Analysis of COVID-19 rapid antigen and PCR detection policy

After the outbreak of COVID-19, Taiwan has implemented rigorous border control and taken specific measures such as virus detection, contact tracing, and quarantine since 2020. Its epidemic prevention performance has been quite outstanding. Even in May 2021, when the epidemic situation worsens, the people in Taiwan fully cooperate with the government's control measures so as to successfully alleviate and control the epidemic in less than three months. Among them, the detection policy has played a pivotal role. We analyze and discuss the false positive and false negative problems from rapid antigen and PCR detection in the screening policy as well as the timing of using these two instruments. This paper provides theoretical verification of the appropriateness of screening policy in Taiwan, offering a few feasible suggestions for related policies in other countries or regions at different stages of this and other potential epidemics. (c) 2022 by the authors;licensee Growing Science, Canada.

RETRACTED ARTICLE: Deep learning system to screen coronavirus disease 2019 pneumonia

The Editor in Chief has retracted this article (1) because the data published here was previously presented by a different set of authors at arXiv pre-print server (2) and a large amount of text, some figures and tables have been re-used without appropriate citation or acknowledgment. The pre-print has been published (3). Author Benson Babu agrees to this retraction. Authors Charmine Butt, Jagpal Gill and David Chun did not respond to any correspondence regarding this retraction. © Springer Science+Business Media, LLC, part of Springer Nature 2020.

The Prevalence, Coexistence, and Correlations between Seven Pathogens Detected by a PCR Method from South-Western Poland Dairy Cattle Suffering from Bovine Respiratory Disease.

Bovine respiratory disease (BRD) is a very important disease that contributes to economic losses in dairy and beef cattle breeding worldwide. The molecular testing of material from 296 calves showing BRD symptoms from 74 dairy herds located in south-western Poland was performed in 2019-2021. Molecular tests were performed using a commercial kit "VetMAXTM Ruminant Respiratory Screening Kit" (Thermo Fisher Scientific) for the simultaneous detection of genetic material of seven pathogens responsible for BRD. At least one pathogen was detected in 95.95% of herds. The overall prevalence was: Pasteurella multocida 87.84%, Mannheimia haemolytica 44.59%, bovine coronavirus (BcoV) 32.43%, Mycoplasma bovis 29.73%, Histophilus somni 28.38%, bovine parainfluenza virus type 3 (BPIV-3) 13.51%, and bovine respiratory syncytial virus (BRSV) 10.81%. Twenty-nine configurations of pathogen occurrences were found. Bacterial infections were the most frequently recorded as 56.7% of all results. Coinfections mainly consisted of two pathogens. Not a single purely viral coinfection was detected. The most frequent result was a single P. multocida infection accounting for 18.31% of all results. The statistically significant correlation (p = 0.001) with the highest strength of effect (ϕ 0.38) was between M. bovis and H. somni.

Analysis of COVID-19 rapid antigen and PCR detection policy

After the outbreak of COVID-19, Taiwan has implemented rigorous border control and taken specific measures such as virus detection, contact tracing, and quarantine since 2020. Its epidemic prevention performance has been quite outstanding. Even in May 2021, when the epidemic situation worsens, the people in Taiwan fully cooperate with the government’s control measures so as to successfully alleviate and control the epidemic in less than three months. Among them, the detection policy has played a pivotal role. We analyze and discuss the false positive and false negative problems from rapid antigen and PCR detection in the screening policy as well as the timing of using these two instruments. This paper provides theoretical verification of the appropriateness of screening policy in Taiwan, offering a few feasible suggestions for related policies in other countries or regions at different stages of this and other potential epidemics. © 2022 by the authors;licensee Growing Science, Canada.

Coronaviruses in Bats

This article summarizes current detections of coronaviruses in bats in different geographic regions worldwide, and underlines the potential role of bats as ancestor and reservoir hosts of different coronaviruses. Bats can be found in almost all global habitats with the notable exception of the high arctic and a few isolated oceanic islands. First, we give an overview on the potential role of bats as carriers of zoonotic viruses, underlining the relevance of specific physiological traits of representatives of this mammalian order, that are supporting the transmission of these disease agents. Research in this field was predominantly initiated by the SARS coronavirus event in 2003, and in the meantime, bats are assumed to be the ancestor hosts for numerous alpha and beta coronaviruses. Bat associated coronaviruses have been detected on all continents where bats are distributed. Since the beginning of the COVID-19 pandemic, coronavirus research has again been considerably intensified, leading to a gain of knowledge regarding the interaction between bats and coronaviruses that will be valuable in the management of potential future outbreaks originating from bats. Finally, an outlook is given on research gaps that could be invaluable when dealing with future pandemic events.

Processing Hundreds of SARS-CoV-2 Samples with an In-House PCR-Based Method without Robotics.

The SARS-CoV-2 pandemic has required the development of multiple testing systems to monitor and control the viral infection. Here, we developed a PCR test to screen COVID-19 infections that can process up to ~180 samples per day without the requirement of robotics. For this purpose, we implemented the use of multichannel pipettes and plate magnetics for the RNA extraction step and combined the reverse transcription with the qPCR within one step. We tested the performance of two RT-qPCR kits as well as different sampling buffers and showed that samples taken in NaCl or PBS are stable and compatible with different COVID-19 testing systems. Finally, we designed a new internal control based on the human RNase P gene that does not require a DNA digestion step. Our protocol is easy to handle and reaches the sensitivity and accuracy of the standardized diagnostic protocols used in the clinic to detect COVID-19 infections.

Detection and infectivity of SARS-CoV-2 in exhumated corpses.

Postmortem detection of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) after the exhumation of a corpse can become important, e.g. in the case of subsequent medical malpractice allegations. To date, data on possible detection periods [e.g. by reverse transcription polymerase chain reaction (RT-PCR)] or on the potential infectivity of the virus after an exhumation are rare. In the present study, these parameters were examined in two cases with a time span of approximately 4 months between day of death and exhumation. Using SARS-CoV-2 RT-PCR on swabs of both lungs and the oropharynx detection was possible with cycle threshold (Ct) values of about 30 despite signs of beginning decay. RT-PCR testing of perioral and perinasal swabs and swabs collected from the inside of the body bag, taken to estimate the risk of infection of those involved in the exhumation, was negative. Cell culture-based infectivity testing was negative for both, lung and oropharyngeal swabs. In one case, RT-PCR testing at the day of death of an oropharyngeal swab showed almost identical Ct values as postmortem testing of an oropharyngeal swab, impressively demonstrating the stability of viral RNA in the intact corpse. However, favorable climatic conditions in the grave have to be taken into account, as it was wintertime with constant low temperatures. Nevertheless, it was possible to demonstrate successful postmortem detection of SARS-CoV-2 infection following exhumation even after months in an earth grave.

An optimized and robust PEG precipitation method for detection of SARS-CoV-2 in wastewater.

Wastewater-based epidemiology is currently being utilized to monitor the dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), on a population scale. The detection of SARS-CoV-2 in wastewater is highly influenced by methodologies used for its isolation, concentration and RNA extraction. Although various viral concentration methods are currently employed, including polyethylene glycol (PEG) precipitation, adsorption-extraction, ultracentrifugation and ultrafiltration, to our knowledge, none of these methods have been standardized for use with a variety of wastewater matrices and/or different kits for RNA extraction and quantification. To address this, wastewater with different physical characteristics was seeded with gamma-irradiated SARS-CoV-2 and used to test the efficiency of PEG precipitation and adsorption-extraction to concentrate the virus from three physiochemically different wastewater samples, sourced from three distinct wastewater plants. Efficiency of viral concentration and RNA extraction was assessed by reverse-transcriptase polymerase chain reaction and the recovery yields calculated. As co-purification of inhibitors can be problematic for subsequent detection, two commonly used commercial master mixes were assessed for their sensitivity and efficiency to detect two SARS-CoV-2 target nucleocapsid (N) gene sequences. Recovery rates varied greatly between wastewater matrices and concentration methods, with the highest and most reproducible recovery rates (46.6-56.7%) observed when SARS-CoV-2 was precipitated with PEG and detected by the Luna® Universal master mix. The adsorption-extraction method was less effective (0-21.7%). This study demonstrates that PEG precipitation is the more robust method, which translates well to varying wastewater matrices, producing consistent and reproducible recovery rates. Furthermore, it is compatible with different kits for RNA extraction and quantitation.

Re-positive of SARS-CoV-2 test is common in COVID-19 patients after hospital discharge. Data from high standard post-discharge quarantined patients in Macao SAR, China.

During the pandemic of COVID-19, Macau faces tremendous pressure because it is a famous gambling and tourism city with the world's highest population density. The Macau government implemented decisive public health intervention to control the transmission of COVID-19, and there were only two independent outbreaks in Macau. In the second outbreak, all 35 cases were infected in foreign countries. They were quarantined in airborne infection isolation rooms for at least 14 days with reverse transcription-polymerase chain reaction (RT-PCR) tests after hospital discharge. Twelve (34.3%) of them had re-positive SARS-CoV-2 test results, and none of them presented any COVID-19 signs or symptoms during the entire quarantine period. In this study, the re-positive patients were more likely to be diagnosed in the early stage of the disease with a longer hospital stay. Virus re-infection is impossible in this high standard isolation setting, and reactivation is also unlikely, so that residual virus nucleic acid should be the possible reason for this phenomenon. Due to limited data about the risk of re-positive patients, it is better to quarantine patients after discharge for a prolonged period with repeat RT-PCR tests to minimize the community's potential risk, particularly in the regions with relative plenty of resources and low community infection rate such as Macau.

Conventional PCR assisted single-component assembly of spherical nucleic acids for simple colorimetric detection of SARS-CoV-2.

Continuous identification of suspected infectious cases is crucial to control the recent pandemic caused by the novel human coronavirus SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2). Real-time polymerase chain reaction (real-time PCR) technology cannot be implemented easily and in large scale in some communities due to lack of resources and infrastructures. Here, we report a simple colorimetric strategy derived from linker-based single-component assembly of gold nanoparticle-core spherical nucleic acids (AuNP-core SNAs) for visual detection of PCR products of SARS-CoV-2 ribonucleic acid (RNA) template. A palindromic linker is designed based on SARS-CoV-2 specific E gene to program the identical colloidal SNAs into large assemblies along with a distinct red-to-purple color change. The linker acts as a probe of SARS-CoV-2 RNA in conventional PCR reaction. In the presence of the correct template the palindromic linker, which is complementary to a short region within the target amplicon, is cleaved by 5'-exonuclease activity of deoxyribonucleic acid (DNA) polymerase. Cleavage of the palindromic linker during the amplification process inhibits the single-component assembly formation of SNAs. So, positive and negative viral samples produce simply red and purple colors in the post PCR colorimetric test, respectively. Evaluation of the samples obtained from cases with laboratory-confirmed SARS-CoV-2 infection revealed that our assay can rival with real-time PCR method in sensitivity.

Optimized qRT-PCR Approach for the Detection of Intra- and Extra-Cellular SARS-CoV-2 RNAs.

The novel coronavirus SARS-CoV-2 is the causative agent of the acute respiratory disease COVID-19, which has become a global concern due to its rapid spread. Meanwhile, increased demand for testing has led to a shortage of reagents and supplies and compromised the performance of diagnostic laboratories in many countries. Both the World Health Organization (WHO) and the Center for Disease Control and Prevention (CDC) recommend multi-step RT-PCR assays using multiple primer and probe pairs, which might complicate the interpretation of the test results, especially for borderline cases. In this study, we describe an alternative RT-PCR approach for the detection of SARS-CoV-2 RNA that can be used for the probe-based detection of clinical isolates in diagnostics as well as in research labs using a low-cost SYBR green method. For the evaluation, we used samples from patients with confirmed SARS-CoV-2 infections and performed RT-PCR assays along with successive dilutions of RNA standards to determine the limit of detection. We identified an M-gene binding primer and probe pair highly suitable for the quantitative detection of SARS-CoV-2 RNA for diagnostic and research purposes.

The Issue of Recurrently Positive Patients Who Recovered From COVID-19 According to the Current Discharge Criteria: Investigation of Patients from Multiple Medical Institutions in Wuhan, China.

The current discharge criteria for COVID-19 require that patients have 2 consecutive negative results for reverse transcription polymerase chain reaction (RT-PCR) detection. Here, we observed that recurrent positive RT-PCR test results in patients with 3 consecutive negative results (5.4%) were significantly decreased compared with those in patients with 2 consecutive negative results (20.6%); such patients reported positive RT-PCR test results within 1 to 12 days after meeting the discharge criteria. These results confirmed that many recovered patients could show a positive RT-PCR test result, and most of these patients could be identified by an additional RT-PCR test prior to discharge.