Lower Cycle Threshold Research Articles

Examining Cycle Threshold values in Repeat SARS-CoV-2 Cases: A Retrospective Analysis in Ireland

Abstract Background Effective public health management during the COVID-19 pandemic requires accurate differentiation between re-infections and residual viral RNA detections in repeat positive cases. This study investigates cycle threshold (CT) trends in repeat SARS-CoV-2 cases to discern between persistent viral remnants and active infections. Collaboration between contact tracing and laboratories is essential for shaping robust public health strategies. Methods A retrospective analysis was conducted on 427 individuals with repeat positive PCR tests. Data was collected at a contact tracing centre from March to August 2021. Key variables including age, gender, and biomedical markers (test intervals, CT values) were examined. Regression and correlation techniques assessed CT trends and their implications. Results Younger individuals (<60 years) occasionally exhibited significantly lower CT values in subsequent tests, suggesting potential re-infections, while older adults generally displayed higher CT values indicative of residual RNA. These age-specific CT trends were statistically significant and instrumental in refining testing protocols and isolation guidelines. Further analysis is expected to strengthen these findings Conclusions Collaborative approaches between contact tracing and laboratories are vital in shaping robust public health strategies for distinguishing between types of repeat infections. Revision of health policies is imperative to prevent unnecessary isolation and optimise healthcare workforce allocation during ongoing transmission periods. Key messages • Decisive decision-making skills are crucial in managing repeat SARS-CoV-2 infections. • Revised health policies align testing and isolation practices with empirical CT data, enhancing public health efficacy.

Detection and Molecular Characterization of GI-1 and GI-23 Avian Infectious Bronchitis Virus in Broilers Indicate the Emergence of New Genotypes in Bolivia.

Infectious Bronchitis Virus (IBV) is a major threat to the poultry industry worldwide, causing significant economic losses. While the virus's genetic structure is well understood, the specific strains circulating in Bolivia have remained uncharacterized until now. This study aimed to identify and characterize new IBV strains in Bolivia. Tissue samples from broilers exhibiting clinical signs of Infectious Bronchitis were screened to detect IBV using real-time RT-PCR (RT-qPCR). Positive samples with low cycle threshold (Ct) values were selected for sequencing the full S1 gene. Of the 12 samples analyzed, 10 were determined to be positive for IBV. However, only four samples yielded sufficient genetic material for sequencing and subsequent phylogenetic analysis. The results revealed the presence of GI-1 and GI-23 lineages, both belonging to genotype I (GI). The GI-1 lineage showed >99% sequence identity to the H120 and Massachusetts vaccine strains, suggesting a close relationship. In contrast, the GI-23 lineage clustered with other IBV strains, showing a distinct subclade that is genetically distant from Brazilian strains. No evidence of recombination was found. Furthermore, amino acid substitution analysis identified specific mutations in the S1 subunit, particularly in the hypervariable regions 1, 2, and 3. These mutations could potentially alter the virus's antigenicity, leading to reduced vaccine efficacy. The findings of this study highlight the importance of continued and broad genomic surveillance of circulating IBV strains and the need to improve vaccination strategies in Bolivia.

A closer look at the link between cycle threshold, clinical features and biomarkers: An observational study in COVID-19 patients.

Symptoms for severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) appear 2-3 days after exposure to the virus. Being a virus, detection is primarily by polymerase chain reaction as this offers superior sensitivity and specificity. There was a misconception that patients with low cycle threshold (Ct) have severe coronavirus disease (COVID), and for individuals with higher Ct, it is the other way around. The prognosis for COVID was derived from various biomarkers and physicians heavily relied on them. A cross-sectional study spanning a duration of 2 years was conducted at a tertiary care centre in western India. A total of 201 individuals were included and the correlation between Ct, clinical features and biomarkers was studied. In the E-gene, 43.28% had lower Ct values and 40.79% had low Ct values in the RdRp gene. 50% of all patients had diabetes, with 60% being between the ages of 61 and 80. 54.1% of hypertension patients belonged to ages between 61 and 80. 90.54% of COVID-positive individuals had lactose dehydrogenase levels ranging from 440 to 760. 79% of patients had a procalcitonin value of more than one but less than six. 79.1% of patients had an erythrocyte sedimentation rate between 36 and 90. Ct value though has a research value; it is a poor prognostic marker when compared to the various biomarkers that have been studied earlier. We cannot conclusively state that all our findings are accurate due to a lack of data but further research into the prognostic value of Ct should be conducted which will help in the ongoing scenario.

Performance of two rapid antigen tests against SARS-CoV-2 in neighborhoods of socioeconomic vulnerability from a middle-income country.

As new and improved antigen-detecting rapid diagnostic tests for SARS-CoV-2 infection (Ag-RDT) continue to be developed, assessing their diagnostic performance is necessary to increase test options with accurate and rapid diagnostic capacity especially in resource-constrained settings. This study aimed to assess the performance of two Ag-RDTs in a population-based study. We conducted a diagnostic accuracy study in neighborhoods with high socioeconomic vulnerability in Salvador-Brazil, including individuals aged ≥12 years old who attended primary health services, between July and December 2022, with COVID-19 symptoms or who had been in contact with a confirmed case. Two Ag-RDTs were compared in parallel using reverse transcription polymerase chain reaction (RT-PCR) as reference standard, the PanbioTM COVID-19 Ag test (Abbott®) and Immuno-Rapid COVID-19 Ag (WAMA Diagnostic®). Sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated. For the Abbott test the sensitivity was 52.7% (95% CI: 44.3% - 61.0%), specificity 100% (95% CI: 98.7% - 100%), PPV 100% (95% CI: 95.4% - 100%) and NPV 80.4% (95% CI: 75.9% - 84.4%). For the WAMA test, the sensitivity was 53.4% (95% CI: 45.0% - 61.6%), specificity 100% (95% CI: 98.7% - 100%), PPV 100% (95% CI: 95.4% - 100%) and NPV 80.7% (95% CI: 76.2% - 84.6%). Sensitivity for the group with Cycle Threshold (CT) <24 was 82.3% (95%CI: 72.1-90.0, n = 83) for PanbioTM COVID-19 Ag test and 87.3% (95%CI: 77.9-93.8, n = 83) for Immuno-Rapid COVID-19 Ag test. Sensitivity for both Ag-RDT was lower than reported by manufacturers. In the stratified analysis, sensitivity was higher among those with lower CT values <24. Specificity was high for both rapid antigen tests. Both Ag-RDT showed to be useful for rapid diagnostic of potential cases of COVID-19. Negative results must be assessed carefully according to clinical and epidemiological information.

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.

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.

Detection of the SARS-CoV-2 Genome in Peripheral Blood Mononuclear Cells of Hospitalized Patients With COVID-19 Infection

Background: Evaluation of viral pathogenicity is an important part of research in every viral disease, and one of the most important parts of pathogenicity is cell and tissue tropism of viruses, which can help us to have a clear picture of the viral replication cycle and viral disease. Objectives: This study aims to evaluate the possibility of SARS-CoV-2 replication in peripheral blood mononuclear cells (PBMCs) of hospitalized patients with COVID-19. Methods: Twenty-six whole blood samples (5 mL) were collected from 70 hospitalized patients infected with SARS-CoV-2. Plasma and PBMCs were collected and subjected to total RNA extraction using the alcohol-chloroform precipitation method by the RNX solution. After complementary DNA (cDNA) synthesis, all samples were subjected to real-time and nested polymerase chain reactions (PCRs) to detect the viral genome. Results: The nested PCR method showed a higher rate of positivity in plasma samples (42.3%) compared to real-time PCR (30.7%), suggesting nested PCR exhibited better sensitivity. This rate in PBMC samples was 57.7% by nested PCR and 7.7% by real-time PCR. Minus-strand viral genome was detected in PBMCs, demonstrating that these cells can support virus replication and act as a virus transporter through blood. Conclusions: PBMCs can be infected with SARS-CoV-2. Plasma and serum samples are also not useful samples for virus detection because all of the positive plasma samples in this study showed low viral load with a low cycle threshold (Ct) value.

STR genotyping by real-time PCR using QueSTR probes

QueSTR probes provide a feasible alternative for implementing forensic Short Tandem Repeat (STR) genotyping on lab-on-a-chip (LoC) devices. Unlike capillary electrophoresis (CE), which poses challenges for LoC implementation, QueSTR probes have been demonstrated to accurately genotype STRs in a hybridization curve assay after PCR amplification. In this study, we modified the QueSTR probe assay for use as a hydrolysis probe assay during qPCR. An asymmetric real-time PCR was performed with QueSTR probes and RNase H2 in the master mix, during which the fluorescence was recorded. Designed to hybridize with specific STR alleles, QueSTR probes contain a fluorescent dye and quencher molecule, enabling detection of probe hybridization through RNase H2-mediated cleavage of the RNA:DNA duplex, which releases the quencher. Matching probes yielded lower threshold cycle values and steeper incline of fluorescence curves compared to non-matching probes, indicating accurate genotype. The QueSTR qPCR assay was used to successfully genotype four CODIS core loci (D16S539, D7S820, TPOX, and TH01) in 12 samples, with one exception. The integration of amplification and detection in a single reaction supports the use of QueSTR probes for miniaturizing STR genotyping, thereby complementing CE-based analyses in centralized labs.

Clinical Significance of Quantitative Viral Load in Patients Positive for SARS-CoV-2

Cycle threshold (CT) refers to the number of cycles in a reverse transcriptase polymerase chain reaction (RT-PCR) assay needed to amplify viral RNA and can be used to indicate viral load. CT is inversely related to viral load, where lower CT values indicate higher viral levels. Data suggest lower CT scores are associated with worse outcomes in COVID; however, quantitative CT scores are not typically reported to patients. This retrospective analysis examined the use of CT scores in patient counseling for positive COVID results and suggests that higher viral loads were associated with greater need for prescription drug therapy in the outpatient setting. Patient perception of CT score was found to influence masking and quarantine behavior. We hypothesize that a quantitative threshold for viral load such as CT can be useful in patient counseling, estimating need for drug therapy, and influencing behavior toward public health concerns.

The Fatal Clinical Outcome of Severe COVID-19 in Hospitalized Patients: Findings from a Prospective Cohort Study in Dhaka, Bangladesh.

Background and Objectives: The morbidity and mortality associated with COVID-19 have burdened worldwide healthcare systems beyond their capacities, forcing them to promptly investigate the virus characteristics and its associated outcomes. This clinical analysis aimed to explore the key factors related to the fatal outcome of severe COVID-19 cases. Materials and Methods: Thirty-five adult severe COVID-19 patients were enrolled from two COVID-19 hospitals in Dhaka, Bangladesh. Clinical manifestation, comorbid conditions, medications, SARS-CoV-2 RT-PCR related cycle threshold (CT) value, hematology, biochemical parameters with SARS-CoV-2 specific IgG and IgM responses at enrollment were compared between the survivors and deceased participants. Results: Total 27 patients survived and 8 patients died within 3 months of disease onset. Deceased patients suffered longer from shortness of breath than the survived (p = 0.049). Among the severe cases, 62% of the deceased patients had multiple comorbid condition compared to 48% of those who survived. Interestingly, the anti-viral was initiated earlier among the deceased patients [median day of 1 (IQR: 0, 1.5) versus 6.5 (IQR: 6.25, 6.75)]. Most of the survivors (55%) received a combination of anticoagulant (p = 0.034). Liver enzymes, creatinine kinase, and procalcitonin were higher among the deceased patients during enrollment. The median CT value among the deceased was significantly lower than the survivors (p = 0.025). A significant difference for initial IgG (p = 0.013) and IgM (p = 0.030) responses was found between the survivor and the deceased groups. Conclusions: The factors including older age, male gender, early onset of respiratory distress, multiple comorbidities, low CT value, and poor antibody response may contribute to the fatal outcome in severe COVID-19 patients. Early initiation of anti-viral and a combination of anticoagulant treatment may prevent or lower the fatality among severe COVID-19 cases.

Clinical Evaluation of Commercial HARDSON COVID-19 Antigen Rapid Test Kit for Routine COVID-19 Diagnosis.

This study aimed to evaluate the sensitivity, specificity, and accuracy of the commercial HARDSON COVID-19 Antigen Rapid Test Kit for diagnosing COVID-19 among the Iranian population by compared with the results of commercial RT-PCR. Two nasopharyngeal swabs were collected from each patient. One swab was tested with HARDSON COVID-19 Antigen Rapid Test Kit, and the second swab was placed in 3 mL of a virus-transmitted inactivated media for RT-PCR testing. Then, the results of both tests were compared to investigate the diagnostic accuracy of the rapid antigen test. A total of 275 suspected COVID-19 patients' samples were collected to investigate the diagnostic accuracy of HARDSON COVID-19 Antigen Rapid Test Kit. In this study, 162 positive and 113 negative samples were evaluated. As a result, the sensitivity, specificity, and accuracy of HARDSON COVID-19 Antigen Rapid Test Kit were 90%, 100%, and 94%, respectively. The diagnostic kit analyzed in this study indicated excellent specificity and a relatively good overall sensitivity for the diagnosis of COVID-19 when compared with the RT-PCR detection kit. Based on the result of this study, COVID-19 Antigen Rapid Test Kit indicated a good sensitivity (96%) in low cycle threshold (Ct) value, and it would be recommended to be integrated into routine diagnostic laboratories and used as an at-home rapid antigen test.

Diagnostic Performance and Tolerability of Saliva and Nasopharyngeal Swab Specimens in the Detection of SARS-CoV-2 by RT-PCR.

Saliva is a promising alternative for a nasopharyngeal swab (NPS) in specimen collection to detect SARS-CoV-2. We compared the diagnostic performance and tolerability of saliva collection versus NPS in a clinical setting. Paired NPS and saliva specimens were collected sequentially from participants (n = 250) at the Turku University Hospital drive-in coronavirus testing station in the spring of 2022, with Omicron BA.2 as the dominant SARS-CoV-2 variant. Discomfort and preference for the sampling method were assessed. The specimens were analyzed for SARS-CoV-2 using real-time multiplex reverse transcriptase PCR (RT-PCR) with a laboratory-developed test (LDT) and two commercial kits (PerkinElmer SARS-CoV-2 and PerkinElmer SARS-CoV-2 Plus) for several target genes. Among the 250 participants, 246 had respiratory symptoms. With LDT, SARS-CoV-2 was detected in 135 and 134 participants from NPS and saliva, respectively. Of the 250 specimens, 11 gave a discordant outcome, resulting in excellent agreement between the specimen types (Cohen's kappa coefficient of 0.911; P = 0.763). The cycle threshold (CT) values of LDT and commercial kit target genes were significantly lower from NPS than from saliva. A total of 172 (69%) participants assessed saliva sampling as more tolerable than NPS (P < 0.0001). Our findings present saliva as an applicable alternative for SARS-CoV-2 diagnostics. However, the lower CT values obtained from NPS indicate that NPS may be a slightly more sensitive specimen type. Participants preferred saliva sampling, although delivering an adequate volume of saliva was challenging for some participants. IMPORTANCE The extensive testing of SARS-CoV-2 is vital in controlling the spread of COVID-19. The reference standard for specimen collection is a nasopharyngeal swab (NPS). However, the discomfort of NPS sampling, the risk of nosocomial infections, and global material shortages have accelerated the development of alternative testing methods. Our study demonstrates that patients tolerate saliva sampling better than NPS. Of importance, although the RT-PCR qualitative test results seem to correspond between NPS and saliva, we show significantly lower CT values for NPS, compared to saliva, thus contradicting the suggested superiority of the saliva specimen over NPS in the detection of the Omicron variants of SARS-CoV-2. Future research is still required to enable individual planning for specimen collection and to determine the effects of different SARS-CoV-2 variants on the sensitivity of the saliva matrix.

Early detection of Pseudocapillaria tomentosa by qPCR in four lines of zebrafish, Danio rerio (Hamilton 1882).

The intestinal nematode Pseudocapillaria tomentosa in zebrafish (Danio rerio) causes profound intestinal lesions, emaciation and death and is a promoter of a common intestinal cancer in zebrafish. This nematode has been detected in zebrafish from about 15% of the laboratories. Adult worms are readily detected about 3 weeks after exposure by either histology or wet mount preparations of the intestine, and larval worms are inconsistently observed in fish before this time. A quantitative PCR (qPCR) test was recently developed to detect the worm in fish and water, and here we determined that the test on zebrafish intestines was effective for earlier detection. Four lines of zebrafish (AB, TU, 5D and Casper) were experimentally infected and evaluated by wet mounts and qPCR at 8, 15-, 22-, 31- and 44-day post-exposure (dpe). At the first two time points, only 8% of the wet mounts from exposed fish were identified as infected, while the same intestines screened by qPCR showed 78% positivity, with low and consistent cycle threshold (Ct) values at these times. Wet mounts at later time points showed a high prevalence of infection, but this was still surpassed by qPCR.

Temporal Series Analysis of Population Cycle Threshold Counts as a Predictor of Surge in Cases and Hospitalizations during the SARS-CoV-2 Pandemic.

Tools to predict surges in cases and hospitalizations during the COVID-19 pandemic may help guide public health decisions. Low cycle threshold (CT) counts may indicate greater SARS-CoV-2 concentrations in the respiratory tract, and thereby may be used as a surrogate marker of enhanced viral transmission. Several population studies have found an association between the oscillations in the mean CT over time and the evolution of the pandemic. For the first time, we applied temporal series analysis (Granger-type causality) to validate the CT counts as an epidemiological marker of forthcoming pandemic waves using samples and analyzing cases and hospital admissions during the third pandemic wave (October 2020 to May 2021) in Madrid. A total of 22,906 SARS-CoV-2 RT-PCR-positive nasopharyngeal swabs were evaluated; the mean CT value was 27.4 (SD: 2.1) (22.2% below 20 cycles). During this period, 422,110 cases and 36,727 hospital admissions were also recorded. A temporal association was found between the CT counts and the cases of COVID-19 with a lag of 9-10 days (p ≤ 0.01) and hospital admissions by COVID-19 (p < 0.04) with a lag of 2-6 days. According to a validated method to prove associations between variables that change over time, the short-term evolution of average CT counts in the population may forecast the evolution of the COVID-19 pandemic.

Measles Outbreak Response Activity in Japan, and a Discussion for a Possible Strategy of Outbreak Response Using Cycle Threshold Values of Real-Time Reverse Transcription PCR for Measles Virus in Measles Elimination Settings.

Measles is a highly contagious, but vaccine-preventable disease caused by the measles virus (MeV). Although the administration of two doses of measles vaccines is the most effective strategy to prevent and eliminate measles, MeV continues to spread worldwide, even in 2022. In measles-eliminated countries, preparedness and response to measles outbreaks originating from imported cases are required to maintain elimination status. Under these circumstances, real-time reverse transcription (RT) PCR for MeV could provide a diagnostic method capable of strengthening the subnational capacity for outbreak responses. Real-time RT-PCR can detect MeV RNA from patients with measles at the initial symptomatic stage, which can enable rapid public health responses aimed at detecting their contacts and common sources of infection. Furthermore, low cycle threshold (Ct) values (i.e., high viral load) of throat swabs indicate high infectiousness in patients with measles. The high basic reproduction number of measles suggests that patients with high infectiousness can easily become super-spreaders. This opinion proposes a possible strategy of rapid and intensive responses to counter measles outbreaks caused by super-spreader candidates showing low Ct values in throat swabs. Our strategy would make it possible to effectively prevent further measles transmission, thereby leading to the early termination of measles outbreaks.

A two-ward acute care hospital outbreak of SARS-CoV-2 delta variant including a point-source outbreak associated with the use of a mobile vital signs cart and sub-optimal doffing of personal protective equipment

BackgroundWe conducted a detailed epidemiological investigation of a linked 2-ward COVID-19 Delta variant outbreak to elucidate its source, risk factors, and control measures.MethodsInvestigations included epidemiologic analysis, detailed case review serial SARS-CoV-2 RT-PCR testing of patients and healthcare workers (HCWs), viral culture, environmental swabbing, HCW-unaware personal protective equipment (PPE) audits, ventilation assessments, and the use of whole genome sequencing (WGS).ResultsThis linked 2-ward outbreak resulted in 17 patient and 12 HCW cases, despite an 83% vaccination rate. In this setting suboptimal adherence and compliance to PPE protocols, suboptimal hand hygiene, multi-bedded rooms, and a contaminated vital signs cart (VSC) with potential fomite or spread via the hands of HCWs were identified as significant risk factors for nosocomial COVID-19 infection.Sudden onset of symptoms, within 72h, was observed in 79% of all Ward 2 patients and 93% of all cases (patients and HCWs) on Ward 2 occurred within one incubation period, consistent with a point source outbreak. RT-PCR assays showed low Cycle threshold (Ct) values, indicating high viral load from environmental swabs including the VSC. WGS results with ≤ 3 SNP differences between specimens were observed.ConclusionsOutbreaks on both wards settled rapidly, within 3 weeks, using a ‘back-to-basics’ approach without extraordinary measures or changes to standard PPE requirements. Strict adherence to recommended PPE, hand hygiene, education, assistance from infected cases (interviews and testing), and additional measures such as limiting movement of patients and staff temporarily were all deemed to have contributed to prompt resolution of the outbreak.

Effectiveness of Rapid Antigen Testing in Forensic Cases of Severe Acute Respiratory Syndrome Coronavirus 2 Infection, Including Delta Variant.

The polymerase chain reaction is indispensable for diagnosing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in forensic cases. However, studies regarding the effectiveness of rapid antigen testing (RAT) in forensic cases remain limited. Therefore, we investigated the efficacy of RAT compared with reverse-transcription quantitative polymerase chain reaction (RT-qPCR) for confirming SARS-CoV-2 infection (including the delta variant). Before the external examination or autopsy, we collected samples from the nasopharyngeal mucosa, which were then assessed via RAT (QuickNavi COVID-19 Ag kit, QuickNavi-Flu+COVID-19 Ag kit) and RT-qPCR. Reverse-transcription quantitative polymerase chain reaction results were positive in 73 of 1255 cases, and 21 cases were identified as those of delta variants. Low RT-qPCR threshold cycle value cases and delta variant infections were more likely to result in coronavirus disease-related deaths. The sensitivity of the QuickNavi COVID-19 Ag kit was 76.32%, and that of the QuickNavi-Flu+COVID-19 Ag kit was 77.14%. The specificity of both RATs was 100%. In QuickNavi COVID-19 Ag kit cases, delta variant cases showed lower sensitivity than non-delta variant cases, even for a similar viral load. Thus, RAT in forensic cases is sufficiently useful as a screening test for SARS-CoV-2 infection. However, RAT carries a risk of false negatives, especially for delta variant cases.

Performance of High Throughput SARS-CoV-2 Antigen Testing Compared to Nucleic Acid Testing.

ObjectiveIndependent assessment of SARS-CoV-2 antigen (COV2Ag) tests remains important as varying performance between assays is common. We assessed the performance of a new high-throughput COV2Ag test compared to SARS-CoV-2 nucleic acid amplification tests (NAAT).MethodsA total of 347 nasopharyngeal samples collected from January to October 2021 were assessed by NAAT as part of standard-of-care testing (CDC LDT or GeneXpert System, Cepheid) and COV2Ag using the ADVIA Centaur CoV2Ag assay (Siemens Healthineers).ResultsAmong NAAT positive specimens we found 82.4% agreement and in NAAT negative specimens we found 97.3% agreement (overall agreement 85.6%). In symptomatic persons, COV2Ag agreed with NAAT 90.0% (n = 291), and in asymptomatic persons, 62.5% (n = 56). Agreement between positive NAAT and COV2Ag increased at lower cycle threshold (Ct) values.ConclusionThe COV2Ag assay exceeded the World Health Organization minimum performance requirements of ≥ 80% sensitivity and ≥ 97% specificity. The COV2Ag assay is helpful for large scale screening efforts due to high-throughput and reduced wait times.

Accuracy of 2 Rapid Antigen Tests During 3 Phases of SARS-CoV-2 Variants

Variants of SARS-CoV-2 have sequence variations in the viral genome that may alter the accuracy of rapid diagnostic tests. To assess the analytical and clinical accuracy of 2 rapid diagnostic tests for detecting SARS-CoV-2 during 3 phases of variants. This diagnostic study included participants aged 18 years or older who reported onset of COVID-19-like symptoms within the prior 5 days and were tested at multiple COVID-19 testing locations in King County, Washington, from February 17, 2021, to January 11, 2022, during 3 distinct phases of SARS-CoV-2 infection (pre-Delta, Delta, and Omicron). Two anterior nasal swab specimens were collected from each participant-1 for onsite testing by the SCoV-2 Ag Detect Rapid Self-Test and 1 for reverse transcriptase-polymerase chain reaction (RT-PCR) testing. The analytical limit of detection of the 2 rapid diagnostic tests (SCoV-2 Ag Detect Rapid Self-Test and BinaxNOW COVID-19 Ag Card) was assessed using Omicron (B.1.1.529/BA.1), Delta (B.1.617.2), and a wild-type (USA-WA1/2020) variant. Diagnostic sensitivity and specificity of clinical testing for the rapid antigen tests were compared with that of RT-PCR testing. A total of 802 participants were enrolled (mean [SD] age, 37.3 [13.3] years; 467 [58.2%] female), 424 (52.9%) of whom had not received COVID-19 vaccination and presented a median of 2 days (IQR, 1-3 days) from symptom onset. Overall, no significant differences were found in the analytical limit of detection or clinical diagnostic accuracy of rapid antigen testing across SARS-CoV-2 variants. The estimated limit of detection for both rapid nucleocapsid antigen tests was at or below a 50% tissue culture infectious dose of 62.5, and the positive percent agreement of the SCoV-2 Ag Detect Rapid Self-Test ranged from 81.2% (95% CI, 69.5%-89.9%) to 90.7% (95% CI, 77.9%-97.4%) across the 3 phases of variants. The diagnostic sensitivity increased for nasal swabs with a lower cycle threshold by RT-PCR, which correlates with a higher viral load. In this diagnostic study, analytical and clinical performance data demonstrated accuracy of 2 rapid antigen tests among adults with COVID-19 symptoms across 3 phases of SARS-CoV-2 variants. The findings suggest that home-based rapid antigen testing programs may be an important intervention to reduce global SARS-CoV-2 transmission.

Environmental detection of SARS-CoV-2 in hospital rooms in different wards of a university hospital

BackgroundTransmission of SARS-CoV-2 can occur through direct, indirect, or close contact with infected people. However, the extent of environmental contamination is unknown. The nature of the relation between patients’ symptoms and SARS-CoV-2 environmental shedding remains unclear. The aim of this study was to assess the relationship between patient covid-19 status and environmental contamination.MethodsBetween May to November 2020, environmental swabs were taken before and after room disinfection at day 7 after symptom onset in a cohort of patients clinically or biologically diagnosed positive for Covid-19. Twelve surfaces per room were collected in 13 rooms. Sample analysis was performed by RT-PCR for SARS-CoV-2 detection (SARS-CoV-2 R-Gene® (Biomérieux)). Clinical data (day of illness, symptoms, RT-PCR results) was collected from the clinical software.ResultsFive medical units were included in the study. Of 156 samples collected in 13 rooms, 5 rooms (38.5%) presented 11 SARS-CoV-2 positive samples. These positive samples were detected on 8 different surfaces. There was no association between detection of SARS-CoV-2 and patient’s age (p= 1) or patient’s symptoms (p= 0.3).ConclusionViral shedding during Covid-19 appears unrelated to the presence of symptoms, patient’s age, and low value cycle threshold of patient’s test. This study supports the evidence for the environmental shedding of SARS-CoV-2 until at least 7 days after patient’s symptoms onset. It emphasizes the need for strict compliance with contact precaution, hand hygiene, the correct use of personal protective equipment and a room disinfection for the routine care of Covid-19 patients.