Detection Of Viral Nucleic Acids Research Articles

Pediatric Endoscopy During the COVID-19 Pandemic: Addressing the Implications of Universal Preprocedural Testing for PPE Utilization.

Pediatric Endoscopy During the COVID-19 Pandemic: Addressing the Implications of Universal Preprocedural Testing for PPE Utilization.

A water-soluble membrane for SARS-CoV-2 viral nucleic acid sampling and detection.

This communication describes a novel water-soluble membrane prepared from chitosan intended for SARS-CoV-2 viral nucleic acid collection and detection. The CSH membrane formed from nanofibers shows promising potential in the quantitative determination of the SARS-CoV-2 viral nucleic acids at a concentration of 102 copies per L in air. The sponge-like structure which allows gas to pass through for collection of viral nucleic acids potentially provides simple, fast, and reliable sampling as well as detection of various types of airborne viruses.

Overview of SARS-CoV-2 viral load and its clinical value

During the unprecedented COVID-19 pandemic, detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral nucleic acids in diverse specimens from patients, people with suspected infection and their close contacts has been widely implemented in infection confirmation, patient management and transmission prevention. Viral nucleic acid detection results not only reflect the infection status in suspected cases, but also are used to quantify positive specimens in most assays. For most viral diseases, viral loads are theoretically associated with potent infectivity and prognostic outcomes in patients. In this review, the principles and characteristics of SARS-CoV-2 viral load detected by real time reverse transcription PCR (RT-PCR) and digital PCR are summarized. The distribution and dynamic changes in viral load in humans are discussed, and the correlations of viral load with disease severity, infectivity and prognostic indicators are analyzed. The comprehensive descriptions of COVID-19 characterization will help guide current practices in infection control, patient treatment and management, and combating the ongoing COVID-19 pandemic.

Swine influenza: Newer data on diagnosis, interpretation of the results, and control

Influenza viruses are among the major causes of acute respiratory disease outbreaks in pigs. In most cases, infections are subclinical. Until 2009, three subtypes of IAV-S circulated in the pig population in Europe, with some geographical restrictions regarding their prevalence: avian-like (av) H1N1, reassortant (r) H3N2, and human (hu) H1N2. Viruses of the H1N1av lineage appeared to be responsible for the majority of swine infections in Europe. In 2009, a fourth subtype entered the pig population: the human pandemic H1N1 2009 influenza A virus (H1N1pdm). Due to the expression of receptors with α-2-6 or α-2-3-linked terminal sialic acids in the porcine upper respiratory tract, swine appear to be susceptible to influenza A viruses of both avian and human origin. A clinical diagnosis of swine influenza is not easy, since there are no observable pathognomonic clinical signs, and the disease must be distinguished from a variety of other respiratory conditions in pigs. A final diagnosis can be made by the following methods: detection of viral proteins or nucleic acid, isolation of virus, or demonstration of virus-specific antibodies. IAV-S is most likely to be found in nasal and pharyngeal secretions during the fever period of illness. Serological tests are used to demonstrate the presence of influenza-specific antibodies. Serology is the most useful technique to determine the immune status of the herd, to assess the levels of maternally derived antibodies in young piglets and their profile, as well as post-vaccination antibody titers, and to perform pre-movement testing of pigs. The interpretation of serological data is often complex and may be further confounded by concurrent circulation of different virus subtypes and gene lineages. In control of IAV-S, vaccination appears to be the primary tool for preventing influenza. The efficacy of vaccination may be various and is correlated with homology between vaccine and field IAV-S strains. There is no treatment available for IAV-S. The administration of aspirin via the water system or of paracetamol in feed may play a role as a support therapy. To avoid subsequent bacterial infections, treatment with an antibiotic is essential.

Droplet Microarray Based on Nanosensing Probe Patterns for Simultaneous Detection of Multiple HIV Retroviral Nucleic Acids.

Multiplexed detection of viral nucleic acids is important for rapid screening of viral infection. In this study, we present a molybdenum disulfide (MoS2) nanosheet-modified dendrimer droplet microarray (DMA) for rapid and sensitive detection of retroviral nucleic acids of human immunodeficiency virus-1 (HIV-1) and human immunodeficiency virus-2 (HIV-2) simultaneously. The DMA platform was fabricated by omniphobic–omniphilic patterning on a surface-grafted dendrimer substrate. Functionalized MoS2 nanosheets modified with fluorescent dye-labeled oligomer probes were prepatterned on positively charged amino-modified omniphilic spots to form a fluorescence resonance energy transfer (FRET) sensing microarray. With the formation of separated microdroplets of sample on the hydrophobic–hydrophilic micropattern, prepatterned oligomer probes specifically hybridized with the target HIV genes and detached from the MoS2 nanosheet surface due to weakening of the adsorption force, leading to fluorescence signal recovery. As a proof of concept, we used this microarray with a small sample size (<150 nL) for simultaneous detection of HIV-1 and HIV-2 nucleic acids with a limit of detection (LOD) of 50 pM. The multiplex detection capability was further demonstrated for simultaneous detection of five viral genes (HIV-1, HIV-2, ORFlab, and N genes of SARS-COV-2 and M gene of Influenza A). This work demonstrated the potential of this novel MoS2-DMA FRET sensing platform for high-throughput multiplexed viral nucleic acid screening.

Spontaneous Pneumothorax: A Complication of Coronavirus Disease 2019 (COVID-19) Patients

At present, the coronavirus disease 2019 (COVID-19) is spreading all over the world. The occurrence of spontaneous pneumothorax in these patients might be higher than the fact, and we should pay high clinical attention to them. Data regarding clinical investigation, laboratory investigation, diagnosis, and treatment measures of 21 COVID-19 patients with spontaneous pneumothorax from January to March of 2020 were collected and analyzed in this study. Seven patients had a history of basic lung diseases. All patients used different methods of oxygen therapy before the occurrence of spontaneous pneumothorax according to the severity of the COVID-19, including 18 patients with ventilator-assisted breathing, 2 patients with bilevel positive airway pressure assisted breathing, and 1 patient with mask oxygen inhalation. All patients were confirmed cases of COVID-19 by chest CT (computed tomography) and virus nucleic acid detection and were found to have spontaneous pneumothorax through physical examination, bedside X-ray, and/or bedside ultrasound. 13 of 21 patients combined with pleural effusion at the same time. All the patients underwent closed thoracic drainage for spontaneous pneumothorax and the pleural effusion, if any. Nine patients died, and 12 patients recovered smoothly. Spontaneous pneumothorax might be an overlooked complication of COVID-19 patients and may be associated with poor prognosis.

Abstract 16323: Ruling Out Sars-cov-2 Infection With a Fast-track 0/1,5-hour Molecular Testing in Patients With Acute Coronary Syndromes

Introduction: Timely reperfusion with PCI remains the main goal in STEMI patients. In the Covid 19 period, the fear of contagion reduced the number of hospitalized patients for myocardial infarction, and a substantial delay in the hospitalizations was also observed. The fear of the operators to treat non-tested patients did not facilitate the hospitalization and treatment of ACS patients with suspected Covid-19. Hypothesis: This study aimed to evaluate the feasibility of a fast-tract protocol to rule out SARS-CoV-2 infection in patients with suspected acute coronary syndrome. Methods: In fifty-one patients (mean age 65 ± 12 y) the real-time PCR to extract RNA for SARS-CoV-2 detection was performed with an automated FDA-approved molecular test (Xpert Xpress Cepheid, Sunnyvale, USA). With its 45-minute processing time and with very high sensitivity, this system was tested in patients transferred from spokes centers or from the field to our HUB center. As soon as the patient had arrived in the Cath Lab, the interventional cardiologist, with adequate protection systems, performed the nasopharyngeal swab that was processed with the rapid Cepheid system. In the meanwhile, coronary angiography (CA) or PCI were performed. At the end of the CA/PCI, based on the test response, patients were transferred to the Covid or non Covid areas. Results: The time to perform the nasopharyngeal swab in the cath lab was: 10 + 10 minutes. The time spent to transport the nasopharyngeal test to the laboratory was: 30 + 20 minutes. The time to load 300 ul of biological material and extraction, amplification, and detection of viral nucleic acid, targeting the viral genomic regions N2 and E using the rapid test was 68 + 15 minutes. The result of the test was immediately made readily available through the hospital computer system. The total time from the execution of the nasopharyngeal swab to the result was 109 ± 26 minutes. Conclusions: This study demonstrates the feasibility of a fast-track protocol with a rapid test to ruling out SARS-CoV-2 Infection in ACS. This pathway allows quick patient stratification and it could of particular importance to detect asymptomatic Covid-19 positive patients with ACS.

One-step rapid quantification of SARS-CoV-2 virus particles via low-cost nanoplasmonic sensors in generic microplate reader and point-of-care device

The spread of SARS-CoV-2 virus in the ongoing global pandemic has led to infections of millions of people and losses of many lives. The rapid, accurate and convenient SARS-CoV-2 virus detection is crucial for controlling and stopping the pandemic. Diagnosis of patients in the early stage infection are so far limited to viral nucleic acid or antigen detection in human nasopharyngeal swab or saliva samples. Here we developed a method for rapid and direct optical measurement of SARS-CoV-2 virus particles in one step nearly without any sample preparation using a spike protein specific nanoplasmonic resonance sensor. As low as 370 vp/mL were detected in one step within 15 min and the virus concentration can be quantified linearly in the range of 0 to 107 vp/mL. Measurements shown on both generic microplate reader and a handheld smartphone connected device suggest that our low-cost and rapid detection method may be adopted quickly under both regular clinical environment and resource-limited settings.

Seroprevalence of SARS-CoV-2 immunoglobulin antibodies in Wuhan, China: part of the city-wide massive testing campaign

ObjectivesThe outbreak of the 2019 coronavirus disease (COVID-19) pandemic in Wuhan, China, has subsided after being hard hit by the disease and subsequent city lockdown. Information on the number of people involved in Wuhan is still inadequate. This study aimed to describe the screening results of 61 437 community members in Wuchang District, Wuhan. MethodsIn mid-May 2020, Wuhan launched a population-scale city-wide SARS-CoV-2 testing campaign, which aimed to perform nucleic acid and viral antibody testing for citizens in Wuhan. Here we show the screening results of cluster sampling of 61 437 residents in Wuchang District, Wuhan, China. ResultsA total of 1470 (2.39%, 95% CI 2.27–2.52) individuals were detected positive for at least one antiviral antibody. Among the positive individuals, 324 (0.53%, 95% CI 0.47–0.59) and 1200 (1.95%, 95% CI 1.85–2.07) were positive for immunoglobulin IgM and IgG, respectively, and 54 (0.08%, 95% CI 0.07–0.12) were positive for both antibodies. The positive rate of female carriers of antibodies was higher than those of male counterparts (male-to-female ratio of 0.75), especially in elderly citizens (ratio of 0.18 in 90+ age subgroup), indicating a sexual discrepancy in seroprevalence. In addition, viral nucleic acid detection using real-time PCR had showed 8 (0.013%, 95% CI 0.006–0.026) asymptomatic virus carriers. DiscussionThe seroprevalence of SARS-CoV-2 in Wuhan was low. Most Wuhan residents are still susceptible to this virus. Precautions, such as wearing mask, frequent hand hygiene and proper social distance, are necessary before an effective vaccine or antiviral treatments are available.

The application of viral nucleic acid detection and lung CT in COVID-19 diagnosis

The Corona Virus Disease 2019 (COVID-19) has the characteristics of fast propagation speed and strong pathogenicity and has attracted wide attention of people, medical workers, and researchers around the world. Accurate, rapid, and timely screening and diagnosis of COVID-19 is of great significance to control the development of the epidemic situation and save the lives of patients. Currently, the detection of viral nucleic acid and lung CT is the main screening and diagnostic methods of COVID-19. Nucleic acid detection has the advantages of fast, strong specificity and high sensitivity, but there is a certain false-negative rate. CT result of lung examination is visual, but it is not typical due to the uncertain time of clinical symptoms and the early medical intervention. Therefore, the diagnosis of COVID-19 should include a combination of epidemiological history, clinical symptoms, imaging, and laboratory tests.

Clinical Challenges in an Outbreak of COVID-19 in Wuhan, China: Concerns from Frontiers.

Purpose of ReviewIn December 2019, outbreaks of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infected pneumonia patients were discovered in Wuhan City, Hubei Province, China. With the spread of the epidemic, new cases have been found in other regions of China and abroad. This review summarizes the current coronavirus disease 2019 (COVID-19) transmission pathways, high-risk factors, diagnostic points, pathogenesis, and therapeutic drugs to provide the prevention and treatment theories for COVID-19.Recent FindingsCOVID-19 is highly contagious and is transmitted mainly through the respiratory tract. Most patients with SARS-CoV-2 infection have milder symptoms. Some patients will have mild disease in the early stage, suddenly exacerbate later, and eventually die of multiple organ failure caused by cytokine storm. The basis for clinical diagnosis of COVID-19 is not just the detection of viral nucleic acids as the gold standard. The diagnostic accuracy improves when viral nucleic acid test is combined with clinical symptoms, CT results, and biochemical tests. For the treatment of COVID-19, the specific antiviral agents have not been developed, except for symptomatic supportive treatments.SummaryThe methods for detecting SARS-CoV-2 infection have become increasingly mature, but specific antiviral drugs for the treatment of COVID-19 have not yet been developed. Treatment must pay attention to the cytokine storm that leads to the seriousness of COVID-19.

Characteristics and roles of severe acute respiratory syndrome coronavirus 2-specific antibodies in patients with different severities of coronavirus 19.

The diagnosis of coronavirus 19 (COVID-19) relies mainly upon viral nucleic acid detection, but false negatives can lead to missed diagnosis and misdiagnosis;severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific antibody detection is convenient, safe and highly sensitive. Immunoglobulin (Ig)M and IgG are commonly used to serologically diagnose COVID-19; however, the role of IgA is not well known. We aimed to quantify the levels of SARS-CoV-2-specific IgM, IgA and IgG antibodies, identify changes in them based on COVID-19 severity, and establish the significance of combined antibody detection. COVID-19 patients, divided into a severe and critical group and a moderate group, and non-COVID-19 patients with respiratory disease were included in this study. A chemiluminescence method was used to detect the levels of SARS-CoV-2-specific IgM, IgA and IgG in the blood samples from the three groups. Epidemiological characteristics, symptoms, blood test results and other data were recorded for all patients. Compared to the traditional IgM-IgG combined antibodies, IgA-IgG combined antibodies are more effective for diagnosing COVID-19. During the disease process, IgA appeared first and disappeared last. All three antibodies had significantly higher levels in COVID-19 patients than in non-COVID-19 patients. IgA and IgG were also higher for severe and critical disease than for moderate disease. All antibodies were at or near low levels at the time of tracheal extubation in critical patients. Detection of SARS-CoV-2-specific combined IgA-IgG antibodies is advantageous in diagnosing COVID-19. IgA detection is suitable during early and late stages of the disease. IgA and IgG levels correspond to disease severity.

Capacity Evaluation of Diagnostic Tests For COVID-19 Using Multicriteria Decision-Making Techniques.

In December 2019, cases of pneumonia were detected in Wuhan, China, which were caused by the highly contagious coronavirus. This study is aimed at comparing the confusion regarding the selection of effective diagnostic methods to make a mutual comparison among existing SARS-CoV-2 diagnostic tests and at determining the most effective one. Based on available published evidence and clinical practice, diagnostic tests of coronavirus disease (COVID-19) were evaluated by multi-criteria decision-making (MCDM) methods, namely, fuzzy preference ranking organization method for enrichment evaluation (fuzzy PROMETHEE) and fuzzy technique for order of preference by similarity to ideal solution (fuzzy TOPSIS). Computerized tomography of chest (chest CT), the detection of viral nucleic acid by polymerase chain reaction, cell culture, CoV-19 antigen detection, CoV-19 antibody IgM, CoV-19 antibody IgG, and chest X-ray were evaluated by linguistic fuzzy scale to compare among the diagnostic tests. This scale consists of selected parameters that possessed different weights which were determined by the experts' opinions of the field. The results of our study with both proposed MCDM methods indicated that the most effective diagnosis method of COVID-19 was chest CT. It is interesting to note that the methods that are consistently used in the diagnosis of viral diseases were ranked in second place for the diagnosis of COVID-19. However, each country should use appropriate diagnostic solutions according to its own resources. Our findings also show which diagnostic systems can be used in combination.

Clinical features of coronavirus disease 2019 in children: a systemic review of severe acute respiratory syndrome, Middle East respiratory syndrome, and coronavirus disease 2019

To systematically summarize the clinical features of coronavirus disease 2019 (COVID-19) in children. PubMed, Embase, Web of Science, The Cochrane Library, CNKI, Weipu Database, and Wanfang Database were searched for clinical studies on COVID-19 in children published up to May 21, 2020. Two reviewers independently screened the articles, extracted data, and assessed the risk of bias of the studies included. A descriptive analysis was then performed for the studies. Related indices between children with COVID-19 and severe acute respiratory syndromes (SARS) or Middle East respiratory syndrome (MERS) were compared. A total of 75 studies were included, with a total of 806 children with COVID-19. The research results showed that the age of the children ranged from 36 hours after birth to 18 years, with a male-female ratio of 1.21 : 1. Similar to SARS and MERS, COVID-19 often occurred with familial aggregation, and such cases accounted for 74.6% (601/806). The children with COVID-19, SARS, and MERS had similar clinical symptoms, mainly fever and cough. Some children had gastrointestinal symptoms. The children with asymptomatic infection accounted for 17.9% (144/806) of COVID-19 cases, 2.5% (2/81) of SARS cases, and 57.1% (12/21) of MERS cases. The children with COVID-19 and MERS mainly had bilateral lesions on chest imaging examination, with a positive rate of lesions of 63.4% (421/664) and 26.3% (5/19) respectively, which were lower than the corresponding positive rates of viral nucleic acid detection, which were 99.8% and 100% respectively. The chest radiological examination of the children with SARS mainly showed unilateral lesion, with a positive rate of imaging of 88.9% (72/81), which was higher than the corresponding positive rate of viral nucleic acid detection (29.2%). Viral nucleic acid was detected in the feces of children with COVID-19 or SARS, with positive rates of 60.2% (56/93) and 71.4% (5/7) respectively. The children with COVID-19 had a rate of severe disease of 4.6% (31/686) and a mortality rate of 0.1% (1/806), the children with SARS had a rate of severe disease of 1.5% (1/68) and a mortality rate of 0%, and those with MERS had a rate of severe disease of 14.3% (3/21) and a mortality rate of 9.5% (2/21). Children with COVID-19 have similar symptoms to those with SARS or MERS, mainly fever and cough. Asymptomatic infection is observed in all three diseases. Children with COVID-19 or SARS have milder disease conditions than those with MERS. COVID-19 in children often occurs with familial aggregation. Epidemiological contact history, imaging examination findings, and viral nucleic acid testing results are important bases for the diagnosis of COVID-19.

Development of two TaqMan real-time reverse transcription-PCR assays for the detection of severe acute respiratory syndrome coronavirus-2

The outbreak of coronavirus disease 2019 (COVID-19) in Wuhan, China, was caused by a novel coronavirus (CoV), named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The rapid detection of viral nucleic acids is critical for the early identification of infected cases. We have developed two TaqMan real-time reverse transcription-PCR assays to detect SARS-CoV-2. The designed primers target the nucleocapsid (N) and open reading frame (ORF) 1b gene regions, where the probes discriminate SARS-CoV-2 from other human and animal CoVs. The sensitivities are one genomic copy per reaction for the N gene assay and ten copies for the ORF 1b gene assay. The overall linear detection ranges are 1–106 and 10–106 copies per reaction for the N gene assay and the ORF 1b gene assay, respectively. Surveillance of 23 suspected COVID-19 patients demonstrated that SARS-CoV-2 could be detected from 100% (23/23) and 62.5% (16/23) of clinical specimens by the N gene assay and the ORF 1b gene assay, respectively. All of the samples not detected by the ORF 1b gene assay were throat swabs, indicating a lower viral load in the upper respiratory tract and the relatively lower sensitivity of the ORF 1b gene assay. The assays developed in the present study offer alternative diagnostic tests for COVID-19.

Noninvasive strategies for surveillance of swine viral diseases: a review.

In view of the intensive development of the swine industry, monitoring and surveillance of infectious diseases require low-cost, effective, and representative population sampling methods. We present herein the state of knowledge, to date, in the use of alternative strategies in the monitoring of swine health. Blood sampling, the most commonly used method in veterinary medicine to obtain samples for monitoring swine health, is labor-intensive and expensive, which has resulted in a search for alternative sampling strategies. Oral fluid (OF) is a good alternative to serum for pooled sample analysis, especially for low-prevalence pathogens. Detection of viral nucleic acids or antiviral antibodies in OF is used to detect numerous viruses in the swine population. Meat juice is used as an alternative to serum in serologic testing. Processing fluid obtained during processing of piglets (castration and tail-docking) may also be used to detect viruses. These matrices are simple, safe, cost-effective, and allow testing of many individuals at the same time. The latest methods, such as snout swabs and udder skin wipes, are also promising. These alternative samples are easy to acquire, and do not affect animal welfare negatively.

Efficacy and Safety of First-Line Therapeutic Drugs with Potential for the Treatment of Novel Coronavirus Pneumonia: A Meta-Analysis

Background: Novel coronavirus pneumonia is spreading all over the world. While countries are actively dealing with the epidemic situation, the lack of specific drugs also indirectly makes the situation hard to control. At present, the internationally controversial first-line treatment drugs cover Hydroxychloroquine, Favipiravir, Remdesivir and so on. Methods: To evaluate the efficacy and safety of first-line treatment drugs for novel coronavirus pneumonia, this study investigated the turning-negative rate in the viral nucleic acid, survival rate and adverse reactions rate of novel coronavirus pneumonia patients under treated with Hydroxychloroquine, Azithromycin, Favipiravir and Remdesivir by performing meta-analysis. Findings: The result showed that hydroxychloroquine could not improve the turning-negative rate in the viral nucleic acid detection of novel coronavirus pneumonia patients compared with conventional therapy (RR=0·925, 95% CI (0·685, 1·250), P=0·61), and at the same time, it also associated with high adverse reactions rate up to 21·6%. Hydroxychloroquine combined with azithromycin can significantly increase the turning-negative rate to almost 62%, but patients’ survival rate is reduced for serious adverse reaction. The efficacy of favipiravir is stable (RR=2·70, 95% CI (1·25, 5·85), P=0·012) and its adverse reaction is under control (ES=6·10%, 95% CI (0·000, 0·121)). Remdesivir shows its therapeutic potential, but the high rate of adverse reactions (ES=58·20%, 95% CI (0·316, 0·848)) is also worthy of attention. More high-quality clinical trial data is required to investigate the efficacy and safety of first-line treatment drugs in further research. Interpretation: In summary, this article found that the clinical medication regimen for patients with mild and common COVID-19 is hydroxychloroquine 400mg/day or favipiravir 1200mg/day, and could be appropriately combined with conventional treatment, such as oxygen theraphy and carrimyclin, abidol, etc. For patients with severe COVID-19, daily use of remdesivir 100~200mg has the best effect, the treatment of hydroxychloroquine combined with azithromycin is regarded as the a last resort because of its severe adverse reaction and it can be combined with oxygen therapy together with other antibacterial drugs. And we suggest FDA to approve the favipiravir to use for patients and decrease the use of hydroxychloroquine combined with azithromycin. Funding Statement: This research work was supported by the National Natural Science Foundation of China (Grant No. 81973180 and No. 81673298), the Natural Science Foundation of the Jiangsu Higher Education Institutions of China for Excellent Young Talents (Grant No. BK20180077) and the Drug Innovation Major Project (2019ZX09301-142.) Declaration of Interests: The authors declare no competing interests.

Laboratory Testing Methods for Novel Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2).

Following the first reports of coronavirus disease-19 (COVID-19) by China to the World Health Organization (WHO) on 31st December 2019, more than 4,302,774 novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cases have been reported by authorities in 212 countries and territories by 12th May 2020. The outbreak and spread of COVID-19 worldwide, highlights the critical need for developing rapid and accurate diagnostic testing methods for emerging human coronavirus (CoV) infections. Testing is crucial to track the spread of disease during a pandemic, and to swiftly permit public health interventions including isolation, quarantine, and appropriate clinical management of afflicted individuals. The key components of viral diagnostic tests are (1) collection of the appropriate sample (blood, nasal swab, and throat swab), (2) availability of the genetic and proteomic sequences of the novel virus for analysis, and (3) rapid and accurate laboratory testing methods. The current gold standard for the molecular diagnosis of SARS-CoV-2 infection is the real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the qualitative and quantitative detection of viral nucleic acids. Other relevant laboratory methods include enzyme-linked immunoassays (EIA) for viral antibody and antigen detection, and serum viral neutralization (SVN) assays for antibody neutralization determination. The challenges faced in developing a diagnostic test for a novel pathogen are the ability to measure low viral loads for early detection, to provide low or no cross-reactivity with other viral strains and to deliver results rapidly. Several point-of-care molecular devices are currently being integrated for fast and accurate diagnosis of SARS-CoV-2 infections. This review discusses the current laboratory methods available to test for coronaviruses by focusing on the present COVID-19 outbreak.

Effect of Jinhua Qinggan granules on novel coronavirus pneumonia in patients.

To evaluate the effectiveness and safety of Jinhua Qinggan granules in the treatment of patients with novel coronavirus pneumonia (COVID-19). Eighty cases of COVID-19 diagnosed from January 24 to February 17, 2020 in Beijing YouAn Hospital Affiliated to Capital Medical University were retrospectively analyzed. All 80 patients received symptomatic and supportive treatment. Among them, 44 patients took Jinhua Qinggan granules (treatment group) within 24 h of admission, and the remaining 36 patients either did not take Jinhua Qinggan granules or took the granules for less than 2 d (control group). In this study, we compared the duration of viral nucleic acid detection and of pneumonia absorption improvement between the two groups. Among the 80 cases, 37 were male (46%) and 43 were female (54%) with age ranging from 15 to 86 years, with an average age of 51.19 years. The average duration of viral nucleic acid detection was (7 ± 4) d in the Jinhua Qinggan administration group and (10 ± 4) d for the control group (P = 0.010), following which, nucleic acid tests were negative. Of the two groups, 56.82% in the Jinhua Qinggan treatment group and 27.78% in the control group demonstrated negative nucleic acid tests within 7 d or less. The 7-day viral clearance rate was significantly higher in the Jinhua Qinggan group compared with the control group (P = 0.009). Furthermore, the pneumonia recovery time indicated by chest CT was (8 ± 4) d in the Jinhua Qinggan group, which was significantly shorter than the control group, at (10 ± 5) d (P = 0.021). No adverse reactions were found in the treatment group after taking this medicine. In patients with COVID-19, Jinhua Qinggan granules can effectively shorten the duration of nucleic acid detection and promote the absorption of pneumonia inflammatory exudate without obvious adverse reactions.

Two Detailed Plaque Assay Protocols for the Quantification of Infectious SARS-CoV-2.

Severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) has been identified as the causal agent of COronaVIrus Disease‐19 (COVID‐19), an atypical pneumonia‐like syndrome that emerged in December 2019. While SARS‐CoV‐2 titers can be measured by detection of viral nucleic acid, this method is unable to quantitate infectious virions. Measurement of infectious SARS‐CoV‐2 can be achieved by tissue culture infectious dose−50 (TCID50), which detects the presence or absence of cytopathic effect in cells infected with serial dilutions of a virus specimen. However, this method only provides a qualitative infectious virus titer. Plaque assays are a quantitative method of measuring infectious SARS‐CoV‐2 by quantifying the plaques formed in cell culture upon infection with serial dilutions of a virus specimen. As such, plaque assays remain the gold standard in quantifying concentrations of replication‐competent lytic virions. Here, we describe two detailed plaque assay protocols to quantify infectious SARS‐CoV‐2 using different overlay and staining methods. Both methods have several advantages and disadvantages, which can be considered when choosing the procedure best suited for each laboratory. These assays can be used for several research purposes, including titration of virus stocks produced from infected cell supernatant and, with further optimization, quantification of SARS‐CoV‐2 in specimens collected from infected animals. © 2019 The Authors. Basic Protocol: SARS‐CoV‐2 plaque assay using a solid double overlay method Alternate Protocol: SARS‐CoV‐2 plaque assay using a liquid overlay and fixation‐staining method