Detection Of Viral Nucleic Acids Research Articles

A direct capture method for purification and detection of viral nucleic acid enables epidemiological surveillance of SARS-CoV-2

Studies have demonstrated that SARS-CoV-2 RNA can be detected in the feces of infected individuals. This finding spurred investigation into using wastewater-based epidemiology (WBE) to monitor SARS-CoV-2 RNA and track the appearance and spread of COVID-19 in communities. SARS-CoV-2 is present at low levels in wastewater, making sample concentration a prerequisite for sensitive detection and utility in WBE. Whereas common methods for isolating viral genetic material are biased toward intact virus isolation, it is likely that a relatively low percentage of the total SARS-CoV-2 RNA genome in wastewater is contained within intact virions. Therefore, we hypothesized that a direct unbiased total nucleic acid(TNA) extraction method could overcome the cumbersome protocols, variability and low recovery rates associated with the former methods. This led to development of a simple, rapid, and modular alternative to existing purification methods. In an initial concentration step, chaotropic agents are added to raw sewage allowing binding of nucleic acid from free nucleoprotein complexes, partially intact, and intact virions to a silica matrix. The eluted nucleic acid is then purified using manual or semi-automated methods. RT-qPCR enzyme mixes were formulated that demonstrate substantial inhibitor resistance. In addition, multiplexed probe-based RT-qPCR assays detecting the N1, N2 (nucleocapsid) and E (envelope) gene fragments of SARS-CoV-2 were developed. The RT-qPCR assays also contain primers and probes to detect Pepper Mild Mottle Virus (PMMoV), a fecal indicator RNA virus present in wastewater, and an exogenous control RNA to measure effects of RT-qPCR inhibitors. Using this workflow, we monitored wastewater samples from three wastewater treatment plants (WWTP) in Dane County, Wisconsin. We also successfully sequenced a subset of samples to ensure compatibility with a SARS-CoV-2 amplicon panel and demonstrated the potential for SARS-CoV-2 variant detection. Data obtained here underscore the potential for wastewater surveillance of SARS-CoV-2 and other infectious agents in communities.

Detection of African swine fever virus in feed dust collected from experimentally inoculated complete feed using quantitative PCR and virus titration assays.

African swine fever virus (ASFV) is a current threat to global pork production due to its high case fatality rate, lack of efficacious vaccine and recent transboundary spread into new regions of the world. Preventing introduction and further spread of ASFV is critical for countries currently negative for the virus. ASFV is stable in feed ingredients subjected to transoceanic conditions and transmission occurs through the natural consumption of contaminated feed. In this study, we investigated the use of feed dust collected from experimentally inoculated feed as a novel diagnostic sample type for ASFV detection. Moist swabs were used to collect dust from creep feeders after natural consumption of feed inoculated with 3.1-5.4 log10 TCID50 /g ASFV Georgia 2007 in the presence and absence of antimicrobial feed additives. Results validate the potential use of feed dust swabs as a novel diagnostic surveillance tool for detection and quantification of viral nucleic acid and infectious virus titre in ASFV-contaminated feed.

Emerging point-of-care biosensors for rapid diagnosis of COVID-19: current progress, challenges, and future prospects.

Coronavirus disease 2019 (COVID-19) pandemic is currently a serious global health threat. While conventional laboratory tests such as quantitative real-time polymerase chain reaction (qPCR), serology tests, and chest computerized tomography (CT) scan allow diagnosis of COVID-19, these tests are time-consuming and laborious, and are limited in resource-limited settings or developing countries. Point-of-care (POC) biosensors such as chip-based and paper-based biosensors are typically rapid, portable, cost-effective, and user-friendly, which can be used for COVID-19 in remote settings. The escalating demand for rapid diagnosis of COVID-19 presents a strong need for a timely and comprehensive review on the POC biosensors for COVID-19 that meet ASSURED criteria: Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable to end users. In the present review, we discuss the importance of rapid and early diagnosis of COVID-19 and pathogenesis of COVID-19 along with the key diagnostic biomarkers. We critically review the most recent advances in POC biosensors which show great promise for the detection of COVID-19 based on three main categories: chip-based biosensors, paper-based biosensors, and other biosensors. We subsequently discuss the key benefits of these biosensors and their use for the detection of antigen, antibody, and viral nucleic acids. The commercial POC biosensors for COVID-19 are critically compared. Finally, we discuss the key challenges and future perspectives of developing emerging POC biosensors for COVID-19. This review would be very useful for guiding strategies for developing and commercializing rapid POC tests to manage the spread of infections.Graphical abstract

Low saliva pH can yield false positives results in simple RT-LAMP-based SARS-CoV-2 diagnostic tests.

Diagnosis of any infectious disease is vital for opportune treatment and to prevent dissemination. RT-qPCR tests for detection of SARS-CoV-2, the causative agent for COVID-19, are ideal in a hospital environment. However, mass testing requires cheaper and simpler tests, especially in settings that lack sophisticated machinery. The most common current diagnostic method is based on nasopharyngeal sample collection, RNA extraction, and RT-qPCR for amplification and detection of viral nucleic acids. Here, we show that samples obtained from nasopharyngeal swabs in VTM and in saliva can be used with or without RNA purification in an isothermal loop-mediated amplification (LAMP)-based assay, with 60–93% sensitivity for SARS-CoV-2 detection as compared to standard RT-qPCR tests. A series of simple modifications to standard RT-LAMP published methods to stabilize pH fluctuations due to salivary acidity resulted in a significant improvement in reliability, opening new avenues for efficient, low-cost testing of COVID-19 infection.

Ultrasensitive detection and quantification of viral nucleic acids with Raindance droplet digital PCR (ddPCR).

Sensitive detection of viral nucleic acids is critically important for diagnosis and monitoring of the progression of infectious diseases such as those caused by SARS-CoV2, HIV-1, and other viruses. In HIV-1 infection cases, assessing the efficacy of treatment interventions that are superimposed on combination antiretroviral therapy (cART) has benefited tremendously from the development of sensitive HIV-1 DNA and RNA quantitation assays. Simian immunodeficiency virus (SIV) infection of Rhesus macaques is similar in many key aspects to human HIV-1 infection and consequently this non-human primate (NHP) model has and continues to prove instrumental in evaluating HIV prevention, treatment and eradication approaches. Cell and tissue associated HIV-1 viral nucleic acids have been found to serve as useful predictors of disease outcome and indicators of treatment efficacy, highlighting the value of and the need for sensitive detection of viruses in cells/tissues from infected individuals or animal models. However, viral nucleic acid detection and quantitation in such sample sources can often be complicated by high nucleic acid input (that is required to detect ultralow level viruses in, for example, cure research) or inhibitors, leading to reduced detection sensitivity and under-quantification, and confounded result interpretation. Here, we present a step-by-step procedure to quantitatively recover cell/tissue associated viral DNA and RNA, using SIV-infected Rhesus macaque cells and tissues as model systems, and subsequently quantify the viral DNA and RNA with an ultrasensitive SIV droplet digital PCR (ddPCR) assay and reverse transcription ddPCR (RT-ddPCR) assay, respectively, on the Raindance ddPCR platform. The procedure can be readily adapted for a broad range of applications where highly sensitive nucleic acid detection and quantitation are required.

In pursuit of sensitivity: Lessons learned from viral nucleic acid detection and quantification on the Raindance ddPCR platform.

Sensitive PCR detection of viral nucleic acids plays a critical role in infectious disease research, diagnosis and monitoring. In the context of SARS-CoV-2 detection, recent reports indicate that digital PCR-based tests are significantly more sensitive than traditional qPCR tests. Numerous factors can influence digital PCR reaction sensitivity. In this review, using a model for human HIV infection and the Raindance ddPCR platform as an example, we describe technical aspects that contribute to sensitive viral signal detection in DNA and RNA from tissue samples, which often harbor viral reservoirs and serve as better predictors of disease outcome and indicators of treatment efficacy.

Inflammatory Profiles and Clinical Features of Coronavirus 2019 Survivors 3 Months After Discharge in Wuhan, China.

BackgroundPost-discharge immunity and its correlation with clinical features among patients recovered from COVID-19 are poorly described. This prospective cross-sectional study explored the inflammatory profiles and clinical recovery of COVID-19 patients at 3 months post-discharge.MethodsCOVID-19 patients discharged from four hospitals in Wuhan, recovered asymptomatic patients (APs) from an isolation hotel, and uninfected healthy controls (HCs) were recruited. Viral nucleic acid and antibody detection, laboratory examination, computed tomography, pulmonary function assessment, multiplex cytokine assay, and flow cytometry were performed.ResultsThe 72 age-, sex- and body mass index-matched participants included 19 severe/critical patients (SPs), 20 mild/moderate patients (MPs), 16 APs, and 17 HCs. At 3 months after discharge, levels of pro-inflammatory cytokines and factors related to vascular injury/repair in recovered COVID-19 patients had not returned to those of the HCs, especially among recovered SPs compared to recovered MPs and APs. These cytokines were significantly correlated with impaired pulmonary function and chest CT abnormalities. However, levels of immune cells had returned to nearly normal levels and were not significantly correlated with abnormal clinical features.ConclusionVascular injury, inflammation, and chemotaxis persisted in COVID-19 patients and were correlated with abnormal clinical features 3 months after discharge, especially in recovered SPs.

Rapid point-of-care testing for SARS-CoV-2 virus nucleic acid detection by an isothermal and nonenzymatic Signal amplification system coupled with a lateral flow immunoassay strip.

An outbreak of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), began in December 2019. Accurate, rapid, convenient, and relatively inexpensive diagnostic methods for SARS-CoV-2 infection are important for public health and optimal clinical care. The current gold standard for diagnosing SARS-CoV-2 infection is reverse transcription-polymerase chain reaction (RT-PCR). However, RTPCR assays are designed for use in well-equipped laboratories with sophisticated laboratory infrastructure and highly trained technicians, and are unsuitable for use in under-equipped laboratories and in the field. In this study, we report the development of an accurate, rapid, and easy-to-implement isothermal and nonenzymatic signal amplification system (a catalytic hairpin assembly (CHA) reaction) coupled with a lateral flow immunoassay (LFIA) strip-based detection method that can detect SARSCoV-2 in oropharyngeal swab samples. Our method avoids RNA isolation, PCR amplification, and elaborate result analysis, which typically takes 6–8 h. The entire CHA-LFIA detection method, from nasopharyngeal sampling to obtaining test results, takes less than 90 min. Such methods are simple and require no expensive equipment, only a simple thermostatically controlled water bath and a fluorescence reader device. We validated our method using synthetic oligonucleotides and clinicalsamples from 15 patients with SARS-CoV-2 infection and 15 healthy individuals. Our detection method provides a fast, simple, and sensitive (with a limit of detection (LoD) of 2000 copies/mL) alternative to the SARS-CoV-2 RT-PCR assay, with 100 % positive and negative predictive agreements.

Estimation of conjunctival swab and nasopharyngeal swab specimens for viral nucleic acid detection in Coronavirus disease 2019 patients to compare the viral load

Objectives: The purpose of the study was to detect the presence of viral ribonucleic acid of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in conjunctival swab along with nasopharyngeal swab specimens of Coronavirus disease 2019 (COVID-19) patients. Material and Methods: Thirty COVID-19 patients with at least one sample positive for real-time reverse transcription-polymerase chain reaction for SARS-CoV-2 in nasopharyngeal swab with the presence or absence of ocular manifestations were included in the study. The conjunctival swab along with nasopharyngeal swab of each patient was collected and sent to microbiology lab for evaluation and analysis of viral nucleic acid to assess the viral load. Results: Out of 30 patients, 21 patients (70%) were males and the remaining nine patients (30%) were females. Mean age of the patients in the study was 44.80 ± 15.37 years. One patient had conjunctivitis as ocular manifestation. Two (6.7%) out of 30 patients were positive for RT-PCR SARS-CoV-2 in the conjunctival swab. There was no statistical correlation between nasopharyngeal swab and conjunctival swab positivity using Pearson’s correlation coefficient (r) = 0.010; P = 0.995 (>0.05). Conclusion: The results of the study revealed that SARS-CoV-2 can also be detected in conjunctival swabs of confirmed cases of COVID-19 patients. Although, in comparison to nasopharyngeal and throat swabs the rate of detection of SARS-CoV-2 in conjunctival swabs is relatively less, still diligent care and precautions should be practiced during the ophthalmic evaluation of COVID-19 patients.

Early Viral Clearance and Antibody Kinetics of COVID-19 Among Asymptomatic Carriers.

Asymptomatic carriers contribute to the spread of Coronavirus Disease 2019 (COVID-19), but their clinical characteristics, viral kinetics, and antibody responses remain unclear. A total of 56 COVID-19 patients without symptoms at admission and 19 age-matched symptomatic patients were enrolled. RNA of SARS-CoV-2 was tested using transcriptase quantitative PCR, and the total antibodies (Ab), IgG, IgA, and IgM against the SARS-CoV-2 were tested using Chemiluminescence Microparticle Immuno Assay. Among 56 patients without symptoms at admission, 33 cases displayed symptoms and 23 remained asymptomatic throughout the follow-up period. 43.8% of the asymptomatic carriers were children and none of the asymptomatic cases had recognizable changes in C-reactive protein or interleukin-6, except one 64-year-old patient. The initial threshold cycle value of nasopharyngeal SARS-CoV-2 in asymptomatic carriers was similar to that in pre-symptomatic and symptomatic patients, but the positive viral nucleic acid detection period of asymptomatic carriers (9.63 days) was shorter than pre-symptomatic patients (13.6 days). There were no obvious differences in the seropositive conversion rate of total Ab, IgG, and IgA among the three groups, though the rates of IgM varied largely. The average peak IgG and IgM COI of asymptomatic cases was 3.5 and 0.8, respectively, which is also lower than those in symptomatic patients with peaked IgG and IgM COI of 4.5 and 2.4 (p < 0.05). Young COVID-19 patients seem to be asymptomatic cases with early clearance of SARS-CoV-2 and low levels of IgM generation but high total Ab, IgG, and IgA. Our findings provide empirical information for viral clearance and antibody kinetics of asymptomatic COVID-19 patients.

Point-of-care bulk testing for SARS-CoV-2 by combining hybridization capture with improved colorimetric LAMP

Efforts to contain the spread of SARS-CoV-2 have spurred the need for reliable, rapid, and cost-effective diagnostic methods which can be applied to large numbers of people. However, current standard protocols for the detection of viral nucleic acids while sensitive, require a high level of automation and sophisticated laboratory equipment to achieve throughputs that allow whole communities to be tested on a regular basis. Here we present Cap-iLAMP (capture and improved loop-mediated isothermal amplification) which combines a hybridization capture-based RNA extraction of gargle lavage samples with an improved colorimetric RT-LAMP assay and smartphone-based color scoring. Cap-iLAMP is compatible with point-of-care testing and enables the detection of SARS-CoV-2 positive samples in less than one hour. In contrast to direct addition of the sample to improved LAMP (iLAMP), Cap-iLAMP prevents false positives and allows single positive samples to be detected in pools of 25 negative samples, reducing the reagent cost per test to ~1 Euro per individual.

CRISPR/Cas technology as a promising weapon to combat viral infections.

The versatile clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has emerged as a promising technology for therapy and molecular diagnosis. It is especially suited for overcoming viral infections outbreaks, since their effective control relies on an efficient treatment, but also on a fast diagnosis to prevent disease dissemination. The CRISPR toolbox offers DNA‐ and RNA‐targeting nucleases that constitute dual weapons against viruses. They allow both the manipulation of viral and host genomes for therapeutic purposes and the detection of viral nucleic acids in “Point of Care” sensor devices. Here, we thoroughly review recent advances in the use of the CRISPR/Cas system for the treatment and diagnosis of viral deleterious infections such as HIV or SARS‐CoV‐2, examining their strengths and limitations. We describe the main points to consider when designing CRISPR antiviral strategies and the scientific efforts to develop more sensitive CRISPR‐based viral detectors. Finally, we discuss future prospects to improve both applications. Also see the video abstract here: https://www.youtube.com/watch?v=C0z1dLpJWl4

Surveillance of Common Respiratory Infections During COVID-19 Pandemic Demonstrates Preventive Effectiveness of Non-Pharmaceutical Interventions

Background: Following the emergence of a novel coronavirus, SARS-CoV-2, and its spread outside of Wuhan, China, the human society has experienced a pandemic of coronavirus disease 2019 (COVID-19). While the development of vaccine and pharmaceutical treatment have been undergoing, government authorities in China implemented unprecedented non-pharmaceutical interventions as primary barriers to curb the spread of the deadly SARS-CoV-2. Although the decline of COVID-19 cases coincided with this implementation of interventions, evidence is needed to demonstrate the efficacy of these interventions since artifactual factors, such as the environment, the pathogen itself, and the phases of epidemic, may also alter the pattern of case development.Methods: We surveyed common viral respiratory infections that have a similar pattern of transmission, tropism, and clinical manifestation as COVID-19 under such interventions during this season with pandemic, and compared it with historical data in previous seasons without the interventions.Finding: Our survey shows that the rates for common respiratory infections, such as influenza and respiratory syncytial virus infections, decreased dramatically from previous 13.7% and 4.64% to 0.73% and 0.00%, respectively, in this season.Interpretation: This surveillance provides compelling evidence that the interventions are cost-effective ways to curb the spread of contagious agents, and may be the only practical approach to limiting the evolving of epidemic until vaccine and pharmaceutical treatment are available.Funding Statement: This study was supported by West China Hospital (HX-2019-nCoV-17), Sichuan Science and Technology Program (2020YFS0008), and National Natural Science Foundation (81671551). Declaration of Interests: CapitalBio Technology is the manufacture of Respiratory Virus Nucleic Acid Detection Kit and RTisochip™ –W Isothermal Microfluidic Chip Analyzer. XX and DL are employees of CapitalBio Technology. All others declare no competing interest.Ethics Approval Statement: Clinical trial (device) 2020, No. 4; Clinical Trial Committee, West China Hospital of Sichuan University. The Institutional Review Board of the West China Hospitalapproved the study.

A comparative study of SARS-COV-2 nucleic acid assay for nasal swab and sputum samples

Objective To analyze nucleic acid detection results of coronavirus disease 2019 (COVID-19) patients' nasal swabs and sputum specimens, and we provide reference for clinical sampling methods. Methods Totally 170 times of matched nasal swabs and sputum specimens of 78 COVID-19 patients in Shenzhen Third People's Hospital were collected from the same day. The samples were tested with real-time fluorescence PCR nucleic acid. The nasal swabs and sputum samples were analyzed and compared. Results A total of 78 patients were involved, including 33 males and 45 females. Age ranged 1-86 years old, the majority of young and middle-aged (20-<60 years, 59.0%) patients, followed by elderly patients (≥ 60 years, 33.0%), 4 children. Among 170 matched samples, 59 nasal swabs were positive, the positive rate was 34.7%; 79 sputum samples were positive, the positive rate was 46.5%, the difference was statistically significant ( P <0.01). There were 46 times of positive nasal swabs and sputum specimens simultaneously. The results showed that the Ct value of 27 sputum samples was lower than that of nasal swabs (58.7%), 17 sputum samples were higher than that of nasal swabs (37.0%), and 2 sputum samples were equal to that of nasal swabs (4.3%). This suggested a higher viral load in sputum specimens than in nose swabs. Conclusion In the nucleic acid assay of COVID-19 patients, the positive rate of sputum specimens is higher than that of nasal swabs, and the rate of missing detection is low. The viral load of sputum specimens is higher than that of nasal swabs, which is more conducive to the detection of viral nucleic acids, and patients are more receptive. 摘要：目的 分析新型冠状病毒肺炎(coranavims disease 2019, COVID-19)患者鼻拭子和痰标本病毒核酸检测结 果, 为临床采样方式的选择提供参考依据。 方法 收集深圳市第三人民医院新型冠状病毒肺炎78例患者共170例次 同一天鼻拭子与痰配对标本, 进行实时荧光PCR核酸检测, 分析比较鼻拭子和痰标本检测情况。 结果 78例患者中男 性33例, 女性45例, 年龄1∼86岁, 中青年(20∼<60岁) 患者居多 (占59.0%), 其次为老年(≥60岁) 患者 (占33.0%), 4例 为儿童。170例次配对标本中, 鼻拭子和痰标本核酸阳性的阳性率分别为34.7%(59/170)和46.5%(79/170), 差异有统 计学意义( P <0.01)。鼻拭子和痰标本同时阳性共46例次, 结果显示27例痰标本Ct值低于鼻拭子Ct值 (占58.7%), 17 例痰标本Ct值高于鼻拭子Ct值 (占37.0%), 2例痰标本Ct值等于鼻拭子Ct值 (占4.3%), 提示痰标本的病毒载量比鼻拭 子的病毒载量更高。 结论 在新型冠状病毒肺炎患者病毒核酸检测中, 痰标本的阳性率高于鼻拭子标本, 且漏检率低, 痰标本的病毒载量比鼻拭子的病毒载量更高, 更有利于病毒核酸的检出, 同时患者更易接受, 建议有痰患者优先采集 痰标本。

Laboratory evaluation of SARS-CoV-2 in the COVID-19 pandemic.

Laboratory evaluation of SARS-CoV-2 involves the detection of viral nucleic acid, viral protein antigens, and the antibody response. Molecular detection of SARS-CoV-2 is the only diagnostic test currently available in acutely or recently infected individuals. In contrast, serological testing is typically performed once viral RNA has been cleared and symptoms have resolved. This leads to some confusion among clinicians as to which test to order and when each is appropriate. While SARS-CoV-2 assays can suffer from poor sensitivity, all FDA authorized assays to date are intended to be qualitative. Serological tests have multiple assay formats, detect various classes of immunoglobulins, and have a distinct role in seroprevalence studies; however, the association with long-term protection remains unclear. Both molecular and serological testing for SARS-CoV-2 have complementary roles in patient management, and we highlight the challenges faced by clinicians and laboratorians alike in the evaluation and interpretation of the currently available laboratory assays.

Nucleic acid detection and quantitative analysis of influenza virus using exhaled breath condensate

Exhaled breath condensate (EBC) is increasingly being used as a non-invasive method for disease diagnosis and environmental exposure assessment. We previously detected the nucleic acids of bacterial pathogens in EBC. Influenza viruses can be transmitted through aerosols during coughing and exhaling. Existing detection methods for influenza have various limitations. The EBC collection method is convenient, non-invasive, and reduces the risk of exposure. We investigated the detection of influenza virus in EBC using a sensitive nucleic acid testing method and performed quantitative analysis to evaluate the present and content of influenza virus in the breath. We evaluated 30 patients with respiratory tract infection during the 2019 influenza season. The clinical data and samples of nasal swabs were collected for rapid influenza diagnostic (antigen) tests. Pharyngeal swab and EBC samples were used for influenza virus nucleic acid detection. Each EBC sample was assessed twice as well as at one-month follow-up of the patients. The nucleic acid test in the EBC of 30 cases revealed 20 and two cases of influenza A and B, respectively, giving a detection rate of 73.3%. The rapid influenza diagnostic (antigen) tests revealed four and 12 cases of influenza A and B, respectively, with a detection rate of 53.3%. All pharyngeal swab samples evaluated by the nucleic acid test were influenza-positive; 12 cases were positive for both influenza A and B and 18 cases were positive for influenza B alone. The influenza viral load in the EBC was 103–107 copies ml−1. Among the 16 patients followed-up after 1 month, 4 were positive (25%) in EBC samples and 10 were positive (62.5%) in pharyngeal swab samples. It was preliminary exploration that influenza virus could be detected in EBC. EBC is one of the sample types that would be used for molecular diagnosis of influenza.

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.