Rapid Detection of the Monkeypox Virus Genome and Antigen Proteins Based on Surface-Enhanced Raman Spectroscopy.

The conventional detection methods cannot satisfy the need for early and rapid detection of monkeypox virus (MPXV) infection. This is due to complicated pretreatment, time consumption, and complex operation of the diagnostic tests. Based on surface-enhanced Raman spectroscopy (SERS), this study attempted to capture the characteristic fingerprints of the MPXV genome and multiple antigenic proteins without the need to design specific probes. The minimum detection limit of this method is 100 copies/mL, with good reproducibility and signal-to-noise ratio. Therefore, the relationship between characteristic peak intensity and the protein and nucleic acid concentration can be used to construct a concentration-dependent spectral line with a good linear relationship. Additionally, principal component analysis (PCA) could identify the SERS spectra of four different MPXV proteins in serum. Therefore, this rapid detection method in the current outbreak of monkeypox control and the future response to possible new outbreaks has broad application prospects.

Persistent Circulating Severe Acute Respiratory Syndrome Coronavirus 2 Spike Is Associated With Post-acute Coronavirus Disease 2019 Sequelae.

The diagnosis of postacute sequelae of coronavirus disease 2019 (PASC) poses an ongoing medical challenge. To identify biomarkers associated with PASC we analyzed plasma samples collected from PASC and coronavirus disease 2019 patients to quantify viral antigens and inflammatory markers. We detect severe acute respiratory syndrome coronavirus 2 spike predominantly in PASC patients up to 12 months after diagnosis.

Diagnostic accuracy of serological tests for the diagnosis of Chikungunya virus infection: A systematic review and meta-analysis.

BACKGROUND: Chikungunya virus (CHIKV) causes febrile illnesses and has always been misdiagnosed as other viral infections, such as dengue and Zika; thus, a laboratory test is needed. Serological tests are commonly used to diagnose CHIKV infection, but their accuracy is questionable due to varying degrees of reported sensitivities and specificities. Herein, we conducted a systematic review and meta-analysis to evaluate the diagnostic accuracy of serological tests currently available for CHIKV. METHODOLOGY AND PRINCIPAL FINDINGS: A literature search was performed in PubMed, CINAHL Complete, and Scopus databases from the 1st December 2020 until 22nd April 2021. Studies reporting sensitivity and specificity of serological tests against CHIKV that used whole blood, serum, or plasma were included. QUADAS-2 tool was used to assess the risk of bias and applicability, while R software was used for statistical analyses. Thirty-five studies were included in this meta-analysis; 72 index test data were extracted and analysed. Rapid and ELISA-based antigen tests had a pooled sensitivity of 85.8% and 82.2%, respectively, and a pooled specificity of 96.1% and 96.0%, respectively. According to our meta-analysis, antigen detection tests serve as a good diagnostic test for acute-phase samples. The IgM detection tests had more than 90% diagnostic accuracy for ELISA-based tests, immunofluorescence assays, in-house developed tests, and samples collected after seven days of symptom onset. Conversely, low sensitivity was found for the IgM rapid test (42.3%), commercial test (78.6%), and for samples collected less than seven of symptom onset (26.2%). Although IgM antibodies start to develop on day 2 of CHIKV infection, our meta-analysis revealed that the IgM detection test is not recommended for acute-phase samples. The diagnostic performance of the IgG detection tests was more than 93% regardless of the test formats and whether the test was commercially available or developed in-house. The use of samples collected after seven days of symptom onset for the IgG detection test suggests that IgG antibodies can be detected in the convalescent-phase samples. Additionally, we evaluated commercial IgM and IgG tests for CHIKV and found that ELISA-based and IFA commercial tests manufactured by Euroimmun (Lübeck, Germany), Abcam (Cambridge, UK), and Inbios (Seattle, WA) had diagnostic accuracy of above 90%, which was similar to the manufacturers' claim. CONCLUSION: Based on our meta-analysis, antigen or antibody-based serological tests can be used to diagnose CHIKV reliably, depending on the time of sample collection. The antigen detection tests serve as a good diagnostic test for samples collected during the acute phase (≤7 days post symptom onset) of CHIKV infection. Likewise, IgM and IgG detection tests can be used for samples collected in the convalescent phase (>7 days post symptom onset). In correlation to the clinical presentation of the patients, the combination of the IgM and IgG tests can differentiate recent and past infections.

Molecular Epidemiology of Norovirus (NoV) Infection in Mie Prefecture: The Kinetics of Norovirus Antigenemia in Pediatric Patients.

Few studies have shown the presence of norovirus (NoV) RNA in blood circulation but there is no data on norovirus antigenemia. We examined both antigenemia and RNAemia from the sera of children with NoV infections and studied whether norovirus antigenemia is correlated with the levels of norovirus-specific antibodies and clinical severity of gastroenteritis. Both stool and serum samples were collected from 63 children admitted to Mie National Hospital with acute NoV gastroenteritis. Norovirus antigen and RNA were detected in sera by ELISA and real-time RT-PCR, respectively. NoV antigenemia was found in 54.8% (34/62) and RNAemia in 14.3% (9/63) of sera samples. Antigenemia was more common in the younger age group (0-2 years) than in the older age groups, and most patients were male. There was no correlation between stool viral load and norovirus antigen (NoV-Ag) levels (rs = -0.063; Cl -0.3150 to 0.1967; p = 0.6251). Higher levels of acute norovirus-specific IgG serum antibodies resulted in a lower antigenemia OD value (n = 61; r = -0.4258; CI -0.62 to -0.19; p = 0.0006). Norovirus antigenemia occurred more commonly in children under 2 years of age with NoV-associated acute gastroenteritis. The occurrence of antigenemia was not correlated with stool viral load or disease severity.

Performance of Three Tests for SARS-CoV-2 on a University Campus Estimated Jointly with Bayesian Latent Class Modeling.

Accurate tests for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been critical in efforts to control its spread. The accuracy of tests for SARS-CoV-2 has been assessed numerous times, usually in reference to a gold standard diagnosis. One major disadvantage of that approach is the possibility of error due to inaccuracy of the gold standard, which is especially problematic for evaluating testing in a real-world surveillance context. We used an alternative approach known as Bayesian latent class modeling (BLCM), which circumvents the need to designate a gold standard by simultaneously estimating the accuracy of multiple tests. We applied this technique to a collection of 1,716 tests of three types applied to 853 individuals on a university campus during a 1-week period in October 2020. We found that reverse transcriptase PCR (RT-PCR) testing of saliva samples performed at a campus facility had higher sensitivity (median, 92.3%; 95% credible interval [CrI], 73.2 to 99.6%) than RT-PCR testing of nasal samples performed at a commercial facility (median, 85.9%; 95% CrI, 54.7 to 99.4%). The reverse was true for specificity, although the specificity of saliva testing was still very high (median, 99.3%; 95% CrI, 98.3 to 99.9%). An antigen test was less sensitive and specific than both of the RT-PCR tests, although the sample sizes with this test were small and the statistical uncertainty was high. These results suggest that RT-PCR testing of saliva samples at a campus facility can be an effective basis for surveillance screening to prevent SARS-CoV-2 transmission in a university setting. IMPORTANCE Testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been vitally important during the COVID-19 pandemic. There are a variety of methods for testing for this virus, and it is important to understand their accuracy in choosing which one might be best suited for a given application. To estimate the accuracy of three different testing methods, we used a data set collected at a university that involved testing the same samples with multiple tests. Unlike most other estimates of test accuracy, we did not assume that one test was perfect but instead allowed for some degree of inaccuracy in all testing methods. We found that molecular tests performed on saliva samples at a university facility were similarly accurate as molecular tests performed on nasal samples at a commercial facility. An antigen test appeared somewhat less accurate than the molecular tests, but there was high uncertainty about that.

Delayed Influenza Treatment in Children With False-Negative Rapid Antigen Test: A Retrospective Single-Center Study in Korea 2016-2019.

BACKGROUND: We aimed to examine the delay in antiviral initiation in rapid antigen test (RAT) false-negative children with influenza virus infection and to explore the clinical outcomes. We additionally conducted a medical cost-benefit analysis. METHODS: This single-center, retrospective study included children (aged < 10 years) with influenza-like illness (ILI), hospitalized after presenting to the emergency department during three influenza seasons (2016-2019). RAT-false-negativity was defined as RAT-negative and polymerase chain reaction-positive cases. The turnaround time to antiviral treatment (TAT) was from the time when RAT was prescribed to the time when the antiviral was administered. The medical cost analysis by scenarios was also performed. RESULTS: A total of 1,430 patients were included, 7.5% were RAT-positive (n = 107) and 2.4% were RAT-false-negative (n = 20). The median TAT of RAT-false-negative patients was 52.8 hours, significantly longer than that of 4 hours in RAT-positive patients (19.2-100.1, P < 0.001). In the multivariable analysis, TAT of ≥ 24 hours was associated with a risk of severe influenza infection and the need for mechanical ventilation (odds ratio [OR], 6.8, P = 0.009 and OR, 16.2, P = 0.033, respectively). The medical cost varied from $11.7-187.3/ILI patient. CONCLUSION: Antiviral initiation was delayed in RAT-false-negative patients. Our findings support the guideline that children with influenza, suspected of having severe or progressive infection, should be treated immediately.

Low anti-SARS-CoV-2 S antibody levels predict increased mortality and dissemination of viral components in the blood of critical COVID-19 patients.

BACKGROUND: Anti-SARS-CoV-2 S antibodies prevent viral replication. Critically ill COVID-19 patients show viral material in plasma, associated with a dysregulated host response. If these antibodies influence survival and viral dissemination in ICU-COVID patients is unknown. PATIENTS/METHODS: We studied the impact of anti-SARS-CoV-2 S antibodies levels on survival, viral RNA-load in plasma, and N-antigenaemia in 92 COVID-19 patients over ICU admission. RESULTS: Frequency of N-antigenaemia was >2.5-fold higher in absence of antibodies. Antibodies correlated inversely with viral RNA-load in plasma, representing a protective factor against mortality (adjusted HR [CI 95%], p): (S IgM [AUC ≥ 60]: 0.44 [0.22; 0.88], 0.020); (S IgG [AUC ≥ 237]: 0.31 [0.16; 0.61], <0.001). Viral RNA-load in plasma and N-antigenaemia predicted increased mortality: (N1-viral load [≥2.156 copies/ml]: 2.25 [1.16; 4.36], 0.016); (N-antigenaemia: 2.45 [1.27; 4.69], 0.007). CONCLUSIONS: Low anti-SARS-CoV-2 S antibody levels predict mortality in critical COVID-19. Our findings support that these antibodies contribute to prevent systemic dissemination of SARS-CoV-2.

SARS-CoV-2 N-antigenemia in critically ill adult COVID-19 patients: Frequency and association with inflammatory and tissue-damage biomarkers.

The current study aimed at characterizing the dynamics of SARS-CoV-2 nucleocapsid (N) antigenemia in a cohort of critically ill adult COVID-19 patients and assessing its potential association with plasma levels of biomarkers of clinical severity and mortality. Seventy-three consecutive critically ill COVID-19 patients (median age, 65 years) were recruited. Serial plasma (n = 340) specimens were collected. A lateral flow immunochromatography assay and reverse-transcription polymerase chain reaction (RT-PCR) were used for SARS-CoV-2 N protein detection and RNA quantitation and in plasma, respectively. Serum levels of inflammatory and tissue-damage biomarkers in paired specimens were measured. SARS-CoV-RNA N-antigenemia and viral RNAemia were documented in 40.1% and 35.6% of patients, respectively at a median of 9 days since symptoms onset. The level of agreement between the qualitative results returned by the N-antigenemia assay and plasma RT-PCR was moderate (k = 0.57; p < 0.0001). A trend towards higher SARS-CoV-2 RNA loads was seen in plasma specimens testing positive for N-antigenemia assay than in those yielding negative results (p = 0.083). SARS-CoV-2 RNA load in tracheal aspirates was significantly higher (p < 0.001) in the presence of concomitant N-antigenemia than in its absence. Significantly higher serum levels of ferritin, lactose dehydrogenase, C-reactive protein, and D-dimer were quantified in paired plasma SARS-CoV-2 N-positive specimens than in those testing negative. Occurrence of SARS-CoV-2 N-antigenemia was not associated with increased mortality in univariate logistic regression analysis (odds ratio, 1.29; 95% confidence interval, 0.49-3.34; p = 0.59). In conclusion, SARS-CoV-2 N-antigenemia detection is relatively common in ICU patients and appears to associate with increased serum levels of inflammation and tissue-damage markers. Whether this virological parameter may behave as a biomarker of poor clinical outcome awaits further investigations.

What is the true place of the SARS-CoV-2 rapid point-of-care antigen test in the hospital setting? Lessons learned from real life.

To assist in the clinical management of patients and to support infection control, we tested the use of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) point-of-care antigen test (AgPOC) for unplanned hospitalization, coupled with a nucleic acid amplification test (NAAT) using specimens collected at the same time upon arrival. The aim of this study was to assess the performance of the AgPOC in this specific use compared to NAAT for SARS-CoV-2 diagnosis, in the context of the low prevalence of infection. For 5 months (between two peaks in France of the SARS-CoV-2 pandemic), all patients admitted who undertook the AgPOC/NAAT paired tests were included in the study. AgPOC performances were determined considering the clinical status and the delay of symptoms onset. NAAT and AgPOC results were available for 4425 subjects. AgPOC results showed a homogeneous specificity (>97%) but a low sensitivity at 45.8%. Considering the national guidelines, sensitivity dropped to 32.5% in cases of symptomatic patients with symptoms older than 5 days or more. This study shows the poor performance of AgPOC for entry screening of patients in hospitals. AgPOC may represent a useful tool in the hospital setting only if the use is restricted to patients with consistent symptoms less than 4 days old.

Responses to a Neutralizing Monoclonal Antibody for Hospitalized Patients With COVID-19 According to Baseline Antibody and Antigen Levels : A Randomized Controlled Trial.

BACKGROUND: In a randomized, placebo-controlled, clinical trial, bamlanivimab, a SARS-CoV-2-neutralizing monoclonal antibody, given in combination with remdesivir, did not improve outcomes among hospitalized patients with COVID-19 based on an early futility assessment. OBJECTIVE: To evaluate the a priori hypothesis that bamlanivimab has greater benefit in patients without detectable levels of endogenous neutralizing antibody (nAb) at study entry than in those with antibodies, especially if viral levels are high. DESIGN: Randomized, placebo-controlled trial. (ClinicalTrials.gov: NCT04501978). SETTING: Multicenter trial. PATIENTS: Hospitalized patients with COVID-19 without end-organ failure. INTERVENTION: Bamlanivimab (7000 mg) or placebo. MEASUREMENTS: Antibody, antigen, and viral RNA levels were centrally measured on stored specimens collected at baseline. Patients were followed for 90 days for sustained recovery (defined as discharge to home and remaining home for 14 consecutive days) and a composite safety outcome (death, serious adverse events, organ failure, or serious infections). RESULTS: Among 314 participants (163 receiving bamlanivimab and 151 placebo), the median time to sustained recovery was 19 days and did not differ between the bamlanivimab and placebo groups (subhazard ratio [sHR], 0.99 [95% CI, 0.79 to 1.22]; sHR > 1 favors bamlanivimab). At entry, 50% evidenced production of anti-spike nAbs; 50% had SARS-CoV-2 nucleocapsid plasma antigen levels of at least 1000 ng/L. Among those without and with nAbs at study entry, the sHRs were 1.24 (CI, 0.90 to 1.70) and 0.74 (CI, 0.54 to 1.00), respectively (nominal P for interaction = 0.018). The sHR (bamlanivimab vs. placebo) was also more than 1 for those with plasma antigen or nasal viral RNA levels above median level at entry and was greatest for those without antibodies and with elevated levels of antigen (sHR, 1.48 [CI, 0.99 to 2.23]) or viral RNA (sHR, 1.89 [CI, 1.23 to 2.91]). Hazard ratios for the composite safety outcome (<1 favors bamlanivimab) also differed by serostatus at entry: 0.67 (CI, 0.37 to 1.20) for those without and 1.79 (CI, 0.92 to 3.48) for those with nAbs. LIMITATION: Subgroup analysis of a trial prematurely stopped because of futility; small sample size; multiple subgroups analyzed. CONCLUSION: Efficacy and safety of bamlanivimab may differ depending on whether an endogenous nAb response has been mounted. The limited sample size of the study does not allow firm conclusions based on these findings, and further independent trials are required that assess other types of passive immune therapies in the same patient setting. PRIMARY FUNDING SOURCE: U.S. government Operation Warp Speed and National Institute of Allergy and Infectious Diseases.

Advances in laboratory detection methods and technology application of SARS-CoV-2.

At present, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is raging worldwide, and the coronavirus disease 2019 outbreak caused by SARS-CoV-2 seriously threatens the life and health of all humankind. There is no specific medicine for novel coronavirus yet. So, laboratory diagnoses of novel coronavirus as soon as possible and isolation of the source of infection play a vital role in preventing and controlling the epidemic. Therefore, selecting appropriate detection techniques and methods is particularly important to improve the efficiency of disease diagnosis and treatment and to curb the outbreak of infectious diseases. In this paper, virus nucleic acid, protein, and serum immunology were reviewed to provide a reference for further developing virus detection technology to provide better prevention and treatment strategies and research ideas for clinicians and researchers.

Development of a Novel Double Antibody Sandwich ELISA for Quantitative Detection of Porcine Deltacoronavirus Antigen.

Porcine deltacoronavirus (PDCoV) can cause diarrhea and dehydration in newborn piglets. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-ELISA) for detection of PDCoV by using a specific monoclonal antibody against the PDCoV N protein and an anti-PDCoV rabbit polyclonal antibody. Using DAS-ELISA, the detection limit of recombinant PDCoV N protein and virus titer were approximately 0.5 ng/mL and 103.0 TCID50/mL, respectively. A total of 59 intestinal and 205 fecal samples were screened for the presence of PDCoV by using DAS-ELISA and reverse transcriptase real-time PCR (RT-qPCR). The coincidence rate of the DAS-ELISA and RT-qPCR was 89.8%. DAS-ELISA had a sensitivity of 80.8% and specificity of 95.6%. More importantly, the DAS-ELISA could detect the antigen of PDCoV inactivated virus, and the viral antigen concentrations remained unchanged in the inactivated virus. These results suggest that DAS-ELISA could be used for antigen detection of clinical samples and inactivated vaccines. It is a novel method for detecting PDCoV infections and evaluating the PDCoV vaccine.

Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.