up to 28 days after initial exposure. Recovery of infectious virus from naïve tadpoles cohabited with SVCV-infected koi further demonstrated that SVCV transmission can occur between classes of ectothermic vertebrates. Collectively, these results indicated that SVCV, a fish rhabdovirus, can be transmitted to and cause lethal disease in two amphibian species. Therefore, members of all five of the major vertebrate groups (mammals, birds, reptiles, fish, and amphibians) appear to be vulnerable to rhabdovirus infections. Future research studying potential spillover and spillback infections of aquatic rhabdoviruses between foreign and domestic amphibian and fish species will provide insights into the stressors driving novel interclass virus transmission events.

Detection of influenza virus in urban wastewater during the season 2022/2023 in Sicily, Italy.

Seasonal influenza generally represents an underestimated public health problem with significant socioeconomic implications. Monitoring and detecting influenza epidemics are important tasks that require integrated strategies. Wastewater-based epidemiology (WBE) is an emerging field that uses wastewater data to monitor the spread of disease and assess the health of a community. It can represent an integrative surveillance tool for better understanding the epidemiology of influenza and prevention strategies in public health. We conducted a study that detected the presence of Influenza virus RNA using a wastewater-based approach. Samples were collected from five wastewater treatment plants in five different municipalities, serving a cumulative population of 555,673 Sicilian inhabitants in Italy. We used the RT-qPCR test to compare the combined weekly average of Influenza A and B viral RNA in wastewater samples with the average weekly incidence of Influenza-like illness (ILI) obtained from the Italian national Influenza surveillance system. We also compared the number of positive Influenza swabs with the viral RNA loads detected from wastewater. Our study investigated 189 wastewater samples. Cumulative ILI cases substantially overlapped with the Influenza RNA load from wastewater samples. Influenza viral RNA trends in wastewater samples were similar to the rise of ILI cases in the population. Therefore, wastewater surveillance confirmed the co-circulation of Influenza A and B viruses during the season 2022/2023, with a similar trend to that reported for the weekly clinically confirmed cases. Wastewater-based epidemiology does not replace traditional epidemiological surveillance methods, such as laboratory testing of samples from infected individuals. However, it can be a valuable complement to obtaining additional information on the incidence of influenza in the population and preventing its spread.

Insights into SARS-CoV-2 Surveillance among Prison Populations in Mato Grosso do Sul, Brazil, in 2022.

This study examines the epidemiological and genomic characteristics, along with the transmission dynamics, of SARS-CoV-2 within prison units I and II in Campo Grande, Mato Grosso do Sul, Brazil. Conducted between May and October 2022, it reveals how the virus spreads in the confined settings of prisons, emphasizing the roles of overcrowded cells, frequent transfers, and limited healthcare access. The research involved 1927 participants (83.93% of the total prison population) and utilized nasopharyngeal swabs and RT-qPCR testing for detection. Contact tracing monitored exposure within cells. Out of 2108 samples, 66 positive cases were identified (3.13%), mostly asymptomatic (77.27%), with the majority aged 21-29 and varying vaccination statuses. Next-generation sequencing generated 28 whole genome sequences, identifying the Omicron variant (subtypes BA.2 and BA.5) with 99% average coverage. Additionally, the study seeks to determine the relationship between immunization levels and the incidence of SARS-CoV-2 cases within this enclosed population. The findings underscore the necessity of comprehensive control strategies in prisons, including rigorous screening, isolation protocols, vaccination, epidemiological monitoring, and genomic surveillance to mitigate disease transmission and protect both the incarcerated population and the broader community.

Mayaro Virus as the cause of Acute Febrile Illness in the Colombian Amazon Basin.

Mayaro Fever (MF) is a tropical disease caused by the Mayaro virus (MAYV), with outbreaks documented in Latin America. A hospital-based fever surveillance in Leticia, Colombian Amazon, collected sera from 1,460 patients aged 5-89 between December 2020 and April 2023. Dengue and malaria were the main diagnoses (19.4 and 5.8%, respectively), leaving 71.4% of cases unidentified after testing. Metagenomic sequencing and real-time RT-qPCR testing identified MAYV in two patients (25-year-old male and an 80-year-old female) exhibiting typical symptoms, of MF including rash, joint pain, and fever. Phylogenetics analysis of these two viruses revealed a close relationship to Peruvian strains within the MAYV D genotype. The study of AFI in Leticia, Colombia, identified dengue as prevalent, with malaria, COVID-19, Influenza, and Zika viruses also detected. Despite extensive testing, most cases remained unexplained until metagenomic sequencing revealed MAYV, previously unseen in Colombia but known in neighboring countries. This study presents the first near full-length genomes of MAYV in Colombia, highlighting the need for further seroprevalence studies and enhanced surveillance to understand and control the spread of the virus in the region.