Cycle Threshold Values Research Articles

Diagnostic performance of rapid antigen tests for SARS-CoV-2 transmission risk based on cycle threshold values in the emergency department

BackgroundThis study aimed to investigate the diagnostic performance of the rapid antigen test (RAT) for screening patients with cycle threshold (Ct) values of SARS-CoV-2 reverse transcription-polymerase chain reaction (RT-PCR) in the emergency department. Previous studies have shown that Ct values could be used as indicators of infectiousness. Therefore, we considered the Ct value an indicator of potential infectiousness. MethodsThis single-center retrospective observational study was conducted between January 1, 2020, and March 31, 2022. Patients who underwent both RT-PCR and RAT for the diagnosis of COVID-19 were included. Patients with negative RT-PCR results were excluded. Patients with Ct values lower than 26 and 30 were considered potentially infectious for COVID-19. ResultA total of 386 patients were analyzed. At Ct value cutoffs of 26 and 30, the result of the RAT showed a sensitivity of 82% and 74%, specificity of 84% and 89%, and area under the curve (AUC) of 0.829 and 0.813, respectively, in the receiver operating characteristic curve. However, the NPV was relatively low at 55% and 25%. ConclusionThe RAT might be a rapid screening tool for detecting patients with the infectiousness of SARS-CoV-2. However, considering the low NPV, it is challenging to depend only on a negative test result from an antigen test to terminate quarantine. Clinicians should consider additional factors, such as the duration of symptoms and the immunocompromised state, for SARS-CoV-2 transmission.

A-259 Detecting Human RNase P Gene Prevents False-Negative of SARS-CoV-2

Abstract Background Real-time Reverse transcriptase—polymerase chain reaction (RT-PCR) is a regular technique for detecting SARS-CoV-2 infection. Human RNase P sequence is using as internal control for PCR assay usually. This article discusses the correlation between SARS-CoV-2 PCR result and human RNase P gene result. Methods Nasopharyngeal swabs were collected from two patients within three consecutive days. We detected for SARS-CoV-2 target gene and human RNase P gene by using real-time RT-PCR assay, which SARS-CoV-2 target gene includes E gene, Rarp gene and N gene . And the threshold cycle values (Ct) were determined by cobas® z 480. Evaluate the results of SARS-CoV-2 and RNase P gene detection. All samples were also analysis for SARS-CoV-2 infection in Taiwan Centers for Disease Control. Results Results of SARS-CoV-2 PCR assay were consistent in both laboratories. SARS-CoV-2 target genes were detected from specimens of both patients on day1. Ct values of E gene were 29.63 and 29.50. Ct values of Rdrp gene were 32.23 and 32.50. Ct values of N gene were 33.09 and 32.68. Ct values of RNase P gene were 21.21 and 21.22. On day 2 and 3, all specimens were negative detected for SARS-CoV-2 PCR. Ct values of RNase P gene were 25.73 and 26.16 on day 2 and 29.82 and 30.28 on day 3. Conclusion The Ct values of human RNase P gene detection can be correlated with the numbers of collected cells. Comparing the results of two patients for three consecutive days, the Ct value of human RNase P gene related to SARS-CoV-2 PCR result. Collection technique can be a factor to affect Ct value of human RNase P gene detection. Develop acceptance range for Ct value of RNase P gene detection can be a quality standard for sample collection to prevent false-negative of SARS-CoV-2.

Comparative detection of syndecan-2 methylation in preoperative and postoperative stool DNA in patients with colorectal cancer.

Early detection of colorectal cancer (CRC) is essential to reduce cancer-related morbidity and mortality. Stool DNA (sDNA) testing is an emerging method for early CRC detection. Syndecan-2 (SDC2) methylation is a potential biomarker for the sDNA testing. Aberrant DNA methylation is an early epigenetic event during tumorigenesis and can occur in the normal colonic mucosa during aging, which can compromise the sDNA test results. To determine whether methylated SDC2 in sDNA normalizes after surgical resection of CRC. In this prospective study, we enrolled 151 patients with CRC who underwent curative surgical resection between September 2016 and May 2020. Preoperative stool samples were collected from 123 patients and postoperative samples were collected from 122 patients. A total of 104 samples were collected from both preoperative and postoperative patients. Aberrant promoter methylation of SDC2 in sDNA was assessed using linear target enrichment quantitative methylation-specific real-time polymerase chain reaction. Clinicopathological parameters were analyzed using the results of SDC2 methylation. Detection rates of SDC2 methylation in the preoperative and postoperative stool samples were 88.6% and 19.7%, respectively. Large tumor size (3 cm, P = 0.019) and advanced T stage (T3-T4, P = 0.033) were positively associated with the detection rate of SDC2 methylation before surgery. Female sex was associated with false positives after surgery (P = 0.030). Cycle threshold (CT) values were significantly decreased postoperatively compared with preoperative values (P < 0.001). The postoperative negative conversion rate for preoperatively methylated SDC2 was 79.3% (73/92). Our results suggested that the SDC2 methylation test for sDNA has acceptable sensitivity and specificity. However, small size and early T stage tumors are associated with a low detection rate of SDC2 methylation. As the cycle threshold values significantly decreased after surgery, SDC2 methylation test for sDNA might have a diagnostic value for CRC.

A-339 Utomated Flow of a Non-invasive Fetal Gender Determination Lab-developed Molecular Test

Abstract Background The discovery of cell-free fetal DNA in maternal circulation during pregnancy fostered the introduction of non-invasive tests. Fetal gender determination may be determined at early pregnancy by real-time PCR, identifying the presence or absence of Y chromosome markers in maternal plasma. Dasa has been offering this test for more than 10 years. In 2022, the assay was adapted to an automated flow. Methods The Flow Solution platform, powered by Roche®, was adapted to the in-house test, allowing the processing of 94 samples. Whole blood is obtained from pregnant women from the 8th week of pregnancy onwards. Each maternal plasma sample is extracted once and the DNA eluate submitted to real-time amplification targeting the DYS14 gene from the Y chromosome, in triplicate. The human RNAseP gene is co-amplified as an internal control. Full process takes 5 hours, with the possibility of overlapping a new routine every 2 hours, with a daily capacity of 12 routines (1128 samples/day). Results In 2022, 129 849 fetal gender determination results were released. Of these, 49% of the results indicated male and 51% female fetuses.A few samples displayed indeterminate results, showing amplification curves but with cycle threshold values that are above the cutoff. Other samples do not reach internal quality metrics and the analysis is repeated from the plasma or a new collection is requested. Results that, after the second collection, remain indeterminate, are forwarded with the patient's clinical history to the medical board. In contact with the patient, possible causes for inconclusive results are explained. In 2022, for every 10 000 tests there were 8 were medical contacts.The automated methodology developed and implemented, allowed a high processing volume, with 129 849 tests released in 2022, an increase of 33% compared to 2021 when the method had manual steps. The Total Service Time (TST) from collection to results release in 2022 was approximately 5 days, a 20% reduction compared to 2021, and the repetition rate of 2.43% in 2022 was 0.9% lower than 2021. Recollections also diminished from 1% in 2021 falling to 0.8% in 2022. Conclusion A new automated flow was successfully implemented in 2022 improving the productivity and quality of the service, achieving a high accuracy and leading to a significant reduction in errors and repetitions while reducing the turnaround time and recollections.

A-301 Analytical and Clinical Performance of the Molecular Method for Monkeypox Virus Detection

Abstract Background Monkeypox (MPX) is a zoonose caused by the Monkeypox virus (MPXV) belonging to the Orthopoxvirus genus of the Poxviridae family. Currently, MPXV is responsible for more than 84 000 infections worldwide, resulting in major social impacts. Although the reservoir is unknown, the main hosts are small rodents, typical of the tropical forests of West and Central Africa. The transmission occurs by interactions of infected animal-to-person (direct contact with the blood, bodily fluids, or cutaneous/mucosal lesions) and person-to-person (close contact with respiratory secretions, skin lesions, or recently contaminated objects). After infection, there is an incubation period of approximately 1–2 weeks (asymptomatic and non-transmissible), followed by the development of skin rashes over the body, lymphadenopathy, and systemic symptoms (fever, malaise, headache, myalgia, and fatigue). The rapid and accurate diagnosis of MPX is extremely important for combating the virus and also for epidemiological tracking. In this sense, molecular methods have been shown essential for the accurate detection and characterization of MPXV. Therefore, this study aims to describe the analytical and clinical validation of real-time polymerase chain reaction (qPCR) assay for MPXV detection. Methods The qPCR assay used in this validation was based on recommendations published by the Center for Disease Control and Prevention (CDC). The primer/probe sequence was commercially acquired and included the analysis of a MPXV-specific genomic region (CADT ID APNKYAD; Thermo Fisher, USA). Assay performance was evaluated using inactivated viral isolates and positive samples with known MPXV positive results. The analysis parameters included: (i) assay verification (performance evaluation of probes and reagents); (ii) Analytical sensitivity; (iii) Limit of detection (LOD); (iv) Analytical specificity (interference study with Herpes simplex virus and Varicella-Zoster virus); and (v) clinical validation (10 positive skin rashes samples for the MPXV). Results The assay demonstrated good performance visualized by the qPCR amplification curves (low cycle threshold (Ct) value and high fluorescence level) with an efficiency &amp;gt;92%. Analytical sensitivity and LOD tests were 100% and 50 copies/µL, respectively, considering a 95% confidence interval. The regression equations obtained show satisfactory reaction conditions with a positive correlation between the variables (coefficient of determination (r2) = 0.99). The assays indicated no cross-reactions with closely related viruses, demonstrating 100% of specificity. Finally, clinical validation showed 100% agreement with the expected results for all samples evaluated. Conclusion qPCR can play an important role in the rapid and accurate diagnosis of several viruses. The analytical and clinical validation provided data indicating high sensitivity and specificity for MPX detection. This study allowed the implementation of a sensitive, specific, and accurate molecular test for viral agent identification and allow prompt surveillance action by health such as epidemiological monitoring and control measures of these infections.

Combination treatment of persistent COVID-19 in immunocompromised patients with remdesivir, nirmaltrevir/ritonavir and tixegavimab/cilgavimab

We present a retrospective study on the treatment outcomes of severely immunocompromised patients with persistent COVID-19. The study analyzed data from 14 patients who received a combination of tixegavimab/cilgavimab and antiviral medications. Response was evaluated based on symptom improvement, PCR cycle-threshold values, and C-reactive protein levels. Eleven patients achieved complete clinical and virological resolution, while three showed partial responses. The study suggests a potential association between non-response and tixegavimab/cilgavimab neutralization. The findings underscore the need for tailored treatment approaches and further research on optimal strategies for managing persistent COVID-19, as well as the development of antivirals and variant-specific monoclonal antibodies.

Extraction-Free Detection of SARS-CoV-2 Viral RNA Using LumiraDx's RNA Star Complete Assay from Clinical Nasal Swabs Stored in a Novel Collection and Transport Medium.

Background: The rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is vital for patient care. The LumiraDx™ SARS-CoV-2 RNA Star Complete (RSC) is an Emergency Use Authorization-recognized molecular test using nasal/nasopharyngeal swabs immersed in a viral/universal transport medium (VTM/UTM). However, there is a critical need for an alternative medium for point-of-care testing (POCT). This study aimed to investigate Xtract-Free (XF), a novel collection medium for transport and direct (extraction-free) use with nucleic acid tests. Methods: Using serially diluted SARS-CoV-2 viral RNA (vRNA) in a routine UTM and XF, a limit of detection (LOD) was established via an RSC test and a quantitative reverse transcription PCR (RT-qPCR). Additionally, the results obtained from a panel of 108 clinical "car-side" nasal swabs collected in XF during the coronavirus pandemic and assessed using the "gold-standard" RT-qPCR assay were compared to Lumira's RSC assay. Results: The average replicate RT-qPCR cycle threshold (CT) values for vRNA in XF and UTM were observed to be equivalent. An LOD for which five out of five replicates were detected using XF or VTM was approximately 2000 copies/mL. The nasal swabs collected in XF exhibited 93.9% positive percent agreement (sensitivity) and 100% negative percent agreement (specificity) compared to the RT-qPCR. Three specimens tested positive via an RT-qPCR were negative when tested via RSC; however, all three samples had CT values ≥ 36.4. Conclusions: XF is equivalent to VTM/UTM and is compatible for use with the RSC test. Furthermore, XF can be used directly with RT-qPCRs and rapid antigen testing without the requirement for separate nucleic acid extraction (an extraction-free process), making it ideal for cost-effective high-throughput and decentralized respiratory testing. Impact Statement: This study is the first to evaluate LumiraDx's SARS-CoV-2 RNA Star Complete assay in concert with Xtract-Free (XF), a novel collection medium containing a proprietary RNase-inactivating technology for the rapid, "extraction-free" detection of SARS-CoV-2 RNA from clinical nasal swabs. Specimens collected in XF combined with rapid LumiraDx detection provide a safe and sensitive alternative to VTM/UTM, and Molecular Transport medium (MTM) for high throughput, "extraction-free" molecular detection.

Sensitivity of rapid antigen tests against SARS-CoV-2 Omicron and Delta variants.

Rapid antigen tests (RATs) have become an invaluable tool for combating the COVID-19 pandemic. However, concerns have been raised regarding the ability of existing RATs to effectively detect emerging SARS-CoV-2 variants. We compared the performance of 10 commercially available, emergency use authorized RATs against the Delta and Omicron SARS-CoV-2 variants using both individual patient and serially diluted pooled clinical samples. The RATs exhibited lower sensitivity for Omicron samples when using PCR cycle threshold (CT) value (a rough proxy for RNA concentration) as the comparator. Interestingly, however, they exhibited similar sensitivity for Omicron and Delta samples when using quantitative antigen concentration as the comparator. We further found that the Omicron samples had lower ratios of antigen to RNA, which offers a potential explanation for the apparent lower sensitivity of RATs for that variant when using C T value as a reference. Our findings underscore the complexity in assessing RAT performance against emerging variants and highlight the need for ongoing evaluation in the face of changing population immunity and virus evolution.

Clinical Outcomes of Acutely Ill Children According to Cycle Threshold Values of Respiratory Viruses Detected by Multiplex PCR Testing.

In this cohort study of 800 children attending a pediatric emergency department at Oulu University Hospital, Finland with fever or respiratory symptoms, the cycle threshold values of point-of-care multiplex polymerase chain reaction testing for respiratory viruses were not associated with hospitalization, respiratory support, or need for intensive care.

Clinical characteristics and prognostic nomograms of 12555 non-severe COVID-19 cases with Omicron infection in Shanghai

BackgroundOmicron variant of the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has rapidly become a global threat to public health. Numerous asymptomatic and mild cases had been admitted in shelter hospitals to quickly win the fight against Omicron pandemic in Shanghai. However, little is known about influencing factors for deterioration and length of stay (LOS) in hospitals among these non-severe cases.MethodsThis study included 12,555 non-severe cases with COVID-19 in largest shelter hospital of Shanghai, aiming to explore prognostic factors and build effective models for prediction of LOS.ResultsData showed that 75.0% of participants were initially asymptomatic. In addition, 94.6% were discharged within 10 days, only 0.3% with deterioration in hospitals. The multivariate analysis indicated that less comorbidities (OR = 1.792, P = 0.012) and booster vaccination (OR = 0.255, P = 0.015) was associated with the decreased risk of deterioration. Moreover, age (HR = 0.991, P < 0.001), number of symptoms (HR = 0.969, P = 0.005), time from diagnosis to admission (HR = 1.013, P = 0.001) and Cycle threshold (CT) values of N gene (HR = 1.081, P < 0.001) were significant factors associated with LOS. Based on these factors, a concise nomogram model for predicting patients discharged within 3 days or more than 10 days was built in the development cohort. In validation cohort, 0.75 and 0.73 of Areas under the curve (AUC) in nomograms, similar with AUC in models of simple machine learning, showed good performance in estimating LOS.ConclusionCollectively, this study not only provides important evidence to deeply understand clinical characteristics and risk factors of short-term prognosis in Shanghai Omicron outbreaks, but also offers a concise and effective nomogram model to predict LOS. Our findings will play critical roles in screening high-risk groups, providing advice on duration of quarantine and helping decision-makers with better preparation in outbreak of COVID-19.

SARS-CoV-2 Screening on the Multiplex Real-Time RT-PCR Gene Cycle Threshold - A Machine Learning Approach

Early diagnosis of the lethal SARS-CoV-2 virus determines a patient’s survival rate. Highly transmissible novel coronavirus prevention is possible with effective, rapid diagnostic strategies. The reverse transcription polymerase chain reaction (RT-PCR), a globally adopted SARS-CoV-2 detection technique, provides better diagnosis results. The output of the RT-PCR test produces the amplified gene scores of ORF1a/b, S, N, E and RdRp. This study intends to evaluate the performance of the RT-PCR-based COVID-19 diagnosis using machine learning models. The confirmatory genes ORF1b, E and RdRp and their cycle threshold (Ct) values are the main parameters used to build the machine learning model for SARS-CoV-2 screening. The real-time dataset collected from the Indian Council of Medical Research (ICMR) database containing missing, redundant information is processed and eliminated. Statistical interpretations are performed with demographic information to understand the dynamics of the disease prevalence in India. Binary classification models delivered promising results in discriminating the samples of two classes. The models were examined further to scrutinize their performance via evaluation metrics such as balanced accuracy, f1-score, ROC curve, precision and recall. This algorithmic assessment exhibits a better outcome on the RT-PCR-based SARS-CoV-2 disease diagnosis.

Diagnostic evaluation of Panbio™ antigen rapid diagnostic test for SARS‐CoV‐2: A systematic review and meta-analysis

IntroductionThe reverse transcriptase polymerase chain reaction (RT-PCR) is the reference diagnostic method for the confirmation of SARS-CoV-2 infected cases. However, various antigen rapid diagnostic tests (Ag-RDTs) have been developed. The purpose of this meta-analysis study was to assess the diagnostic performance of Panbio™ Ag-RDT (Abbott Point of Care) in identifying the SARS-CoV-2 virus. MethodsWe systematically searched eight databases from March 2020 until March 2023 to look for potentially eligible articles. Diagnostic meta-analysis of Panbio™ Ag-RDT used diverse evaluation indicators, including sensitivity, specificity, Diagnostic Odds Ratio (DOR), and the area under the curve (AUC) value. ResultsOf the 794 articles identified, 49 studies met the inclusion criteria. The pooled estimates of Panbio™ Ag-RDT for the diagnosis of SARS-CoV-2 were 0,65 (95% CI: 0,64–0,66), 0,99 (95% CI: 0,99–1,00), 578,03 (95% CI: 333,37–1002,26) for sensitivity, specificity, and DOR, respectively. Moreover, the summary receiver operating characteristic (SROC) curve revealed an AUC value of 0,942 (95% CI: 0,941–0,943), suggesting an outstanding diagnostic accuracy. Subgroup and meta-regression analyses showed that continent, study period, age, study population and cycle threshold (Ct) values constituted a source of heterogeneity. Furthermore, we demonstrated proof of publication bias for DOR values analyzed using Deek’s test (p = 0,001) and funnel plot. ConclusionPanbio™ Ag-RDT presented an outstanding diagnostic accuracy in the detection of the SARS-CoV-2 virus in both adults and children with or without symptoms.

Clinical characteristics of abruptly increased paediatric patients with Omicron BF.7 or BA.5.2 in Beijing

BackgroundThe coronavirus disease 2019 outbreak has hit Beijing since mid-Nov, 2022, with soaring growth of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among children. Therefore, it is vital to determine the clinical manifestations of epidemic SARS-CoV-2 strains in paediatric patients.MethodsIn this study, nucleic acid tests (NATs) for SARS-CoV-2 were performed in paediatric outpatients with symptoms of acute respiratory tract infection during 18 Nov–6 Dec, 2022. Half of the outpatients positive for SARS-CoV-2 were randomly selected to screen for other respiratory pathogens, whereas those with low cycle threshold values in SARS-CoV-2 NATs were amplified and sequenced to determine the SARS-CoV-2 variants. Finally, children positive for SARS-CoV-2 with clinical information in detail were enrolled in a follow-up study to identify potential factors significantly associated with long recovery.ResultsAmong 9625 paediatric outpatients tested for nucleic acid of SARS-CoV-2, 733 (7.62%, 733/9625) were identified as SARS-CoV-2 NAT positive, with only three (0.82%, 3/366) co-infected with other pathogens among 366 randomly selected patients, and 71 (62.83%) determined as Omicron subvariant BF.7 and 42 (37.22%) as BA.5.2 among 113 successfully sequenced. Among the 681 patients with complete clinical information, fever was the most common symptom (96.8%). In a follow-up study of 592 patients, 46.96% became asymptomatic on the third day and 65.71% on the fifth day. Only 1.7% of infected children experienced febrile seizures. Combined with abnormal C-reactive protein, a higher percentage of antibiotics administration was observed. More co-living members and longer duration of first symptoms served as independent risk factors for long-term recovery, especially in children vaccinated for SARS-CoV-2.ConclusionsBF.7 and BA.5.2 were the dominate Omicron subvariants and caused milder infections during the SARS-CoV-2 outbreak in Beijing. The number of co-living members and duration of first symptoms were independent risk factors for long-term recovery.

Surveillance of Vermont wildlife in 2021–2022 reveals no detected SARS-CoV-2 viral RNA

Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American’s wildlife populations.

Assessment and verification of chemical inactivation of peste des petits ruminants virus by virus isolation following virus capture using Nanotrap magnetic virus particles.

Research including diagnosis on highly contagious viruses at the molecular level such as PCR and next-generation sequencing requires complete inactivation of the virus to ensure biosafety and biosecurity so that any accidental release of the virus does not compromise the safety of the susceptible population and the environment. In this work, peste des petits ruminants virus (PPRV) was inactivated with chemical agents, and the virus inactivation was confirmed by virus isolation (VI) using Vero cells. Since the chemical agents are cytotoxic, inactivated virus (PPRV) was diluted 1:100 to neutralize cytotoxicity, and the residual viruses (if any) were captured using Nanotrap magnetic virus particles (NMVPs). The NMVPs and the captured viruses were subjected to VI. No CPE was observed, indicating complete inactivation, and the results were further supported by real-time RT-PCR. This new protocol to verify virus inactivation can be applicable to other viruses.

Prolonged existence of SARS-CoV-2 RNAs in the extracellular vesicles of respiratory specimens from patients with negative reverse transcription-polymerase chain reaction

Background and aimSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is primarily in the respiratory tract, particularly in patients with underlying comorbidities. This study aimed to investigate the presence of the virus inside the extracellular vesicles (EVs) in patients with and without chronic liver disease (CLD). MethodsEighty patients with positive SARS-CoV-2, including twenty-four patients with CLD and fifty-six patients without CLD, and five healthy controls with negative SARS-CoV-2 were enrolled. Nasal swab specimens were tested for the detection of SARS-CoV-2 using reverse transcription-polymerase chain reaction (RT-PCR). Patients with coronavirus disease 2019 (COVID-19) were followed up on days 7 and 14. Nasal swab, collected in viral transport media (VTM), and plasma samples were investigated at each time point. EVs were isolated from the nasal swabs (collected in VTM) and plasma using differential ultracentrifugation and estimated at each time point. The transmission or replication by the EVs was assessed in Vero E6 cells. ResultsIn patients with baseline RT-PCR positive, SARS-CoV-2 RNAs inside the EVs were found in 68/80 (85%) patients with higher viral load in the nasal swabs than in the EVs (cycle threshold (Ct) value, 23.4 ± 5.7 vs. 30.3 ± 5.0, P < 0.001). On follow-up at day 7, of the 32 patients negative for COVID-19, 15 (46.9%) had virus persistence in the EVs (Ct value, 30.7 ± 2.7), and on day 14, of the 56 patients with negative SARS-CoV-2, 16 patients (28.6%) had positive SARS-CoV-2 RNAs in the EVs (Ct value, 31.4 ± 3.0). The mean viral load decreased on days 7 and 14 compared to baseline in the nasal swabs (P < 0.001) but not in the EVs. Additionally, SARS-CoV-2 RNAs were undetectable in the plasma, but 12.5% of patients were positive in the plasma EVs. Significantly prolonged and high viral load was found in the EVs on day 14 in COVID-19 patients combined with CLD compared with COVID-19 patients (P = 0.0004). We found significant higher levels of EV-associated with endothelial cells and hepatocytes in the COVID-19 + CLD group than COVID-19 group (P = 0.032 and P = 0.002, respectively), suggesting more endothelial cells and hepatocytes cellular injury in liver disease patients with COVID-19. Interestingly, we also found EVs could transmit SARS-CoV-2 RNAs into Vero E6 cells at 24 h post-infection. ConclusionsThe identification of SARS-CoV-2 RNAs in the EVs in patients with negative RT-PCR indicates the persistence of infection and likely recurrence of the infection. It is suggestive of another route of transmission as EVs harbor SARS-CoV-2 RNAs. EV-associated RNAs may determine the ongoing inflammation and clinical course of subjects with undetectable SARS-CoV-2 virus and this may have relevance to better management of patients with CLD.

Comparison of two assays to diagnose herpes simplex virus in patients with central nervous system infections.

Multiplex PCR panels are frequently used for detecting the diagnosis of central nervous system infection, but have demonstrated lower sensitivity for herpes simplex virus (HSV) compared to targeted PCRs. Providers in our institution frequently order both multiplex (BioFire Meningitis/Encephalitis Panel (MEP)) and targeted HSV PCR (Diasorin Simplexa HSV-1/2 Direct), allowing comparison of assay performance in practice and a unique clinical characterization of patients with concordant positive or discordant results. We retrospectively analyzed all HSV testing performed using the two assays simultaneously in a large multi-center institution between 12/10/2018 and 6/6/2022. We described and compared laboratory, diagnostic and therapeutic data between patients with positive results on both assays with patients with discordant results. 1,387 patients were tested with both assays, of which 25 were positive on at least one assay. Eleven (44%) had discordant results; 9 were targeted-PCR-positive/MEP-negative and 2 were targeted-PCR-negative/MEP-positive. Compared with patients with concordant positive results, discordant targeted PCR-positive/MEP-negative patients had a higher PCR cycle threshold value (i.e., >35) and milder illness characterized by lower CSF WBC counts (4vs 152; p=0.032), fewer MRI abnormalities (37.5% vs. 87.5%; p=0.039), HSV meningoencephalitis as primary discharge diagnosis (33% vs. 100%; p<0.0001) and were more often treated with oral medication or none at all. Patients with discordant targeted PCR-positive/MEP-negative results represent a population with milder clinical disease. These findings will help clinicians and laboratorians determine when both targeted HSV and multiplex assays should be performed simultaneously and identify patients for whom a multiplex PCR may suffice, despite lower assay sensitivity.

Efficacy of Nirmatrelvir-Ritonavir versus Azvudine for COVID-19 Treatment in Tibet: A Retrospective Study.

Nirmatrelvir-ritonavir, also known as paxlovid, is a widely used antiviral drug against coronavirus disease 2019 (COVID-19). Azvudine, a drug previously used to treat human immunodeficiency virus-1, has also been used to treat COVID-19 in China. However, only a few clinical studies have evaluated the effects of azvudine. Additionally, studies comparing nirmatrelvir-ritonavir with azvudine have been limited in number. We carried out a retrospective case‒control analysis at the Third People's Hospital of the Tibet Autonomous Region. Eighty-two eligible patients with COVID-19 who received azvudine treatment were included. A total of 145 control patients who received nirmatrelvir-ritonavir treatment were selected by propensity score matching for age, sex, the severity of disease, and initial cycle threshold values. A comparison of the nucleic acid test negative conversion time, the length of hospitalization, and mortality rate was conducted. Overall, the mean nucleic acid test negative conversion time was comparable between the nirmatrelvir-ritonavir and azvudine groups (7.0 [11.0, 15.0] vs 9.0 [6.0, 12.0] days, P=0.064). However, for patients with mild COVID-19, the nucleic acid test negative conversion time was significantly shorter in the nirmatrelvir-ritonavir group than in the azvudine group (6.0 [5.0, 8.0] vs 8.0 [6.0, 11.0] days, P=0.029). The nirmatrelvir-ritonavir group and the azvudine group did not differ significantly in length of hospitalization (8.0 [5.5,10.5] vs 8.0 [5.0,10.0] days, P=0.378). Regarding the mortality rate, there were 4 (2.8%) deaths in the nirmatrelvir-ritonavir group and 3 (3.7%) in the azvudine group (P=0.706). Azvudine is generally as effective as nirmatrelvir-ritonavir, but for patients with mild COVID-19, nirmatrelvir-ritonavir could suppress the virus more rapidly. For those who cannot be treated with nirmatrelvir-ritonavir, azvudine might be an effective therapy for COVID-19.

&lt;i&gt;In vitro&lt;/i&gt; antiviral activity of a double-stranded RNA sodium salt-based medicinal product against SARS-CoV-2

Scientific relevance. Innate immune activation in the early phases of COVID-19 infection and subsequent interferon induction may help control viral replication and protect cells not yet infected with SARS-CoV-2. Thus, immunostimulants that induce interferon (IFN), including double-stranded RNA-based agents, are a promising means of post-exposure prophylaxis and treatment of COVID-19 at early stages.Aim. The study evaluated the in vitro antiviral activity of a double-stranded RNA sodium salt-based medicinal product against SARS-CoV-2.Materials and methods. The authors analysed the double-stranded RNA sodium salt-based medicinal product RADAMIN®VIRO using Vero cells and the Delta variant of SARS-CoV-2 (B.1.617). The virus titre was calculated as the tissue cytopathic dose that caused 50% cell death. The authors measured the content of IFN-α and IFN-γ in the culture fluid by enzyme immunoassay and assessed the viral load by real-time polymerase chain reaction (using the cycle threshold value) and by titration (using Vero cells).Results. The studied double-stranded RNA sodium salt-based medicinal product at a concentration of 250 or 500 μg/mL induced IFN-α and IFN-γ expression by Vero cells, thus increasing their resistance to SARS-CoV-2. The authors evaluated the antiviral activity of the medicinal product based on the virus titre, viral load, and cell monolayer damage. The antiviral activity became clear 24 h after treatment, which confirmed the ability of the medicinal product to inhibit the replication of the SARS-CoV-2 virus in vitro as early as the first day after infection.Conclusions. The double-stranded RNA sodium salt-based medicinal product induced IFN-α and IFN-γ synthesis in Vero cells, increasing their resistance to SARS-CoV-2 infection in vitro. These results demonstrate the immunomodulatory and antiviral potential of the medicinal product.

Antiviral efficacy of RAY1216 monotherapy and combination therapy with ritonavir in patients with COVID-19: a phase 2, single centre, randomised, double-blind, placebo-controlled trial

This study aimed to evaluate the efficacy and safety of RAY1216, a novel inhibitor of 3-chymotrypsin-like cysteine protease (3CLpro), in adults with coronavirus disease 2019 (COVID-19). This phase 2, single centre, randomised, double-blind, placebo-controlled trial included hospitalised patients between August 14, 2022, and September 26, 2022, in Sanya Central Hospital (The Third People's Hospital of Hainan Province) in China with no severe symptoms if they had laboratory-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection for not more than 120h (5 days) and a real-time quantitative polymerase chain reaction (qPCR) cycle threshold (Ct) value of ≤30 for both the open reading frames 1ab (ORF1ab) and nucleocapsid (N) genes within 72h before randomisation. Half of the participants (n=30) were randomly assigned (2:1) to receive either RAY1216 or a matched placebo three times a day (TID) for 5 days (15 doses in total), while the other half received RAY1216 plus ritonavir (RAY1216 plus RTV) or a matched placebo every 12h for 5 days (10 doses in total). The primary endpoint was the time of viral clearance. Secondary outcomes included the changes of the SARS-CoV-2 RNA viral load, the positivity rate of the nucleic acid test, and the recovery time of clinical symptoms. A safety evaluation was performed to record and analyse all adverse events that occurred during and after drug administration as well as any cases in which dosing was halted because of these events. Clinicaltrials.gov identifier: ChiCTR2200062889. The viral shedding times in the RAY1216 and RAY1216 plus RTV groups were 166h (95% confidence interval (CI): 140-252) and 155h (95%CI: 131-203), respectively, which were 100h (4.2 days) and 112h (4.6 days) shorter than that of the placebo group, respectively (RAY1216 group vs. Placebo p=0.0060, RAY1216 plus RTV group vs. Placebo p=0.0001). At 24h, 72h, and 120h after administration, the viral RNA loads in the RAY1216 and RAY1216 plus RTV groups were significantly less than those of the placebo groups. At 280h (11.5 days) after administration, the nucleic acid test results in the RAY1216 and RAY1216 plus RTV groups were both negative. The common adverse events related to the investigational drugs were mild and self-limiting laboratory examination abnormalities. Our findings suggest that RAY1216 monotherapy and RAY1216 plus ritonavir both demonstrated significant antiviral activity and reduced the duration of COVID-19 while maintaining a satisfactory safety profile. Considering the limited clinical application of RTV, it is recommended to use RAY1216 alone to further verify its efficacy and safety. This study was sponsored by the Key Research and Development Program of China (2022YFC0868700).