Journal of Clinical Virology Plus

AbstarctO_ST_ABSBackgroundC_ST_ABSThe COVID-19 serological tests for IgG and IgM have been developed with several methodologies: Immunoenzymatic Assay (ELISA), Chemiluminescence, Electro Chemiluminescence, Fluorescent Lateral Flow Immunoassays and Immunochromatography. None of these tests should be used for the diagnosis or population screening of the disease, considering that the antibodies appear only on the 8th - 14th day of the disease onset. The present study evaluates a sample of immunofluorescent and immunochromatographic rapid tests to show their agreement in relation to Chemiluminescence. MethodsA diagnostic test evaluation assay was performed to establish the performance of five "rapid" tests (4 immunochromatographic and 1 immunofluorescent tests) for IgG and IgM serology for SARS-CoV-2 using a panel of 30 serum samples from patients received in the laboratory analysis routine. For the evaluation of clinical performance, the qualitative results of the "rapid" tests were compared against those obtained by chemiluminescence, dichotomized as positives ([&ge;] 10 AU / mL) or negative (<10 UA / mL). FindingsThe best agreement is seen in the immunofluorescent assay, for the IgG contrast, with a particularly good kappa index (0.85), without positive disagreements and a negative disagreement of about 15%. In the immunochromatographic methods Kappa index was 0.61 at best, with disagreements in negative findings of {approx}35% and in positive cases of up to {approx}70%. The IgM concordance behavior, on the other hand, reflects a weak to moderate Kappa concordance value (Kappa 0.2 to 0.6), with negative disagreements reaching up to 55% and positives of up to 84%, without any evaluated test reaching Kappa performance equal to or greater than 0.8. InterpretationSerological studies should be used in the clinical and epidemiological context and of other diagnostic tests. Given the high demand and supply in the market of "rapid serological tests", its evaluation against panels of serologically positive or negative samples established by Chemiluminescence or Electro chemiluminescence is essential to authorize its extensive use in populations FundingNone

The clinical and epidemiological use of SARS-CoV-2 antibody assays is under debate with urgent need to validate and verify the performance of SARS-CoV-2 serologic assays. We aim to assess the clinical and analytical performance of three commercial serological assays of SARS-CoV-2, comparing three anti-SARS-CoV-2-IgG ELISA and identifying the seroconversion and seroprevalence in our population. A cross sectional study conducted from April 2020 to July 2020 at National Institute of Blood disease and Bone Marrow Transplantation Karachi, Pakistan with sample size of 404, enrolled consecutively. Participants were categorized into four groups namely convalescent plasmadonors (CPDs n=239), health care professionals (HCPs n=44), healthy blood donors (HBDs n=70) and from community (n=51). We evaluated the performance of Elecsys anti-SARS-CoV-2 electrochemiluminescence (ECLIA) assay on Cobas-e411 by Roche, three qualitative anti-SARS-CoV-2-IgG enzyme linked imunosorbant assay (ELISA) by (Generic assays, Euroimmun & Omega diagnostics), one quantitative ELISA assay by AESKU Diagnostics and two immune chromatography(ICT) kits namely InstaTest by CORTEZ and TEST IT by TURKLAB. From total 404 subjects, 322 (83.5%) were males. Mean age was 36.79{+/-}11.95 years. Among 239 in CPDs group, 202(84.5%) showed positive antibodies by ECLIA. The qualitative anti-SARS-CoV-2 IgG ELISA was positive in 174 (72.8%) and quantitative IgG in 180(75.3%) with mean titer of 56.7 {+/-}39.7 U/ml. Sensitivity and specificity of ECLIA were 97.44& 99%, ELISA by Generic assays were 67.85% and 89.9%; Euroimmun had 90.38% and 94.9%; Omega Diagnostics 96.4% and 95% and the AESKULISA 93.75% and 100% respectively. Seroconversion was found to be 53.8% and 77.77% within 7 -8 days and 12 to 14 days post onset of symptoms respectively. ICT had more specificity but less sensitivity. Seroprevalence was found to be 84.5%, 40.9% and 21.4% in CPDs, HCPs and HBDs respectively. The Roche ECLIA, qualitative ELISA by Omega Diagnostics & Euroimmun showed higher sensitivity as well as higher specificity. Quantitative ELISA has higher specificity and relatively high sensitivity. Significant numbers of COVID patients do not have detectable antibodies by all assays.

A commercially available antibody, Cox mAB 31A2, raised against the VP1 protein of coxsackievirus B3 (CVB3) has been reported as suitable for the detection of CVB3 in diagnostic samples (Ettischer-Schmid 2016). The authors compared this antibody with the widely used, multi-specific, monoclonal anti-VP1 antibody marketed by Dako (clone 5D8/1) and concluded that clone 5D8/1 should not be used to identify enterovirus infections in diagnostic samples. Rather they suggested that Cox mAB 31A2 is preferable for this purpose. Here we address these issues and show that Cox mAB 31A2 can be used successfully to diagnose CVB3 infection in various cell and tissue samples but we demonstrate that it fails to detect many clinically relevant enterovirus types, thereby limiting its use as a general diagnostic reagent for clinical specimens. Rather, we propose that, when used under carefully controlled conditions, clone 5D8/1 should remain the reagent of choice for such purposes.

Introduction: Herpes simplex virus type 1 (HSV-1) is highly prevalent worldwide, making accurate serological testing essential for both clinical diagnosis and epidemiological surveillance. Automated chemiluminescent immunoassays (CLIAs) offer operational advantages over enzyme-linked immunosorbent assays (ELISAs); however, their diagnostic performance relative to Western blot (WB) confirmation in high-prevalence settings remains insufficiently characterized. Hypothesis/Gap Statement: The comparative diagnostic accuracy of CLIA- and ELISA-based assays for HSV-1 IgG detection, when benchmarked against a WB reference standard in endemic populations, remains unclear. Aim: This study aimed to evaluate HSV-1 IgG seroprevalence and diagnostic performance of one CLIA and two ELISA platforms using Western blot as the reference method. Methodology: Four hundred archived serum samples from adult male craft and manual workers in Qatar were tested using the Mindray CL-900i CLIA, HerpeSelect ELISA, NovaLisa ELISA, and Euroimmun Western blot. Seroprevalence, diagnostic accuracy, and interassay agreement were assessed using WB as the reference standard, with equivocal and indeterminate results excluded from analysis. Results: HSV-1 IgG seroprevalence estimates were comparable across assays: HerpeSelect 72.5%, Mindray 70.5%, NovaLisa 66.3%, and Western blot 66.5%, with no statistically significant differences (all p > 0.05). The Mindray CLIA demonstrated the highest diagnostic performance (sensitivity 95.7%, specificity 88.9%, accuracy 93.4%) and strong agreement with Western blot ({kappa} = 0.85). HerpeSelect showed substantial agreement ({kappa} = 0.81), while NovaLisa exhibited lower specificity. Conclusion: CLIA- and ELISA-based assays produced comparable HSV-1 seroprevalence estimates in this high-prevalence population; however, diagnostic accuracy varied across platforms. The CLIA platform demonstrated the strongest agreement with Western blot, supporting its use in high-throughput settings, while confirmatory testing remains important to minimize misclassification.

ObjectiveTo investigate the performance of the HIV RDTs used in Zambia. Method2,564 participants aged between 15 and 95 years from two sites in Lusaka province years were tested on OraQuick ADVANCE, Abbot Determine, and then confirmed on Uni-Gold Recombigen(R). The data from the participants were analyzed using SPSS version 25.0. ResultsThe 3 RDTs when compared to the 4th generation Abbot Architect results had the following results: OraQuick ADVANCE(R), Alere Determine and Uni-Gold Ultra, at 95% CI had Sensitivities of: 91.8%, 93.3% and 92.5% respectively. The specificities of OraQuick ADVANCE(R) and Uni-Gold were the same (100.0%; 95% CI: 98.8 -100.0) but slightly different from Alere Determine (99.8%). Positive predictive values at 95% CI were 100% for OraQuick ADVANCE(R) and Uni-Gold and 98.4% for Alere Determine. Negative predictive values (at 95% CIs) were 99.1, 99.2 and 99.1 for OraQuick ADVANCE(R), Alere Determine, and Uni-Gold Ultra respectively. The results showed that these RDTs could only detect 12 out of every 13 HIV positive results. ConclusionThird generation RDTs are not effective in detecting acute positive cases. Fourth generation Rapid Tests are required to capture the positive cases being missed out.

SARS-CoV-2 seroprevalence in an antenatal population in Kingston, Jamaica was assessed for September-November 2020 in a repeated cross-sectional study using the Abbott Architect SARS-CoV-2 IgG assay. After adjusting for test performance, seroprevalence was 6.9% for September, 16.9% for October, and 24.0% for November. Of the 37 pregnant women testing SARS-CoV-2 IgG positive, only 3 were symptomatic. One symptomatic woman testing SARS-CoV-2 IgG positive had multiple co-morbidities and succumbed to COVID-19 pneumonia. Up to January 31, 2021, 8 women identified as SARS-CoV-2 IgG positive delivered, all without complications. Comparison of test adjusted seroprevalence data with cumulative PCR-confirmed COVID-19 cases within the Kingston Metropolitan Area indicated that as many as 44.4 times more people were infected with SARS-CoV-2 than identified with PCR testing. These findings provide the first evidence for the extent of SARS-CoV-2 infections in Jamaica and will inform future SARS-CoV-2 testing strategies.

BackgroundViral culture is currently the most accurate method to demonstrate viability and infectivity of Severe acute respiratory syndrome Coronavirus (SARS-2 CoV). Routine clinical diagnosis, however, is mostly performed by PCR - based assays that do not discriminate between infectious and non-virus. Herein, we aimed to determine the correlation between positive viral cultures and either PCR positivity, the Cycle Threshold (Ct) or the number of viral copies. MethodsA systematic electronic literature search was performed and studies that reported both viral SARS-CoV-2 culture and PCR-based assays were included. A separate search for samples from blood, urine, stool, breast milk and tears were performed. To convert Ct values reported in the reviewed studies were to viral genomic copies, calibration experiments with four different reaction performed, using quantified RNA molecules. ResultsA total 540 articles were reviewed, and 38 studies were included in this review. Out of 276 positive-culture of non-severe patients, 272 (98.55%) were negative ten days after symptoms onset, while PCR assays remained positive for up to 67 days. In severely ill or immunocompromised patients positive-culture was obtained up to 32 days and out of 168 cultures, 31 (18.45%) stayed positive after day 10. In non-severe patients, in Ct value greater than 30 only 10.8% were still culture-positive while in Ct >35 it was nearly universally negative. The minimal calculated number of viral genome copies in culture-positive sample was 2.5 x 103 copies / mL. These findings were similar in immunocompromised patients. Recovering positive culture from non-respiratory samples was sporadically obtained in stool or urine samples. Conversion of Ct values to viral genome copies showed variability between different PCR assays and highlighted the need to standardize reports to correctly compare results obtained in different laboratories. ConclusionDuring the pandemic phase, non-severe COVID-19 patients who are recovering and are not immuno-suppressed, can be regarded as non-infectious, within 10 days from symptom onset, or with Ct value greater than 35 (or a calculated viral load lower than 1.2x103 copies / mL). These findings have important implications for recovering patients and asymptomatic patients, with respect to isolation criteria. The conversion of Cq values to viral genome copies described herein may be useful in future work, enabling a more standardized comparison between results reported in different studies from different laboratories.

IntroductionNasopharyngeal (NP) swab is an invasive procedure that is difficult to perform in pediatric cases and those with special needs. On the other hand, saliva has been a proposed sample given the ease of collection, comfort and the ability to self-collect. The research project aims to study the presence of SARS-CoV-2 in the saliva of suspected COVID-19 patients in comparison to its presence in NP swabs. MethodologyA cross-sectional study was conducted in October 2020 in COVID19 clinic in the Bahrain Defense Force Hospital. The study compared the presence of SARS-CoV2 by PCR in saliva samples to nasopharyngeal samples. COVID-19 Clinic tests symptomatic, staff, close contacts and pre-operation patients. ResultsThe saliva PCR has shown a sensitivity of 72.9% (95% CI: 58.2% - 84.7%) and a specificity of 98.8% (95% CI: 97.8% - 99.4%). The PPV was 74.5% (95% CI 59.7% to 86.1%) and the NPV was 98.6% (95% CI 97.7% to 99.3%). Kappa coefficient of agreement between saliva and NP was 0.723 (95% CI 0.62 to 0.82, p<0.001). Moreover, when restricting cases to symptomatic only, the sensitivity of saliva increased to 86.7% (95% CI 59.5% to 98.3%) while specificity remained high at 97.2%. ConclusionThe findings of the study suggest that saliva samples have the potential to be used as a screening tool for SARS-CoV-2, especially in symptomatic individuals. This is especially important when it is difficult to collect NP samples. Saliva samples are however at risk of producing more false negative tests.

BackgroundThere are conflicting results about the duration of antibodies induced by SARS-CoV-2, but several studies show a rapid decay in a few months after infection. To evaluate antibody decline, we re-evaluated the presence of anti-SARS-CoV-2 antibodies among individuals found seropositive in a first population survey conducted 4 months before. MethodsAll individuals above ten years of age resident in 5 municipalities of the Autonomous Province of Trento, northern Italy, who resulted IgG positive for anti-SARS-CoV-2 nucleocapsid (NC) antibodies in a serosurvey conducted on May 2020 were retested after 4 months. Anti-SARS-CoV-2 antibodies were detected using the Abbott SARS-CoV-2 IgG assay (Abbott Diagnostics, USA) detecting anti-NC antibodies. Samples that gave a negative result were re-tested using the same test plus Liaison SARS-CoV-2 IgG assay (DiaSorin, Italy) to assess anti-spike (S) S1/S2 IgG antibodies. Seroprevalence was calculated as the proportion of positive people on the total number of tested. A neutralizing assay was performed on a subgroup of formerly positives sera using fifty-percent tissue culture infective dose (TCID50) as endpoint dilution to produce a cytopathic effect in 50% of inoculated Vero E6 cells culture. In all the analyses a p value < 0.05 were considered statistically significant. Statistical analysis was performed by STATA version 16.1 (STATA Corp., College Station, Texas, USA). FindingsOverall, 1159 out of 1402 initially anti-NC seropositive participants were enrolled in the study. Of them, 480 (41.1%) became seronegative for anti-NC IgG antibodies. When 479 negative sera were tested for anti-S IgG, 373 samples (77.9%) resulted positives. A functional neutralization assay was performed on 106 sera showing high concordance with anti-S antibodies positivity. InterpretationA decline of anti-NC IgG values was recorded 4 months after the first evaluation. Worth of note, a high proportion of anti-NC seronegative individuals were positive for anti-spike IgG antibodies, which appear to persist longer and to better correlate with neutralization activity.

BackgroundReflex Cryptococcal antigen (CrAg) testing in HIV-positive patients is done routinely at 47 laboratories in South Africa on samples with a confirmed CD4 count <100 cells/l, using the IMMY Lateral Flow Assay (LFA) as the standardized predicate method. ObjectiveThis study aimed to verify the diagnostic performance of newer CrAg LFA assays against the predicate method. MethodsRemnant CD4 samples collected between February and June 2019, with confirmed predicate LFA CrAg results, were retested on settled plasma with the (i) IMMY CrAg semi-quantitative (SQ) LFA; (ii) Bio-Rad RDT CryptoPS SQ; and (iii) Dynamiker CrAg SQ assays, within 24 hours of predicate testing. Sensitivity/ specificity analyses were conducted comparing predicate versus the newer assays, with McNemars tests p-values reported for comparative results (p values <0.05 significant). Positivity grading was noted for the IMMY SQ and Bio-Rad assays. ResultsOf the 254 samples tested, 228 had comparative CrAg results across all assays. The predicate method reported 85 CrAg positive (37.2%), compared to between 35.08 and 37.28% for the Bio-Rad, IMMY SQ and Dynamiker assays. The IMMY CrAg SQ grading (+1 to +5) showed 67% of CrAg positive results had a grading [&ge;]3, indicative of higher CrAg concentration (infection severity). False-negative results across all assays were <2%, with sensitivity >95% for all. False-positive results were highest for the Dynamiker LFA (14%) with a specificity of 77% (p=0.001). IMMY SQ and Bio-Rad assays specificities exceeded 90% (p=0.6 and 0.12). Internal quality control showed 100% accuracy for all assays. ConclusionPerformance verification of newer CrAg LFA assays under typical laboratory conditions varied, with best results by IMMY SQ and Bio-Rad. The high burden of HIV and cryptococcal disease in South Africa requires high specificity and - sensitivity (>90%) to prevent unnecessary treatment/hospitalization. The added value of positivity grading for patient management needs confirmation.

IntroductionPost-acute sequelae of COVID-19 (PASC) is causing a silent pandemic in the U.S. Gulf South, a part of flyover U.S. where patients are quietly withdrawing from the workforce and largely disconnected from the advocacy resources growing in more affluent regions[1]. To date, there is no clinical test to diagnose PASC and PASC risk factors and etiology remain unclear. MethodsThis prospective study investigates PASC alongside pre-COVID-19 medical history, acute COVID-19 course, and a panel of 25 blood biomarkers collected from 100 COVID-19 patients in New Orleans, LA, in a 52.5% Black cohort, providing a unique opportunity to describe PASC symptoms and associations within a comorbidity-rich population. 107 participants recruited from the ClinSeqSer COVID-19 study at University Medical Center (UMC) or Tulane Medical Center (TMC) in New Orleans underwent PASC symptom questionnaires at 3-month intervals. 100 blood samples from patients at their initial post-COVID follow-up visit were analyzed for cardiac, metabolic, inflammatory, coagulation, chemistry, and hematologic markers in a clinical laboratory. Results were analyzed in SPSS for associations with PASC positivity which was defined as presence of three or more new-since-COVID symptoms present at a visit 12 or more weeks after COVID diagnosis. PASC prevalence was also analyzed alongside demographics and past medical history. ResultsEnrolled participants ranged from 21-87 years old (median 53, mean 52.1, STD 13.7). 63% of participants were female, 52.5% Black, 44% White, and 3% Asian. 52% of participants were hospitalized during their acute COVID-19 course. Severity of participants prior acute COVID was known for most subjects. For 82% of subjects, nasal swab and or saliva SARS CoV-2 qRT-PCR value was known and PCR values did not predict later PASC. Maximum severity scores were assigned to 100 out of 105 participants from whom acute COVID-19 data was collected. On average, patients reported over 5 new-since-COVID symptoms and 75% of patients who completed a questionnaire at time of blood draw were PASC positive. Questionnaire results identified common new-since-COVID symptoms including fatigue (64%), dyspnea (53%), myalgias (48%), trouble concentrating (48%), and memory problems (50%). Over one third of participants reported new-since-COVID arthralgias (34%), headaches (40%), and problems sleeping (40%). For all patients reporting these common symptoms, average frequency and severity of symptoms were reported on a scale of 1 (mild) to 5 (severe) as follows (frequency; severity): fatigue (3.3; 3.3), myalgia (3.4, 3.4), memory problems (3.1, 3.2). Comparison of means analysis indicates that hemoglobin, hematocrit and calcium are lower in PASC positive patients but still within normal range. Analysis of demographics indicates that females in this study are 4.8 times more likely to be classified as PASC positive than males. Serology identified a mild trend toward higher anti-N concentration, and plasma proximity extension proteome detected higher IL-6 and TNF, among PASC vs non-PASC. DiscussionPASC is highly prevalent among post-COVID subjects in this 52.5% Black cohort. A panel of commonly ordered clinical labs was unable to distinguish PASC vs non-PASC subjects, indicating an ongoing need for diagnostic biomarkers relevant across diverse patient groups.

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.

SARS-CoV-2 is a novel coronavirus that mainly affects the upper airways. Approximately one third of all detected cases is asymptomatic. We report an asymptomatic individual who tested positive for SARS-CoV-2 over a period of nine months. Of this individual, whole mouth saliva was tested by a novel fluorescence in situ hybridization-based assay which detects only the active form of the virus. During the observation period of nine months, there was a possible co-infection with a second SARS-CoV-2 variant accompanied by none or very low antibody production until the possible co-infection. We suspect that the SARS-CoV-2 infection in this individual is limited to the salivary glands and does not spread (much) throughout the systemic compartment(s) of the body.

BackgroundRapid antigen tests (RATs) for SARS-CoV-2 have been used to combat the still ongoing Covid-19 pandemic. This study is the extension of the COVAG study originally performed from February 1 to March 31, 2021. We compared two RATs, the Panbio COVID-19 Ag Rapid Test (Abbott) and the SD Biosensor Q SARS-CoV-2 Rapid Antigen Test (Roche), against RT-PCR on the foil of new variants. MethodsWe included 888 all-comers at a diagnostic center between October 20, 2021, and March 18, 2022. RT-PCR-positive samples with a Ct value [&le;] 32 were examined for SARS-CoV-2 variants. FindingsThe sensitivity of the Abbott-RAT and Roche-RAT were 65% and 67%, respectively. For both RATs, lower Ct values were significantly correlated with higher sensitivity. For samples with Ct values [&le;] 25, the sensitivities of the Roche-RAT and of the Abbott-RAT were 96% and 95%, for Ct values 25-30 both were 19%, and for Ct values [&ge;] 30 they were 6% and 2%, respectively. The RATs had substantially higher sensitivities in symptomatic than asymptomatic participants (76, 77%, vs. 29, 31%, for Abbott-RAT, Roche-RAT, respectively) and in participants referred to testing by their primary care physician (84%, 85%) compared to participants who sought testing due to referral by the health department (55%, 58%) or a warning by the Corona-Warn-App (49%, 49%). In persons with self-reported previous Covid-19 sensitivities were markedly lower than in patients without previous Covid-19: 27% vs. 75% for Roche-RAT and 27% vs. 73% for Abbott-RAT. Depending on the vaccination status, the sensitivity of the RATs is 67.6%, 61.5% and 70.6% for non-vaccinated, vaccinated and boostered participants, respectively. For the considered subpopulation of 888 participants, we find no significant correlation between vaccination status and sensitivity. The Omicron variant was detected with a sensitivity of 94% and 92%, the delta variant with a sensitivity of 80% and 80% for Abbott-RAT and Roche-RAT, respectively. This difference is attributable to the lower Ct values of the Omicron samples compared to the Delta samples. When adjusted for the Ct value, a multivariate logistic regression did not show a significant difference between Omicron and Delta. In terms of sensitivity, we found no significant difference between the wild-type and the Omicron and Delta variants, but a significantly lower sensitivity to the alpha variant compared to the other variants. For a Ct value [&le;] 25 the sensitivities were 95.2% and 96.0% for the Abbott-RAT and the Roche-RAT, respectively (Table 4). For a Ct value of 25-30 both RATs had a sensitivity of 18.8%. For a Ct value of 30-32, the sensitivities were 0.0% and 7.1% respectively, for Ct values [&ge;]32 the sensitivities were 3.0% and 6.0% for Abbott-RAT and Roche-RAT, respectively. The specificities were >99% overall. Interpretation: The sensitivity of the RATs for asymptomatic carriers is unsatisfactory questioning their use for screening. When used in symptomatic patients or when requested by a primary care physician the sensitivities were higher. Our study does not suggest that the vaccination status influences the sensitivity of RATs.

BackgroundWith the impact of SARS-CoV-2 upon public health directly and socioeconomically, further information was required to inform policy decisions designed to limit virus spread. This study sought to contribute to serosurveillance work within Northern Ireland to track SARS-CoV-2 progression and guide health strategy. MethodsSera/plasma samples from clinical biochemistry laboratories were analysed for anti-SARS-CoV-2 immunoglobulins (Ig). Samples were assessed using an Elecsys anti-SARS-CoV-2 or anti-SARS-CoV-2 S ECLIA (Roche) on an automated Cobas-e-analyser. Samples were also assessed via ELISA (Euroimmun). A subset of samples assessed via Roche Elecsys anti-SARS-CoV-2 IgG assay were subsequently analysed in an ACE2 pseudoneutralisation assay using a V-PLEX SARS-CoV-2 Panel 7 for IgG and ACE2 by MesoScale Diagnostics Inc. ResultsAcross three testing rounds (June-July 2020, November-December 2020 and June-July 2021 (rounds 1-3 respectively)), 4844 residual sera/plasma specimens were assayed for SARS-CoV-2 Ig. Seropositivity rates increased across the study, peaking at 11.6% during round 3. Varying trends in SARS-CoV-2 seropositivity were noted based on demographic factors. For instance, highest rates of seropositivity shifted from older to younger demographics across the study period. In round 3, alpha (B.1.1.7) variant neutralising antibodies were most frequently detected across age groups, with median concentration of anti-spike protein antibodies elevated in 50-69 year olds and anti-S1 RBD antibodies elevated in over 70s, relative to other age groups. ConclusionsWith seropositivity rates of <15% across the assessment period, it can be concluded that the significant proportion of the Northern Ireland population had not yet naturally contracted the virus by mid-2021.

BackgroundThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Delta lineage (B.1.617.2) was implicated in the SARS-CoV-2 surge in India. We sought to describe the rapid expansion of the Delta lineage in Ontario, Canada (population 15 million) using mutation profile information and confirmatory whole genome sequencing. MethodsAll laboratory-confirmed SARS-CoV-2 cases reported to Public Health Ontario between April 1st and June 12th 2021, with cycle threshold values [&le;]35, were eligible for screening for the N501Y and the E484K mutations. We classified cases via mutation screening as: (1) N501Y-/E484K- (wild-type/Delta), (2) Alpha (N501Y+/E484K-), (3) Beta/Gamma (N501Y+/E484K+), or (4) N501Y-/E484K+ (predominantly B.1.525, and B.1.1.318). ResultsThe N501Y-/E484K- mutation profile went from having a 29% transmission deficit relative to Alpha (relative Re = 0.71, 95%CI: 0.64, 0.77) on April 1st to having a 50% transmission advantage on June 12th (relative Re = 1.50, 95%CI: 1.31, 1.71). Whole genome sequencing of N501Y-/E484K-cases (N=583) confirmed that the pattern of increasing relative reproduction number coincided with the replacement of wild-type with Delta variant (from 2.2% in early April, to 83% in late May). DiscussionDelta is rapidly overtaking other SARS-CoV-2 variants in Ontario, and has a substantial transmission advantage. An inflection in the proportion of cases missing the N501Y mutation from rapidly decreasing to rapidly increasing,3 may be an early warning signal for Delta lineage expansion.

ObjectiveTo detect N501Y mutation of the SARS-CoV-2 spike protein by RT-PCR to distinguish (B.1.1.7) UK and (501Y.V2) South African strains from others in the population of Telangana and to determine its clinical implications. MethodsA primer-probe mix that specifically detects the mutated N501Y strain by real time RT-PCR was designed. 93 samples that were reported positive for COVID-19 by our laboratory in the month of February 2021 were tested using our own primer-probe mix for the presence of N501Y by RT-PCR. The results of RT-PCR were validated by Sanger sequencing in representative samples. Sanger sequencing of other defining spike mutations of B.1.1.7 (del 69-70, del 144, N501Y, A570D, D614G, P681H, T716I, S982A and D1118H) and 501Y.V2 (K417N, E484K, N501Y and D614G) was also investigated. FindingsOut of 93 COVID-19 positive samples, 12 samples are detected positive for N501Y by RT-PCR. Sanger sequencing of these 12 samples further confirmed the presence of N501Y and other mutations that are characteristic of UK strain (B.1.1.7). The South African strain (501Y.V2) is not detected in any of our samples in this study. But, the E484K mutation that is characteristic of 501Y.V2 is detected in one N501Y negative sample. ConclusionStrain-specific RT-PCR for N501Y was developed and validated with Sanger sequencing. Such strategy facilitates quick surveillance for more transmissible and more vaccine resistant strains.

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.

Since the emergence of SARS-CoV-2, global monitoring of the virus using whole genome sequencing has identified mutations occurring across the viral genome. Whilst the majority have little impact on the virus, they are used effectively to monitor the movement of the virus globally and to inform locally on transmission chains. In late 2020, a variant of SARS-CoV-2 (B.1.1.7 - VOC 202012/01) was identified in the UK with a distinct constellation of mutations, including in the spike gene that increased transmissibility. A deletion in spike also affected one of the screening qPCR tests being used in the UK outside of Wales, causing a failure to detect the target. This quickly became a surrogate marker for the variant to allow rapid monitoring of the virus as it seeded into new regions of the UK. A screening study using this assay as a proxy marker, was undertaken to understand the prevalence of the variant in Wales. Secondary analysis of a screening qPCR that didnt target the S gene and also included an endogenous control, was also performed to understand viral load excretion in those infected with the variant compared to other circulating lineages. Using a combination of analytical methods based on the Ct values of two gene targets normalised against the endogenous control, there was a difference in the excreted viral load. Those with the variant excreting more virus than those not infected with the variant. Supporting not only increased infectivity but offering a plausible reason why increased transmission was associated with this particular variant. Whilst there are limitations in this study, the method using Ct as a proxy for viral load can be used at the population level to determine differences in viral excretion kinetics associated with different variants.

Introduction: The Cycle Threshold (CT) value in Real-time Polymerase Chain Reaction (RT-PCR) is where a target specific amplification signal becomes detectable and can infer viral load, risk of transmission and recovery in SARS-CoV-2 infections. Adoption into routine practice is however uncommon. Gap Statement: The lack of inclusion of CT values when reporting SARS-CoV-2 RT-PCR results in routine practice. Aim: To use CT values when reporting SARS-CoV-2 RT-PCR results in Qatar to aid clinical interpretation and patient management. Methodology: Routine CT values across 3 different RT-PCR platforms were reviewed for concordance at presentation and clearance in patients with COVID-19. An Indicative Threshold of CT 30 based on viral clearance kinetics categorized low and high CT values. Results: There was very high Correlation and Kappa Score agreement between the different gene targets in each platform (p&lt;0.001). Using the Indicative Threshold it was possible to autoverify and add average CT values and append Interpretive Comments to all RT-PCR reports. The new reporting algorithm impacted immediately and safely on: physician interpretation of SARS-CoV-2 results; patient management; staff surveillance protocols; length of stay in quarantine; a redefinition of patient recovery. Conclusion: Incorporation of CT values into routine practice is possible across different RT-PCR platforms and adds useful information for patient management. The use of an Indicative Threshold and interpretive comments improves clinical interpretation of the result and could be a model for reporting other respiratory infections. The current accepted practice of withholding CT values should be reviewed by the profession, accreditation bodies and regulators.