Journal of Clinical Virology

ObjectivesThis multicentre study investigated the utility of next-generation sequencing (NGS) to detect and generate hepatitis B virus (HBV) genomes in samples of low viral load (from 0.2 to 6207 IU/ml). Methods23 HBV DNA positive plasma samples of genotypes A-E and one HBV-negative control sample were assayed blindly via 9 established NGS methods from 6 European laboratories. Methods included untargeted metagenomics, pre-enrichment by probe-capture followed by Illumina sequencing, and HBV-specific PCR pre-amplification followed by sequencing with Nanopore or Illumina. ResultsFull HBV genomes were obtained only from samples with viral loads >1000 IU/ml using probe-capture methods, >200 IU/ml using PCR-Illumina methods, >10 IU/ml using PCR-Nanopore methods, and in no samples using metagenomic methods. Contamination was observed in the negative control and samples with very low viral loads in all PCR-based methods. Probe-capture and metagenomic methods detected additional viruses not routinely screened in blood donations, including polyomaviruses and herpesviruses; positive results were confirmed by PCR. ConclusionsNGS may delineate whole-genome sequences at low viral loads if supported by a PCR pre-amplification step. Probe-capture methods also reliably detect HBV without pre-amplification but achieve limited genome characterisation at low viral loads; they may additionally detect a wide range of blood-borne viruses.

BACKGROUNDFast identification of severe acute respiratory syndrome Coronavirus-2 (SARS-CoV-2) infected individuals is a strategically vital task to ensure correct management and quarantine. Rapid antigen test could be a supplement to the standard-of-care Nucleic Acid Amplification Test (NAAT). The aim of this study was to determine the accuracy of the BD Veritor SARS-CoV-2 antigen test as a screening instrument in a hospital setting. METHODSA cohort of prospective samples were collected from hospital staff and patients at the Emergency, Infectious Diseases and Pediatrics and Adolescent Medicine departments at Hvidovre Hospital. All samples were collected using oropharyngeal swabs, and BD Veritor Antigen test results were paired with routine NAAT test results. Sensitivity, specificity, positive and negative predictive values of the antigen test were calculated using NAAT as reference. RESULTSOverall, 809 samples from 674 individuals were included (average age 45 years, range 0-98 years). Among all samples, 8% were SARS-CoV-2 positive by NAAT testing and 5.3% by BD Veritor. The sensitivity of the antigen test was 63.1% and specificity 99.7%. The positive predictive value was 95.3%. False-positive rate was 4%. The cycle threshold value was significantly higher among individuals with false negative antigen tests compared to true positives. CONCLUSIONThe sensitivity, specificity and positive predictive values show that the BD Veritor antigen test from oropharyngeal collected specimens performs well. Antigen testing may be a supplement, but not substitute, to NAAT testing as the primary diagnostic modality in hospital settings where fast turnaround test results may assist in decisions regarding isolation and quarantine.

BackgroundThere is a need to confirm the clinical performance of point-of-care (POC) testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) in emergency departments and time-sensitive outpatient settings. This study aimed to compare the clinical performance of the Cobas(R) SARS-CoV-2 nucleic acid test for use on the Cobas Liat System (POC SARS- CoV-2) with the Cobas SARS-CoV-2 Qualitative Assay for use on the Cobas 6800/8800 System (68/8800) when used to detect SARS-CoV-2 infection in individuals at POC settings. MethodsThis prospective, two-site, non-interventional study, conducted in the US and Germany, collected fresh POC nasopharyngeal samples according to local procedures for upper respiratory swab sampling. Pairwise agreement was evaluated by estimating positive, negative, and overall percent agreement (PPA, NPA, and OPA, respectively) between POC SARS-CoV-2 and 68/8800 assays. Site-specific analyses were also conducted. FindingsOverall, 317 evaluable samples were collected from March 30 to July 2023 (US site) and from November 7, 2023 to April 4, 2024 (German site). Relative to 68/8800, POC SARS-CoV-2 had a PPA of 98.8% (169/171) and an NPA of 90.4% (132/146). The OPA was 95.0%. Site-specific analyses were broadly comparable to the overall results, although enrichment for positive samples at the US site resulted in a PPA and NPA of 98.6% (139/141) and 55.6% (5/9), respectively. InterpretationPOC SARS-CoV-2 used in real-world POC settings showed good overall clinical performance relative to 68/8800, a widely used and accurate laboratory-based test, for detecting SARS-CoV-2. Efficient POC testing will help improve the timely management of SARS-CoV-2 infections. Key pointsO_LIThere are limited clinical studies evaluating the clinical performance of SARS-CoV-2 assays in real-world populations C_LIO_LIThe Cobas(R) SARS-CoV-2 nucleic acid test for use on the Cobas Liat System showed good clinical performance relative to a widely used laboratory-based test for detecting SARS-CoV-2 at two point-of-care hospital sites. C_LIO_LIEfficient point-of-care testing will help improve the timely management of SARS- CoV-2 infections C_LI

Background2019-Novel coronavirus (2019-nCoV) outbreaks create challenges for hospital laboratories because thousands of samples must be evaluated each day. Sample types, interpretation methods, and corresponding laboratory standards must be established. The possibility of other infections should be assessed to provide a basis for clinical classification, isolation, and treatment. Accordingly, in the present study, we evaluated the testing methods for 2019-nCoV and co-infections. MethodsWe used a fluorescence-based quantitative PCR kit urgently distributed by the Chinese CDC to detect 8274 close contacts in the Wuhan region against two loci on the 2019-nCoV genome. We also analyzed 613 patients with fever who underwent multiple tests for 13 respiratory pathogens; 316 subjects were also tested for 2019-nCoV. FindingsAmong the 8274 subjects, 2745 (33.2%) had 2019-nCoV infection; 5277 (63.8%) subjects showed negative results in the 2019-nCoV nucleic acid test (non-2019-nCoV); and 252 cases (3.0%) because only one target was positive, the diagnosis was not definitive. Eleven patients who originally had only one positive target were re-examined a few days later; 9 patients (81.8%) were finally defined as 2019-nCoV-positive, and 2 (18.2%) were finally defined as negative. The positive rates of nCoV-NP and nCovORF1ab were 34.7% and 34.7%, respectively. nCoV-NP-positive only and nCovORF1ab-positive cases accounted for 1.5% and 1.5%, respectively. In the 316 patients with multiple respiratory pathogens, 104 were positive for 2019-nCov and 6/104 had co-infection with coronavirus (3/104), influenza A virus (2/104), rhinovirus (2/104), and influenza A H3N2 (1/104); the remaining 212 patients had influenza A virus (11/202), influenza A H3N2 (11/202), rhinovirus (10/202), respiratory syncytial virus (7/202), influenza B virus (6/202), metapneumovirus (4/202), and coronavirus (2/202). InterpretationClinical testing methods for 2019-nCoV require improvement. Importantly, 5.8% of 2019-nCoV infected and 18.4% of non-2019-nCoV-infected patients had other pathogen infections. It is important to treat combined infections and perform rapid screening to avoid cross-contamination of patients. A test that quickly and simultaneously screens as many pathogens as possible is needed. FundingNo founding was received Research in contextO_ST_ABSEvidence before this studyC_ST_ABSWe searched PubMed for articles published up to January 31, 2020 using the keywords "2019 novel coronavirus" or "2019-nCoV". No published study on the characteristics of 2019-nCoV tests or 2019-nCoV co-infections was found. We only noted recent laboratory findings for other tests of patients infected with 2019-nCoV. Added value of this studyPositive detection of nCoV-NP or nCovORF1ab is presented, and individuals with/without 2019-nCoV infections or with inconclusive results were identified. Patients with inconclusive results may be diagnosed with 2019-nCoV infection or found to be negative for the infection after resampling and retesting in the next few days. Approximately 5.8% of the subjects diagnosed with 2019-nCoV had co-infection. Implications of all the available evidenceManagement of the population showing inconclusive results should be given attention; additionally, such results can be minimized by improving the sampling, sample pretreatment, and testing methodologies. When diagnosing 2019-nCoV subjects, the possibility of co-infection should be considered. Finally, better clinical detection methods are needed to simultaneously screen as many pathogens as possible.

IntroductionSARS-CoV2 serology testing is multipurpose provided to choose an efficient test. We evaluated and compared 4 different commercial serology tests, three of them had the Food and Drug Administration (FDA) approval. Our goal was to provide new data to help to guide the interpretation and the choice of the serological tests. MethodsFour commercial tests were evaluated: Cobas(R)Roche(R)(total anti-N antibodies), VIDAS(R)Biomerieux(R)(IgM and IgG anti-RBD antibodies), Mindray(R)(IgM and IgG anti-N and anti-RBD antibodies) and Access(R)Beckman Coulter(R)(IgG anti-RBD antibodies). Were tested: a positive panel (n=72 sera) obtained from COVID-19 confirmed patients and a negative panel (n=119) of pre-pandemic sera. Were determined the analytical performances and was drawn the ROC curve to assess the manufacturers threshold. ResultsA large range of variability between the tests was found. Mindray(R)IgG and Cobas(R) tests showed the best overall sensitivity 79,2%CI95%[67,9-87,8]. Cobas(R) showed the best sensitivity after D14; 85,4%CI95%[72,2-93,9]. The best specificity was noted for Cobas(R), VIDAS(R)IgG and Access(R) IgG(100%CI95%[96,9-100]). Access(R) had the lower sensitivity even after D14 (55,5% CI95%[43,4-67,3]). VIDAS(R)IgM and Mindray(R)IgM tests showed the lowest specificity and sensitivity rates. Overall, only 43 out of 72 sera gave concordant results (59,7%). Retained cut-offs for a significantly better sensitivity and accuracy, without altering significantly the specificity, were: 0,87 for Vidas(R)IgM(p=0,01), 0,55 for Vidas(R)IgG(p=0,05) and 0,14 for Access(R)(p<10-4). ConclusionAlthough FDA approved, each laboratory should realize its own evaluation for commercial tests. Tests variability may raise some concerns that seroprevalence studies may vary significantly based on the used serology test.

Background and AimsHepatitis B virus (HBV) generates a double-stranded linear DNA (dslDNA) byproduct during replication. This dslDNA can undergo intermolecular and intramolecular nonhomologous end-joining (NHEJ) recombination, resulting in viral integration and dslDNA derived covalently closed circular DNAs (dsl-cccDNAs), respectively. The insertions and deletions (INDELs) at the end-joining site around the direct repeat (DR) 1 motif have been used to differentiate dsl-cccDNA from the authentic cccDNA. The frequency and characteristics of dsl-cccDNA in chronic hepatitis B (CHB) patients remain unclear. MethodsHBV-targeted next-generation sequencing (NGS) was used to identify 32 dsl-cccDNA positive candidates, 22 HBeAg(+) and 10 HBeAg(-), from 56 liver biopsies of antiviral treatment-naive CHB patients for dsl-cccDNA confirmation and characterization by cccDNA-PCR NGS. INDELs within the DR2-1 region (nt 1600-1840) of the cccDNA were analyzed. ResultsVarious clonally expanded, heterogenous [~]22-nt deletions in the X-gene were detected in all 32 samples, which are likely quasi-species from the authentic cccDNA. We, therefore, defined dsl-cccDNA only by the presence of INDELs clustered at the DR1 surrounding region (nt 1800-1840). The percentage of dsl-cccDNA in total cccDNA was higher among HBeAg(+) compared to HBeAg(-) samples [11.32 (3.24-26.94)% vs. 7.72 (2.16-28.23)%, p=0.01]. The diversity of dsl-cccDNA species correlated with cccDNA levels (log-transformed; r=0.82, p<0.001), HBeAg(+) CHB (p<0.001), and serum HBV DNA (p<0.001). Conclusionsdsl-cccDNA is more prevalent and heterogenous among the HBeAg(+) CHB subjects, which is likely due to the active viral cccDNA transcription and reverse transcription at the HBeAg(+) phase. The existence of replication-defective dsl-cccDNA may facilitate immune evasion and HBV integration, and complicate HBV pathogenesis. The potential impact of dsl-cccDNA in HBV therapeutic response deserves further assessment.

BackgroundSARS-CoV-2 causes COVID-19, which can be fatal and is responsible for a global pandemic. Variants with increased transmissibility or the potential to evade immunity have emerged and represent a threat to global pandemic control. Variants of concern (VOC) can be identified by sequencing of viral RNA, or by more rapid methods for detection of subsets of signature mutations. MethodsWe developed a multiplex, real-time RT-PCR assay (cobas(R) SARS-CoV-2 Variant Set 1) for the qualitative detection and differentiation of three key SARS-CoV-2 mutations in the viral spike protein: del 69-70, E484K and N501Y. Analytical sensitivity and accuracy were evaluated at three testing sites using clinical specimens from patients infected with SARS-CoV-2 variants belonging to several different lineages, including B.1.1.7, B.1.351, and P.1. ResultsThe limit of detection for E484K was between 180 and 620 IU/mL for the three different isolates tested. For N501Y, the LOD was between 270 and 720 IU/mL (five isolates), while for del 69-70, it was 80 - 92 IU/mL (two isolates). Valid test results were obtained with all clinical specimens that were positive using routine diagnostic tests. Compared to sequencing (Sanger and next-generation), test results were 100% concordant at all three loci; no false positive or false negative results were observed. ConclusionsData collected at three independent laboratories indicates excellent performance and concordance of cobas(R) SARS-CoV-2 Variant Set 1 with sequencing. New sets of primers and probes that target additional loci can be rapidly deployed in response to the identification of other emerging variants.

IntroductionConcerns have been raised regarding the accuracy of diagnostic antigen testing for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron variant. We compared the performance of the LumiraDx SARS-CoV-2 Antigen Test between symptomatic participants recruited prospectively during the Delta to Omicron variant transition in the USA. MethodsTwo paired anterior nasal swabs were collected from each participant (adults and children) within 12 days of symptom onset between November 24th, 2021 and February 1st, 2022, during which time Omicron replaced Delta as the dominant variant in the sample population. Swabs were tested by the LumiraDx SARS-CoV-2 Antigen Test and compared using real-time polymerase chain reaction (RT-PCR) reference testing. Reference samples identified as positive were sequenced to identify the SARS-CoV-2 variant. Positive percent agreement (PPA) was calculated, with results stratified by RT-PCR cycle threshold (Ct). ResultsOf the 38 participants for whom LumiraDx SARS-CoV-2 Antigen Test results were available, 36 were confirmed positive by RT-PCR. Overall, PPA of the LumiraDx SARS-CoV-2 Antigen Test was 94.7% (95% confidence interval: 82.3%, 99.4%) and PPA was 100% for samples with a Ct <33. Sufficient viral load for sequencing was present in nine samples (six Delta, three Omicron), all of which returned a positive result using the LumiraDx SARS-CoV-2 Antigen Test. There were no performance differences observed between participants with the Delta and Omicron variants. ConclusionsSARS-CoV-2 differences between Delta and Omicron variant mutations did not affect the performance of the LumiraDx SARS-CoV-2 Antigen Test which detects the nucleocapsid protein antigen. The LumiraDx SARS-CoV-2 Antigen Test can be a useful antigen test to diagnose emerging variants of coronavirus disease 2019.

The reliable detection of immunoglobulin G (IgG) or total antibodies directed against the novel severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is important for clinical diagnostics and epidemiological studies. Here, we compare the diagnostic accuracy of six commercially available SARS-CoV-2 IgG (Abbott SARS-CoV-2 IgG; Diasorin Liaison(R) SARS-CoV-2 S1/2 IgG; Epitope EDI Novel Coronavirus COVID-19 IgG ELISA Kit; Euroimmun Anti-SARS-CoV-2 ELISA (IgG); Mikrogen recomWell SARS-CoV-2 IgG) or total SARS-CoV-2 antibody assays (Roche Elecsys Anti-SARS-CoV-2). The test sensitivities were analyzed with a set of 34 sera obtained from 26 patients after PCR-confirmed SARS-CoV-2 infection and varied from 76.9% (Euroimmun) to 96.2% (Abbott). The majority of assay results were confirmed in a laboratory-developed plaque reduction neutralization test and by a SARS-CoV-2 IgG-specific line assay including measurement of generally low IgG avidities (Mikrogen recomLine Coronavirus IgG [Aviditat], prototype). Moreover, 100 stored sera collected during summer 2018 (N = 50) and winter season 2018/2019 (N = 50) were included to demonstrate test specificities. These varied from 96.0% (DiaSorin) to 100% (Epitope EDI). A subset of sera were retested with a lateral flow test (STANDARD Q COVID-19 IgM/IgG Duo) and a considerably lower sensitivity was noted. Overall, the diagnostic accuracy of the six SARS-CoV-2 IgG/total antibody assays was good and varied from 92.9% (Euroimmun) to 98.4% (Abbott). Due to the different specificities, results of commercially available SARS-CoV-2 antibody tests should be interpreted with caution. A high proportion of antibody-positive patient sera demonstrated neutralizing capacity against SARS-CoV-2.

BackgroundThe sensitivity of commercially available RT-PCR assays varies over 10,000 fold, ranging from 10 to 20,000 viral copies/ml. The reporting of high Ct value results has been under scrutiny, as the clinical significance of these values is not yet completely understood. The early detection of infected individuals (high Ct results) in the pre-symptomatic phase of the disease using highly sensitive RT-PCR methods has been argued as a strategy to prevent transmission, while on the contrary, the reporting of high Ct has been criticized as false-positive results causing unnecessary testing and having several negative implications. The purpose of this study was to verify the presence of SARS-CoV-2 genomes in samples with a wide range of RT-PCR Ct values including samples with high Ct (37 to 42) using next-generation sequencing (NGS). MethodsThe study evaluated a total of 547 previously positive samples tested with the PerkinElmer(R) New Coronavirus Nucleic Acid Detection RT-PCR kit. The samples included in this study ranged from Ct values of 17-42, with 44 samples having a Ct > 37. Of the 547 samples, 149 were sequenced using PerkinElmer NEXTFLEX Variant-Seq SARS-CoV2 assay on NovaSeq 6000, and 398 samples were sequenced using Illumina SARS-CoV-2 respiratory viral panel kits using the NextSeq 500/550 system. ResultsBetween the two clinical laboratories, a total of [~]1.95 million samples were tested using the FDA-EUA PerkinElmer(R) New Coronavirus RT-PCR assay. Of the 1.95 million samples, [~]1.72 million were negative, [~]250,000 positive, and [~]16,500 in the range of 37-42. Of the 547 samples sequenced, the percentage of sequencing reads that aligned to the SARS-CoV-2 Wuhan-hu-1 reference genome (NC_045512.2) ranged from 25.5% to 99.69%. All samples sequenced showed high sequence specificity to the SARS-CoV-2 virus. Low Ct samples showed complete uniform coverage across the entire 29kb SAR-CoV-2 genome. The average coverage in samples with high Ct (>37) was found to be 55.5% (range 16.1-99.2%). However, as sample Ct increased, a gradual decrease in coverage uniformity was observed for few samples. ConclusionThis study demonstrates for the first time that the viral RNA is present in the high Ct value range of 37-42 and the sequence is unique to SARS-CoV-2 confirmed using two separate sequencing assays. This confirms that the detected Ct values are reflective of the presence of the SARS-CoV-2 virus and they are not an artifact or contamination. In light of the recent work highlighting the majority of transmission being pre-symptomatic/ asymptomatic, and high Ct results being observed at both the early and late phases of infection warrants further investigation into the clinical utility of high Ct results to curtail the spread of the virus.

IntroductionRapid qualitative antigen testing has been widely used for the laboratory diagnosis of COVID-19 with nasopharyngeal samples. Saliva samples have been used as alternative samples, but the analytical performance of those samples for qualitative antigen testing has not been sufficiently evaluated. MethodsA prospective observational study evaluated the analytical performance of three In Vitro Diagnostics (IVD) approved COVID-19 rapid antigen detection kits for saliva between June 2022 and July 2022 in Japan using real-time reverse transcription polymerase chain reaction (RT-PCR) as a reference. A nasopharyngeal sample and a saliva sample were simultaneously obtained, and RT-PCR was performed. ResultsIn total, saliva samples and nasopharyngeal samples were collected from 471 participants (140 RT-PCR-positive saliva samples and 143 RT-PCR-positive nasopharyngeal samples) for the analysis. The median Ct values were 25.5 (interquartile range [IQR]: 21.9-28.8) for saliva samples and 17.1 (IQR: 15.5-18.7) for nasopharyngeal samples (p<0.001). Compared with saliva samples of RT-PCR, the sensitivity and specificity were 46.4% and 99.7% for ImunoAce SARS-CoV-2 Saliva, 59.3% and 99.1% for Espline SARS-CoV-2 N, and 61.4% and 98.8% for QuickChaser Auto SARS-CoV-2, respectively. The sensitivity is >90% for saliva samples with a moderate-to-high viral load (Ct<25), whereas the sensitivity is <70% for high-viral-load nasopharyngeal samples (Ct<20). ConclusionCOVID-19 rapid antigen detection kits with saliva showed high specificities, but the sensitivities varied among kits, and the analytical performance of saliva qualitative antigen detection kits was much worse than that of kits using nasopharyngeal samples.

IntroductionMolecular diagnosis of SARS-CoV-2 is a huge challenge to many countries around the world. The cost of tests to check infected people is inaccessible since specialized teams and equipment are not disposable in remote locations. Herein, we compared the fitness of two primers sets to the SARS-CoV-2 N gene in the molecular diagnosis of COVID-19. Materials and MethodsThe 1029 patient samples were tested to presense/abscence molecular test using in house US CDC protocol. We compared the fitness of two primers sets to two different regions of N gene targets. ResultsBoth targets, N1 and N2 displayed similar fitness during testing with no differences between Ct or measurable viral genome copies. In addition, we verified security ranges Cts related to positive diagnostic with Ct above 35 value failuring in 66,6% after retesting of samples. Main conclusionOur data suggest that it is secure to use just one primer set to the N gene to identify SARS-CoV-2 in samples and the labs should be careful to set positive samples in high Ct values using high cutoffs.

BackgroundDifferent rates of morbidity, mortality, transmission, and immune escape are associated with various strains of the SARS-CoV-2 virus. With the emergence of new strains during seasonal outbreaks, ensuring that antigen-based immunoassays can detect SARS-CoV-2 infections across identified circulating viral variants is a crucial component of infection control efforts. ObjectiveTo validate the performance of the BD Veritor System for Rapid Detection of SARS-CoV-2 Assay (BD Veritor assay) to detect SARS-CoV-2 across variants of concern (VOC) and variants of interest (VOI). MethodsUsing the Illumina NextSeq 2000 Sequencer, viral sequencing was performed on prospectively collected, then frozen, SARS-CoV-2 RT-PCR positive nasal swabs stored in universal transport media (UTM). Specimens from symptomatic and asymptomatic individuals were included in the study. Using the information obtained from the sequencing analysis, the performance of the BD Veritor System assay was evaluated against the highly sensitive molecular RT-PCR Quidel Lyra SARS-CoV-2 assay for each variant. ResultsThe resulting PPA was 97.4% (95% CI: 86.8, 99.5) for detection of SARS-CoV-2 across all variants identified by Next Generation Sequencing (i.e., WHO-labeled variants Alpha, Delta, Gamma, Iota, Lambda, as well as two other non-labeled variants), with a 100% PPA for five of the six variant labels identified. ConclusionThe results demonstrate the robust performance of the BD Veritor assay in detecting SARS-CoV-2 in clinical nasal specimens in selected variants. As new variants emerge, additional studies will be beneficial to ensure the sustained performance of SARS-CoV-2 assays.

ObjectivesDetermine the sensitivity and specificity of a Point-Of-Care test ( COVIDISC) for SARS-COV2. The novelty of the test is to integrate, on the same (low-cost) compact plastic/paper device, solid phase RNA extraction and RT-LAMP amplification, all reagents being freeze-dried on it. MethodRetrospective study with a cohort of 99 patients characterized by real-time RT-PCR. The 37 positive naso-pharyngeal samples cover a broad range of viral loads (from 5 gc /{micro}L to 2 106 gc/ {micro}L of sample). ResultsThe COVIDISC found 36 positives (out of 37 by IP4 RT-PCR protocols) and 63 negatives (out of 62 by RT-PCR). ConclusionThe sensitivity of the COVIDISC, found in this 99-patient retrospective study, is 97% and the specificity 100%.

BackgroundThe increasing number of cases and hospital admissions due to COVID-19 created an urgent need for rapid, reliable testing procedures for SARS-CoV-2 in Emergency Departments (ED) in order to effectively manage hospital resources, allocate beds and prevent nosocomial spread of infection. The ID NOW COVID-19 assay is a simple, user-friendly, rapid molecular test run on an instrument with a small footprint enabling point-of-care diagnostics. MethodsIn the first wave, outsourced RT-PCR testing regularly required 36-48 hours before results were available. This prospective study was conducted in the second wave (October 2020-April 2021) and evaluated the impact the implementation of the ID NOW COVID-19 test in the ED had on clinical care processes and patient pathways. 710 patients were recruited upon arrival at the ED which included those presenting clinical symptoms, asymptomatic individuals or persons fulfilling epidemiological criteria. The first anterior nasal swab was taken by trained nurses in the ambulance or a separate consultation room. The ID NOW COVID-19 test was performed in the ED in strict compliance with the manufacturers instructions and positive or suspected cases were additionally tested with RT_PCR (cobas SARS-COV-2 RT-PCR, Roche) following collection of a second nasopharyngeal NP specimen. ResultsSwabs directly tested with the ID NOW COVID-19 test showed a diagnostic concordance of 98 % (sensitivity 99.59 %, specificity 94.55 %, PPV 97.6 %, NPV 99.05 %) compared to RT-PCR as reference. The 488 patients that tested positive with the ID NOW COVID-19 had a Ct range in RT-PCR results between 7.94 to 37.42 (in 23.2 % > 30). Two false negative results (0.28%) were recorded from patients with Ct values > 30. 14 (1.69%) discordant results were reviewed case-by-case and usually associated with either very early or very advanced stages of infection. Furthermore, patients initially negative with the ID NOW COVID-19 test and admitted to the hospital were tested again on days 5 and 12: no patient became positive. DiscussionThe ID NOW COVID-19 test for detection of SARS-CoV-2 demonstrated excellent diagnostic agreement with RT-PCR under the above-mentioned patients pathways implemented during the second wave. The main advantage of the system was the provision of reliable results within a few minutes. This not only allowed immediate initiative of appropriate therapy and care for COVID-19 (patient benefit) but provided essential information on isolation and thus available beds. This drastically helped the overall finances of the department and additionally allowed more patients to be admitted including those requiring immediate attention; this was not possible during the first wave since beds were blocked waiting for diagnostic confirmation. Our findings also show that when interpreting the results, the clinical condition and epidemiological history of the patient must be taken into account, as with any test procedure. Overall, the ID NOW COVID-19 test for SARS-CoV-2 provided a rapid and reliable alternative to laboratory-based RT-PCR in the real clinical setting which became an acceptable part of the daily routine within the ED and demonstrated that early patient management can mitigate the impact of the pandemic on the hospital.

BackgroundCOVID-19 caused by the novel coronavirus SARS-CoV-2 has caused the greatest public health emergency of our time. Accurate laboratory detection of the virus is critical in order to contain the spread. Although real-time polymerase chain reaction (PCR) has been the cornerstone of laboratory diagnosis, there have been conflicting reports on the diagnostic accuracy of this method. MethodsA retrospective review was performed on all hospitalized patients tested for SARS-CoV-2 (at St. Pauls Hospital in Vancouver, BC) from March 13 - April 12, 2020. Diagnostic accuracy of initial PCR on nasopharyngeal (NP) swabs was determined against a composite reference standard which included a clinical assessment of the likelihood of COVID-19 by medical experts, initial and repeat PCR, and post-hoc serological testing. ResultsA total of 323 patients were included in the study, 33 (10.2%) tested positive and 290 (89.8%) tested negative by initial PCR. Patients testing positive were more likely to exhibit features of cough (66.7% vs 39.3%), shortness of breath (63.6% vs 35.9%), fever (72.7% vs 27.6%), radiographic findings (83.3% vs 39.6%) and severe outcomes including ICU admission (24.2% vs 9.7%) and mortality (21.2% vs 6.2%) compared to patients testing negative. Serology was performed on 90 patients and correlation between serology and PCR was 98.9%. There were 90 patients included in the composite reference standard. Compared to the composite reference standard, initial PCR had sensitivity of 94.7% (95% CI 74.0 to 99.9%), specificity of 100% (95% CI 94.9 to 100%), positive predictive value of 100% (95% CI 81.5 to 100%) and a negative predictive value of 98.6% (95% CI 92.5 to 100%). DiscussionOur study showed high sensitivity of PCR on NP swab specimens when compared to composite reference standard in hospitalized patients. High correlation of PCR with serological testing further increased confidence in the diagnostic reliability of properly collected NP swabs.

BackgroundSeveral serological assays have been developed to detect anti-SARS-CoV-2 IgG antibodies, but evidence about their comparative performance is limited. We sought to assess the sensitivity of four anti-SARS-CoV-2 IgG enzyme-linked immunosorbent assays (ELISA) in individuals with evidence of prior SARS-CoV-2 infection. MethodsWe obtained sera from 36 individuals with PCR-confirmed SARS-CoV-2 infection between March and May 2020. We evaluated samples collected at around 21 days ({+/-}14 days) after their initial PCR test using 3 commercially available ELISA assays, two anti-spike (Ortho- Clinical Diagnostics Vitros, and Euroimmun) and one anti-nucleocapsid (Abbott Architect), and a Yale-developed anti-spike ELISA test. We determined the sensitivity of the tests and compared their results. The Euroimmun and Yale ELISA had an equivocal and indeterminate category, which were considered as both negative and positive. ResultsAmong the 36 individuals with SARS-CoV-2 infection, mean age was 43 ({+/-}13) years and 19 (53%) were female. The sensitivities of the tests were not significantly different (Abbott Architect, Ortho Vitros, Euroimmmun, and Yale assays: 86% (95% confidence interval [CI], 71- 95), 94% (95% CI, 81-99), 86% (95% CI, 71-95), and 94% (95% CI, 81-99), respectively; p- value=0.464). The sensitivities of the Euroimmun and Yale ELISA tests increased when the equivocal/indeterminate results were considered positive (97% [95% CI, 85-100] and 100% [95% CI, 90-100], respectively), but were not significantly different from other tests (p=0.082). The cross-correlation coefficient ranged from 0.85-0.98 between three anti-spike protein assays (Ortho Vitros, Euroimmun, Yale) and was 0.58-0.71 between the three anti-spike protein assays and the anti-nucleocapsid assay (Abbott). ConclusionThe sensitivities of four anti-SARS-CoV-2 protein assays did not significantly differ, although the sample size was small. Sensitivity also depended on the interpretation of equivocal and indeterminate results. The strongest correlations were present for the three anti- spike proteins assays. These findings suggest that individual test characteristics and the correlation between different tests should be considered when comparing or aggregating data across different populations studies for serologic surveillance of past SARS-CoV-2 infection.

BackgroundAccurate and accessible diagnostics for hepatitis E virus (HEV) are essential for outbreak preparedness and surveillance, particularly in low-resource settings. Dried blood spots (DBS) offer a simple, scalable alternative to serum, but their diagnostic performance for HEV remains poorly characterised. MethodsPaired DBS and serum samples were collected from suspected HEV cases during an outbreak in Bentiu, South Sudan. HEV RNA was detected and quantified by real-time RT-PCR, and anti-HEV IgM and IgG antibodies were measured by ELISA. DBS performance was compared to serum across assays, and associations between DBS positivity and serum viral load were analysed using logistic regression. ResultsAmong 100 serum RT-PCR positive samples, 83 were positive by DBS (sensitivity 83.0%, 95% CI: 74.5-89.1). Ct values from DBS and serum were strongly correlated ({rho} = 0.74), and most discordances at low viral loads (Ct > 30). DBS achieved 88.3% sensitivity (95% CI: 77.8-94.2) and 100% specificity (95% CI: 91.2-100) for IgM, while IgG sensitivity and specificity reached 95.0% (95% CI: 86.3-98.6) and 97.5-100%, respectively. Results were consistent across DBS card types and age groups. ConclusionsDBS provide a reliable, practical alternative to serum for HEV diagnostics. Despite slightly reduced sensitivity at low viral loads, DBS maintained high specificity and strong correlation with serum results. Their ease of collection, storage, and transport without cold chain requirements supports their application for HEV surveillance and outbreak response in resource-limited settings. Further optimisation of elution methods to increase analyte concentration, along with improved storage conditions, could enhance diagnostic sensitivity.

BackgroundWith the ability to simultaneously sequence more than 5,000 disease-associated genes, next-generation sequencing (NGS) has replaced Sanger sequencing as the preferred method in the diagnostic field at the laboratory level. However, Sanger sequencing has been used routinely to confirm identified variants prior to reporting results. This validation process causes a turnaround time delay and cost increase. Thus, this study aimed to set a quality threshold that does not require Sanger confirmation by analyzing the characteristics of identified variants from whole exome sequencing (WES). MethodsOur study analyzed data on a total of 694 disease-causing variants from 578 WES samples that had been diagnosed with suspected genetic disease. These samples were sequenced by Novaseq6000 and Exome Research Panel v2. All 694 variants (513 single-nucleotide variants (SNVs) and 181 indels) were validated by Sanger sequencing. ResultsA total of 693 variants included 512 SNVs and 181 indels from 578 patients and 367 genes. Five hundred seven heterozygous SNVs with at > 250 quality score and > 0.3 allele fraction were 100% confirmed by Sanger sequencing. Five heterozygous variants and one homozygous variant were not confirmed by Sanger sequencing, which showed 98.8% accuracy. There were 146 heterozygous variants and 35 homozygous variants among 181 indels, of which 11 heterozygous variants were not confirmed by Sanger sequencing (93.9% accuracy). Five non-confirmed variants with high quality were not identified on the ram .bam file. ConclusionOur results indicate that Sanger confirmation is not necessary for exome-derived SNVs with > 250 quality score and 0.3 > allele fraction set to an appropriate quality threshold. Indels or SNVs that do not meet the quality threshold should be reviewed by raw .bam file and Sanger confirmation should be performed to ensure accurate reporting.

Influenza (Flu) and respiratory syncytial virus (RSV) are responsible for lower respiratory tract infections (LRTIs) associated with significant hospitalization among young children. In the present study, the performances of a triplex PCR assay detecting Flu A/B and RSV were compared with our in-house single-plex assays using 160 stored respiratory specimens previously tested using a panel of laboratory-developed real-time RT-PCR. Of them, 61 were positive for FluA, 41 for FluB, and 58 for RSV. All samples were retrospectively quantified with Respiratory Viral (RV) ELITe MGB(R) Panel (ELITechGroup Molecular Diagnostics, Puteaux, France) processed using ELITe InGenius(R) system. Overall, the total percentage agreement observed was 93.4% (57/61) for FluA, 92.7% (38/41) for FluB, and 86.2% (50/58) for RSV. A significant correlation of VL values was observed between the two methods for FluA and RSV ({rho}= 0.91 and 0.84). This finding was supported by the strength of agreement between the two methods, as showed by the linear regression analysis (R2 =0.84 and 0.80). FluB viral load values measured by RV Panel were less significantly correlated ({rho}= 0.77 and R2 =0.56). The bland-Altman analysis showed how 84.2% (48/57) of FluA and 86.0% of RSV (43/50) samples fell within {+/-}1.0 Log10 variation from our laboratory results, while only 21.1% (8/38) of FluB results fell within this range. The great majority of FluB samples (29/30) outside range had values higher than +1.0 Log10 (median +2.1 Log10 range +1.0 to +3.5 Log10). In conclusion, RV ELITe MGB(R) Panel constitutes a valid and robust system for simultaneous detection and quantification of Flu A/B and RSV.