Antigen tests for COVID-19.

PCR diagnosis has been considered as the gold standard for coronavirus disease 2019 (COVID-19) and other many diseases. However, there are many problems in using PCR, such as non-specific (i.e., false-positive) and false-negative amplifications, the limits of a target sample volume, deactivation of the enzymes used, complicated techniques, difficulty in designing probe sequences, and the expense. We, thus, need an alternative to PCR, for example an ultrasensitive antigen test. In the present review, we summarize the following three topics. (1) The problems of PCR are outlined. (2) The antigen tests are surveyed in the literature that was published in 2020, and their pros and cons are discussed for commercially available antigen tests. (3) Our own antigen test on the basis of an ultrasensitive enzyme-linked immunosorbent assay (ELISA) is introduced. Finally, we discuss the possibility that our antigen test by an ultrasensitive ELISA technique will become the gold standard for diagnosis of COVID-19 and other diseases.

RT-qPCR Assays for Rapid Detection of the N501Y, 69-70del, K417N, and E484K SARS-CoV-2 Mutations: A Screening Strategy to Identify Variants With Clinical Impact.

Background: Several variants of the SARS-CoV-2 have been documented globally during the current COVID-19 pandemic. The N501Y, 69-70del, K417N, and E484K SARS-CoV-2 mutations have been documented among the most relevant due to their potential pathogenic biological effects. This study aimed to design, validate, and propose a fast real-time RT-qPCR assay to detect SARS-CoV-2 mutations with possible clinical and epidemiological relevance in the Mexican population. Methods: Targeting spike (S) gene mutations of SARS-CoV-2 (N501Y, 69-70del, K417N, and E484K), specific primers, and probes for three specific quantitative reverse transcription PCR (RT-qPCR) assays were designed, and validated using Sanger sequencing. These assays were applied in clinical samples of 1060 COVID-19 patients from Jalisco Mexico. Results: In silico analyzes showed high specificity of the three assays. Amplicons of samples were confirmed through sequencing. The screening of samples of COVID-19 patients allowed the identification of the E484K mutation in nine individuals and the identification of P.2 Brazilian variant in Mexico. Conclusion: This work provides low-cost RT-qPCR assays for rapid screening and molecular surveillance of mutations with potential clinical impact. This strategy allowed the detection of E484K mutation and P.2 variant for the first time in samples from the Mexican population.

Serologic Screening of Severe Acute Respiratory Syndrome Coronavirus 2 Infection in Cats and Dogs during First Coronavirus Disease Wave, the Netherlands.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animal species, including minks, cats, and dogs. To gain insights into SARS-CoV-2 infections in cats and dogs, we developed and validated a set of serologic assays, including ELISA and virus neutralization. Evaluation of samples from animals before they acquired coronavirus disease and samples from cats roaming SARS-CoV-2-positive mink farms confirmed the suitability of these assays for specific antibody detection. Furthermore, our findings exclude SARS-CoV-2 nucleocapsid protein as an antigen for serologic screening of cat and dog samples. We analyzed 500 serum samples from domestic cats and dogs in the Netherlands during April-May 2020. We showed 0.4% of cats and 0.2% of dogs were seropositive. Although seroprevalence in cats and dogs that had unknown SARS-CoV-2 exposure was low during the first coronavirus disease wave, our data stress the need for development of continuous serosurveillance for SARS-CoV-2 in these 2 animal species.

Development of a multiplex microsphere immunoassay for the detection of antibodies against highly pathogenic viruses in human and animal serum samples.

Surveillance of highly pathogenic viruses circulating in both human and animal populations is crucial to unveil endemic infections and potential zoonotic reservoirs. Monitoring the burden of disease by serological assay could be used as an early warning system for imminent outbreaks as an increased seroprevalance often precedes larger outbreaks. However, the multitude of highly pathogenic viruses necessitates the need to identify specific antibodies against several targets from both humans as well as from potential reservoir animals such as bats. In order to address this, we have developed a broadly reactive multiplex microsphere immunoassay (MMIA) for the detection of antibodies against several highly pathogenic viruses from both humans and animals. To this aim, nucleoproteins (NP) of Ebola virus (EBOV), Marburg virus (MARV) and nucleocapsid proteins (NP) of Crimean-Congo haemorrhagic fever virus, Rift Valley fever virus and Dobrava-Belgrade hantavirus were employed in a 5-plex assay for IgG detection. After optimisation, specific binding to each respective NP was shown by testing sera from humans and non-human primates with known infection status. The usefulness of our assay for serosurveillance was shown by determining the immune response against the NP antigens in a panel of 129 human serum samples collected in Guinea between 2011 and 2012 in comparison to a panel of 88 sera from the German blood bank. We found good agreement between our MMIA and commercial or in-house reference methods by ELISA or IIFT with statistically significant higher binding to both EBOV NP and MARV NP coupled microspheres in the Guinea panel. Finally, the MMIA was successfully adapted to detect antibodies from bats that had been inoculated with EBOV- and MARV- virus-like particles, highlighting the versatility of this technique and potentially enabling the monitoring of wildlife as well as human populations with this assay. We were thus able to develop and validate a sensitive and broadly reactive high-throughput serological assay which could be used as a screening tool to detect antibodies against several highly pathogenic viruses.

Evaluation of the MP Rapid 2019-NCOV IgM/IgG combo POCT test vs. an established platform-based method.

BACKGROUND: Accurate and rapid testing for SARS-COV-2 antibodies could improve the diagnosis and management of COVID-19. In this study, we aim to evaluate the diagnostic accuracy of a commercially available point-of-care lateral flow kit independently and in comparison to an established platform-based system. METHOD: Samples from 144 PCR-confirmed COVID-19 cases and 130 pre-pandemic negative controls were tested in parallel by MP Rapid 2019-NCOV IgM/IgG Combo test and Roche Elecsys. Comparison of results based on serum and capillary blood testing was undertaken. RESULTS: Sensitivity at day 15 onwards was 100% for both methods. Between days 1 and 7 post admission, the IgM/IgG Combo test and Roche Elecsys shown sensitivity of 74% (95%CI: 62%-85%) vs. 67% (95% CI: 55%-79%, P = 0.3947). Combo test specificities were 100% for IgG, 98.5% for IgM vs. Roche Elecsys specificity of 100%. Concordance analysis showed 98.5% agreement to the Roche Elecsys method (Cohen's Kappa 0.96 95% CI [0.92-0.99]). Capillary blood results showed complete agreement with serum samples using the Combo test. CONCLUSION: In comparison to Roche Elecsys, our data show that the MP Rapid 2019-NCOV IgM/IgG Combo test provides a high-confidence assay system for the detection of previous exposure to SARS-COV-2 infection with advantage of affording near-patient testing.

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19.

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N = 80 samples from persons with RT-PCR confirmed SARS-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).

Molecular diagnostic assays for COVID-19: an overview.

The coronavirus disease 2019 (COVID-19) pandemic has highlighted the cardinal importance of rapid and accurate diagnostic assays. Since the early days of the outbreak, researchers with different scientific backgrounds across the globe have tried to fulfill the urgent need for such assays, with many assays having been approved and with others still undergoing clinical validation. Molecular diagnostic assays are a major group of tests used to diagnose COVID-19. Currently, the detection of SARS-CoV-2 RNA by reverse transcription polymerase chain reaction (RT-PCR) is the most widely used method. Other diagnostic molecular methods, including CRISPR-based assays, isothermal nucleic acid amplification methods, digital PCR, microarray assays, and next generation sequencing (NGS), are promising alternatives. In this review, we summarize the technical and clinical applications of the different COVID-19 molecular diagnostic assays and suggest directions for the implementation of such technologies in future infectious disease outbreaks.

Development and application of sensitive, specific, and rapid CRISPR-Cas13-based diagnosis.

Nucleic acid detection is a necessary part of medical treatment and fieldwork. However, the current detection technologies are far from ideal. A lack of timely and accessible testing for identifying cases and close contacts has allowed severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the causative virus of the ongoing coronavirus disease-2019 (COVID-19) pandemic, to spread uncontrollably. The slow and expensive detection of mutations-predictors for chronic diseases such as cancer-form a barrier to personalized treatment. A recently developed diagnostic assay is ideal and field-ready-it relies on CRISPR-Cas13. CRISPR-Cas13 works similarly to other CRISPR systems: Cas13 is guided by a crRNA to cleave next to a specific RNA target sequence. Additionally, Cas13 boasts a unique collateral cleavage activity; collateral cleavage of a fluorescent reporter detects the presence of the target sequence in sample RNA. This system forms the basis of CRISPR-Cas13 diagnostic assays. CRISPR-Cas13 assays have >95% sensitivity and >99% specificity. Detection is rapid (<2 h), inexpensive ($0.05 per test), and portable-a test using lateral flow strips is akin to a pregnancy test. The recent adaptation of micro-well chips facilitates high-level multiplexing and is high-throughput. In this review, we cover the development of CRISPR-Cas13 assays for medical diagnosis, discuss the advantages of CRISPR-Cas13-based diagnosis over the traditional reverse transcription polymerase chain reaction (RT-PCR), and present examples of detection from real patient samples.

Clinical performance of the Panbio assay for the detection of SARS-CoV-2 IgM and IgG in COVID-19 patients.

Following the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, numerous serological tests have been developed, including rapid diagnostic tests. This study aims at assessing the clinical performance of the Panbio immunoglobulin G (IgG)/IgM coronavirus disease 2019 (COVID-19) test (Abbott), a rapid lateral flow assay for the qualitative detection of IgG and IgM against SARS-CoV-2. One hundred and thirty-eight samples from 95 COVID-19 patients with a positive SARS-CoV-2 reverse-transcriptase polymerase chain reaction were analyzed to assess the clinical sensitivity. Seventy-six pre-COVID-19 samples were used to evaluate the clinical specificity. Two independent and blinded raters determined visually the presence or absence of the IgG, IgM, and control lines for each test after 10 and 20 min. The sensitivity obtained from collected samples more than 14 days after the onset of symptoms was 95.2% for IgG. IgM was less frequently detected (highest sensitivity of 20.5%). The specificities obtained were 98.7% and 100% for IgG and IgM, respectively. In addition, the sensitivity of the assay was better when the reading was performed at 20 min than at 10 min, whereas the specificity was unchanged. The Panbio COVID-19 IgG/IgM rapid test detects IgG with high sensitivity 14 days since symptom onset but presents a low sensitivity for IgM. The specificity was excellent for both IgG and IgM.

Specific and Rapid SARS-CoV-2 Identification Based on LC-MS/MS Analysis.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, emerged as the cause of a global crisis. Rapid and reliable clinical diagnosis is essential for effectively controlling transmission. The gold standard assay for SARS-CoV-2 identification is the highly sensitive real-time quantitative polymerase chain reaction (RT-qPCR); however, this assay depends on specialized reagents and may suffer from false results. Thus, additional assays based on different approaches could be beneficial. Here, we present a novel method for SARS-CoV-2 identification based on mass spectrometry. The approach we implemented combines a multistep procedure for the rational down-selection of a set of reliable markers out of all optional in silico derived tryptic peptides in viral proteins, followed by monitoring of peptides derived from tryptic digests of purified proteins, cell-cultured SARS-CoV-2, and nasopharyngeal (NP) swab matrix spiked with the virus. The marker selection was based on specificity to SARS-CoV-2 and on analytical parameters including sensitivity, linearity, and reproducibility. The final assay is based on six unique and specific peptide markers for SARS-CoV-2 identification. The simple and rapid (2.5 h) protocol we developed consists of virus heat inactivation and denaturation, tryptic digestion, and identification of the selected markers by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS/MS). The developed assay enabled the identification of 104 PFU/mL SARS-CoV-2 spiked into buffer. Finally, the assay was successfully applied to 16 clinical samples diagnosed by RT-qPCR, achieving 94% concordance with the current gold standard assay. To conclude, the novel MS-based assay described here is specific, rapid, simple, and is believed to provide a complementary assay to the RT-qPCR method.

Antibody seroprevalence in the epicenter Wuhan, Hubei, and six selected provinces after containment of the first epidemic wave of COVID-19 in China.

BACKGROUND: China implemented containment measures to stop SARS-CoV-2 transmission in response to the COVID-19 epidemic. After the first epidemic wave, we conducted population-based serological surveys to determine extent of infection, risk factors for infection, and neutralization antibody levels to assess the real infections in the random sampled population. METHODS: We used a multistage, stratified cluster random sampling strategy to conduct serological surveys in three areas - Wuhan, Hubei Province outside Wuhan, and six provinces selected on COVID-19 incidence and containment strategy. Participants were consenting individuals >1 year old who resided in the survey area >14 days during the epidemic. Provinces screened sera for SARS-CoV-2-specific IgM, IgG, and total antibody by two lateral flow immunoassays and one magnetic chemiluminescence enzyme immunoassay; positive samples were verified by micro-neutralization assay. FINDINGS: We enrolled 34,857 participants (overall response rate, 92%); 427 were positive by micro-neutralization assay. Wuhan had the highest weighted seroprevalence (4•43%, 95% confidence interval [95%CI]=3•48%-5•62%), followed by Hubei-ex-Wuhan (0•44%, 95%CI=0•26%-0•76%), and the other provinces (<0•1%). Living in Wuhan (adjusted odds ratio aOR=13•70, 95%CI= 7•91-23•75), contact with COVID-19 patients (aOR=7•35, 95%CI=5•05-10•69), and age over 40 (aOR=1•36, 95%CI=1•07-1•72) were significantly associated with SARS-CoV-2 infection. Among seropositives, 101 (24%) reported symptoms and had higher geometric mean neutralizing antibody titers than among the 326 (76%) without symptoms (30±2•4 vs 15±2•1, p<0•001). INTERPRETATION: The low overall extent of infection and steep gradient of seropositivity from Wuhan to the outer provinces provide evidence supporting the success of containment of the first wave of COVID-19 in China. SARS-CoV-2 infection was largely asymptomatic, emphasizing the importance of active case finding and physical distancing. Virtually the entire population of China remains susceptible to SARS-CoV-2; vaccination will be needed for long-term protection. FUNDING: This study was supported by the Ministry of Science and Technology (2020YFC0846900) and the National Natural Science Foundation of China (82041026, 82041027, 82041028, 82041029, 82041030, 82041032, 82041033).

Seroepidemiological Survey of the Antibody for Severe Acute Respiratory Syndrome Coronavirus 2 with Neutralizing Activity at Hospitals: A Cross-sectional Study in Hyogo Prefecture, Japan.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic is spreading rapidly all over the world. The Japanese government lifted the state of emergency, announced in April 2020, on May 25, but there are still sporadic clusters. Asymptomatic patients who can transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause some of these clusters. It is thus urgent to investigate the seroprevalence of antibodies against SARS-CoV-2 and their neutralizing activity. We conducted a cross-sectional study of >10,000 samples at hospitals in Hyogo Prefecture, Japan. METHODS: Between August 6 and October 1, 2020, we collected samples of residual blood from the patients who visited or were admitted to five hospitals and a foundation in Hyogo. We tested the samples for antibodies against SARS-CoV-2 by electrochemiluminescence immunoassay (ECLIA) and chemiluminescent enzyme immunoassay (CLEIA). Sera that were positive by ECLIA or CLEIA were analyzed by an immunochromatographic (IC) test and neutralizing activity assay. RESULTS: We tested 10,377 samples from patients aged between 0 and 99 years old; 27 cases (0.26%) were positive on the ECLIA, and 51 cases (0.49%) were positive on CLEIA. In the 14 cases that tested positive on both ECLIA and CLEIA, the positive rates on the IC test and for neutralizing activity were high (85% and 92%, respectively). In 50 cases (0.48%) that were positive by either ECLIA or CLEIA, the corresponding rates were low (20% and 6%, respectively). The positive rate of neutralizing antibody was 0.15%. CONCLUSIONS: These results indicate that most Hyogo Prefecture residents still do not have antibodies and should avoid the risk of incurring a SARS-CoV-2 infection. Two or more antibody tests should be required for seroepidemiological studies of the antibody for SARS-CoV-2, and a neutralizing activity assay is also essential.

Greater Fibrinolysis Resistance but No Greater Platelet Aggregation in Critically Ill COVID-19 Patients.

BACKGROUND: The hemostatic balance in patients with coronavirus disease 2019 (COVID-19) seems to be shifted toward a hypercoagulable state. The aim of the current study was to assess the associated coagulation alterations by point-of-care-diagnostics, focusing on details of clot formation and lysis in these severely affected patients. METHODS: The authors' prospective monocentric observational study included critically ill patients diagnosed with COVID-19. Demographics and biochemical data were recorded. To assess the comprehensive hemostatic profile of this patient population, aggregometric (Multiplate) and viscoelastometric (CloPro) measures were performed in the intensive care unit of a university hospital at a single occasion. Coagulation analysis and assessment of coagulation factors were performed. Data were compared to healthy controls. RESULTS: In total, 27 patients (21 male; mean age, 60 yr) were included. Impedance aggregometry displayed no greater platelet aggregability in COVID-19 in comparison with healthy controls (area under the curve [AUC] in adenosine diphosphate test, 68 ± 37 U vs. 91 ± 29 U [-27 (Hodges-Lehmann 95% CI, -48 to -1); P = 0.043]; AUC in arachidonic acid test, 102 ± 54 U vs. 115 ± 26 U [-21 (Hodges-Lehmann 95% CI, -51 to 21); P = 0.374]; AUC in thrombin receptor activating peptide 6 test, 114 ± 61 U vs. 144 ± 31 U [-31 (Hodges-Lehmann 95% CI, -69 to -7); P = 0.113]). Comparing the thromboelastometric results of COVID-19 patients to healthy controls, the authors observed significant differences in maximum clot firmness in fibrin contribution to maximum clot firmness assay (37 ± 11 mm vs. 15 ± 4 mm [21 (Hodges-Lehmann 95% CI, 17 to 26); P < 0.001]) and lysis time in extrinsic activation and activation of fibrinolysis by tissue plasminogen activator assay (530 ± 327 s vs. 211 ± 80 s [238 (Hodges-Lehmann 95% CI, 160 to 326); P < 0.001]). CONCLUSIONS: Thromboelastometry in COVID-19 patients revealed greater fibrinolysis resistance. The authors did not find a greater platelet aggregability based on impedance aggregometric tests. These findings may contribute to our understanding of the hypercoagulable state of critically ill patients with COVID-19.

SARS-CoV-2 Antibody Responses Are Correlated to Disease Severity in COVID-19 Convalescent Individuals.

Globally, the COVID-19 pandemic has had extreme consequences for the healthcare system and has led to calls for diagnostic tools to monitor and understand the transmission, pathogenesis, and epidemiology, as well as to evaluate future vaccination strategies. In this study, we have developed novel, to our knowledge, flexible ELISA-based assays for specific detection of human SARS-CoV-2 Abs against the receptor-binding domain, including an Ag sandwich ELISA relevant for large population screening and three isotype-specific assays for in-depth diagnostics. Their performance was evaluated in a cohort of 350 convalescent participants with previous COVID-19 infection, ranging from asymptomatic to critical cases. We mapped the Ab responses to different areas on protein N and S and showed that the IgM, A, and G Ab responses against receptor-binding domain are significantly correlated to the disease severity. These assays and the data generated from them are highly relevant for diagnostics and prognostics and contribute to the understanding of long-term COVID-19 immunity.

SARS-CoV-2 seroprevalence trends in healthy blood donors during the COVID-19 outbreak in Milan.

BACKGROUND: The Milan metropolitan area in Northern Italy was among the most severely hit by the SARS-CoV-2 outbreak. The aim of this study was to examine the seroprevalence trends of SARS-CoV-2 in healthy asymptomatic adults, and the risk factors and laboratory correlates of positive tests. MATERIALS AND METHODS: We conducted a cross-sectional study in a random sample of blood donors, who were asymptomatic at the time of evaluation, at the beginning of the first phase (February 24th to April 8th 2020; n=789). Presence of IgM/IgG antibodies against the SARS-CoV-2-Nucleocapsid protein was assessed by a lateral flow immunoassay. RESULTS: The test had a 100/98.3 sensitivity/specificity (n=32/120 positive/negative controls, respectively), and the IgG test was validated in a subset by an independent ELISA against the Spike protein (n=34, p<0.001). At the start of the outbreak, the overall adjusted seroprevalence of SARS-CoV-2 was 2.7% (95% CI: 0.3-6%; p<0.0001 vs 120 historical controls). During the study period, characterised by a gradual implementation of social distancing measures, there was a progressive increase in the adjusted seroprevalence to 5.2% (95% CI: 2.4-9.0; 4.5%, 95% CI: 0.9-9.2% according to a Bayesian estimate) due to a rise in IgG reactivity to 5% (95% CI: 2.8-8.2; p=0.004 for trend), but there was no increase in IgM+ (p=not significant). At multivariate logistic regression analysis, IgG reactivity was more frequent in younger individuals (p=0.043), while IgM reactivity was more frequent in individuals aged >45 years (p=0.002). DISCUSSION: SARS-CoV-2 infection was already circulating in Milan at the start of the outbreak. The pattern of IgM/IgG reactivity was influenced by age: IgM was more frequently detected in participants aged >45 years. By the end of April, 2.4-9.0% of healthy adults had evidence of seroconversion.

Evaluation of commercially available immuno-magnetic agglutination in comparison to enzyme-linked immunosorbent assays for rapid point-of-care diagnostics of COVID-19.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is caused by Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Fast, accurate, and simple blood-based assays for quantification of anti-SARS-CoV-2 antibodies are urgently needed to identify infected individuals and keep track of the spread of disease. METHODS: The study included 33 plasma samples from 20 individuals with confirmed COVID-19 by real-time reverse-transcriptase polymerase chain reaction and 40 non-COVID-19 plasma samples. Anti-SARS-CoV-2 immunoglobulin M (IgM)/immunoglobulin A (IgA) or immunoglobulin G (IgG) antibodies were detected by a microfluidic quantitative immunomagnetic assay (IMA) (ViroTrack Sero COVID IgM + IgA/IgG Ab, Blusense Diagnostics) and compared to an enzyme-linked immunosorbent assay (ELISA) (EuroImmun Medizinische Labordiagnostika). RESULTS: Of the 33 plasma samples from the COVID-19 patients, 28 were positive for IgA/IgM or IgG by IMA and 29 samples were positive by ELISA. Sensitivity for only one sample per patient was 68% for IgA + IgM and 75% IgG by IMA and 80% by ELISA. For samples collected 14 days after symptom onset, the sensitivity of both IMA and ELISA was around 91%. The specificity of the IMA reached 100% compared to 95% for ELISA IgA and 97.5% for ELISA IgG. CONCLUSION: IMA for COVID-19 is a rapid simple-to-use point-of-care test with sensitivity and specificity similar to a commercial ELISA.

A new positive SARS-CoV-2 test months after severe COVID-19 illness: reinfection or intermittent viral shedding?

We present a case of a patient who had a history of severe coronavirus disease (COVID-19) 4 months prior to this current presentation and, after a long asymptomatic period, subsequently tested positive for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) by a RNA PCR assay, after several interval negative SARS-CoV-2 RNA tests. We present this potential case of SARS-CoV-2 reinfection in order to incite discussion around differentiating persistent infection with intermittent viral shedding and reinfection, as well as to discuss evolving knowledge and approaches to the clinical management, follow-up molecular testing and treatment of COVID-19 reinfection.

SARS-CoV-2 seroprevalence trends in healthy blood donors during the COVID-19 outbreak in Milan.

BACKGROUND: The Milan metropolitan area in Northern Italy was among the most severely hit by the SARS-CoV-2 outbreak. The aim of this study was to examine the seroprevalence trends of SARS-CoV-2 in healthy asymptomatic adults, and the risk factors and laboratory correlates of positive tests. MATERIALS AND METHODS: We conducted a cross-sectional study in a random sample of blood donors, who were asymptomatic at the time of evaluation, at the beginning of the first phase (February 24th to April 8th 2020; n=789). Presence of IgM/IgG antibodies against the SARS-CoV-2-Nucleocapsid protein was assessed by a lateral flow immunoassay. RESULTS: The test had a 100/98.3 sensitivity/specificity (n=32/120 positive/negative controls, respectively), and the IgG test was validated in a subset by an independent ELISA against the Spike protein (n=34, p<0.001). At the start of the outbreak, the overall adjusted seroprevalence of SARS-CoV-2 was 2.7% (95% CI: 0.3-6%; p<0.0001 vs 120 historical controls). During the study period, characterised by a gradual implementation of social distancing measures, there was a progressive increase in the adjusted seroprevalence to 5.2% (95% CI: 2.4-9.0; 4.5%, 95% CI: 0.9-9.2% according to a Bayesian estimate) due to a rise in IgG reactivity to 5% (95% CI: 2.8-8.2; p=0.004 for trend), but there was no increase in IgM+ (p=not significant). At multivariate logistic regression analysis, IgG reactivity was more frequent in younger individuals (p=0.043), while IgM reactivity was more frequent in individuals aged >45 years (p=0.002). DISCUSSION: SARS-CoV-2 infection was already circulating in Milan at the start of the outbreak. The pattern of IgM/IgG reactivity was influenced by age: IgM was more frequently detected in participants aged >45 years. By the end of April, 2.4-9.0% of healthy adults had evidence of seroconversion.

Comparison of clinical and serological features of RT-PCR positive and negative COVID-19 patients.

BACKGROUND: In December 2019, an outbreak of coronavirus disease 2019 (COVID-19) began in Wuhan, China, and led to a global epidemic. We aimed to compare the clinical and serological features of COVID-19 patients with positive and negative reverse transcriptase polymerase chain reaction (RT-PCR) tests. METHODS: This was a retrospective cohort study conducted from 9 February to 4 April 2020. COVID-19 patients at Leishenshan Hospital in Wuhan, China (125 total cases; 87 RT-PCR positive and 38 RT-PCR negative) were included. COVID-19 serology was assessed by colloidal gold assay. All cases were analyzed for demographic, clinical, and serological features. RESULTS: There were no significant differences in most demographic features, clinical symptoms, complications or treatments of RT-PCR positive and negative COVID-19 patients. Serum IgM/IgG was positive in 82 (94%) and 33 (87%) RT-PCR positive and negative cases, respectively. IgM was detectable as early as 3 days after symptom onset and was undetectable 60 days after symptom onset. By contrast, IgG could be detected only 10 days after symptom onset and reached its peak 60 days after symptom onset. CONCLUSIONS: Serological tests performed during the appropriate time window of disease progression could be valuable auxiliary methods to RT-PCR in COVID-19 patients.

Development and performance evaluation of a rapid in-house ELISA for retrospective serosurveillance of SARS-CoV-2.

BACKGROUND: In the ongoing pandemic situation of COVID-19, serological tests can complement the molecular diagnostic methods, and can be one of the important tools of sero-surveillance and vaccine evaluation. AIM: To develop and evaluate a rapid SARS-CoV-2 specific ELISA for detection of anti-SARS-CoV2 IgG from patients' biological samples. METHODS: In order to develop this ELISA, three panels of samples (n = 184) have been used: panel 1 (n = 19) and panel 2 (n = 60) were collected from RT-PCR positive patients within 14 and after 14 days of onset of clinical symptoms, respectively; whereas panel 3 consisted of negative samples (n = 105) collected either from healthy donors or pre-pandemic dengue patients. As a capturing agent full-length SARS-CoV2 specific recombinant nucleocapsid was immobilized. Commercial SARS-CoV2 IgG kit based on chemiluminescent assay was used for the selection of samples and optimization of the assay. The threshold cut-off point, inter-assay and intra-assay variations were determined. RESULTS: The incubation/reaction time was set at a total of 30 minutes with the sensitivity of 84% (95% confidence interval, CI, 60.4%, 96.6%) and 98% (95% CI, 91.1%, 100.0%), for panel 1 and 2, respectively; with overall 94.9% sensitivity (95% CI 87.5%, 98.6%). Moreover, the clinical specificity was 97.1% (95% CI, 91.9%, 99.4%) with no cross reaction with dengue samples. The overall positive and negative predictive values are 96.2% (95% CI 89.2%, 99.2%) and 96.2% (95% CI, 90.6% 99.0%), respectively. In-house ELISA demonstrated 100% positive and negative percent agreement with Elecsys Anti-SARS-CoV-2, with Cohen's kappa value of 1.00 (very strong agreement), while comparing 13 positive and 17 negative confirmed cases. CONCLUSION: The assay is rapid and can be applied as one of the early and retrospective sero-monitoring tools in all over the affected areas.