Molecular epidemiology and genetic diversity of canine coronavirus from domestic dogs in Chengdu, China from 2020 to 2021 using a multiplex RT-PCR.

Recent reports on identification of canine coronavirus (CCoV) in humans have emphasized the urgency to strengthen surveillance of animal CoVs. The fact that recombinations between CCoV with feline, porcine CoVs brought about new types of CoVs indicated that more attention should be paid to domestic animals like dogs, cats and pigs, and the CoVs they carried. However, there are about ten kinds of CoVs that infect above animals, and thus representative CoVs with zoonotic potentials were considered in this study. Multiplex RT-PCR against CCoV, Feline coronavirus (FCoV), porcine deltacoronavirus and porcine acute diarrhea syndrome coronavirus was developed to investigate the prevalence of CoVs from domestic dogs in Chengdu, Southwest China. Samples from a total of 117 dogs were collected from a veterinary hospital, and only CCoV (34.2%, 40/117) was detected. Therefore, this study focused on CCoV and its characteristics of S, E, M, N and ORF3abc genes. Compared with CoVs that are capable of infecting humans, CCoV strains showed highest nucleotide identity with the novel canine-feline recombinant detected from humans (CCoV-Hupn-2018). Phylogenetic analysis based on S gene, CCoV strains were not only clustered with CCoV-II strains, but also closely related to FCoV-II strains ZJU1617 and SMU-CD59/2018. As for assembled ORF3abc, E, M, N sequences, CCoV strains had the closest relationship with CCoV-II (B203_GZ_2019, B135_JS_2018 and JS2103). What's more, specific amino acid variations were found, especially in S and N proteins, and some mutations were consistent with FCoV, TGEV strains. Altogether, this study provided a novel insight into the identification, diversification and evolution of CoVs from domestic dogs. It is of top priority to recognize zoonotic potential of CoVs, and continued comprehensive surveillance will help better understand the emergence, spreading, and ecology of animal CoVs.

Single-Center Experience in Detecting Influenza Virus, RSV and SARS-CoV-2 at the Emergency Department.

Reverse transcription polymerase chain reaction (RT-PCR) on respiratory tract swabs has become the gold standard for sensitive and specific detection of influenza virus, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In this retrospective analysis, we report on the successive implementation and routine use of multiplex RT-PCR testing for patients admitted to the Internal Medicine Emergency Department (ED) at a tertiary care center in Western Austria, one of the hotspots in the early coronavirus disease 2019 (COVID-19) pandemic in Europe. Our description focuses on the use of the Cepheid® Xpert® Xpress closed RT-PCR system in point-of-care testing (POCT). Our indications for RT-PCR testing changed during the observation period: From the cold season 2016/2017 until the cold season 2019/2020, we used RT-PCR to diagnose influenza or RSV infection in patients with fever and/or respiratory symptoms. Starting in March 2020, we used the RT-PCR for SARS-CoV-2 and a multiplex version for the combined detection of all these three respiratory viruses to also screen subjects who did not present with symptoms of infection but needed in-hospital medical treatment for other reasons. Expectedly, the switch to a more liberal RT-PCR test strategy resulted in a substantial increase in the number of tests. Nevertheless, we observed an immediate decline in influenza virus and RSV detections in early 2020 that coincided with public SARS-CoV-2 containment measures. In contrast, the extensive use of the combined RT-PCR test enabled us to monitor the re-emergence of influenza and RSV detections, including asymptomatic cases, at the end of 2022 when COVID-19 containment measures were no longer in place. Our analysis of PCR results for respiratory viruses from a real-life setting at an ED provides valuable information on the epidemiology of those infections over several years, their contribution to morbidity and need for hospital admission, the risk for nosocomial introduction of such infection into hospitals from asymptomatic carriers, and guidance as to how general precautions and prophylactic strategies affect the dynamics of those infections.

Development and Validation of ScriptTaq COVID PCR: An In-House Multiplex rRT-PCR for Low-Cost Detection.

The COVID-19 pandemic necessitated an extensive testing for active SARS-CoV-2 infection. However, securing affordable diagnostic tests is a struggle for low-resource settings. We report herein the development and validation of an in-house multiplex real-time RT-PCR diagnostic test for the detection of active COVID-19 infection (ScriptTaq COVID PCR). Furthermore, we describe two methods for RNA extraction using either an in-house silica column or silica-coated magnetic beads to replace commercial RNA extraction kits. Different buffer formulations for silica column and silica-coated magnetic beads were tested and used for RNA isolation. Taq polymerase enzyme and thermostable reverse transcriptase enzyme were purified from bacterial clones. Primers/probes sequences published by the WHO and CDC were used for the qualitative detection of the RNA-dependent RNA polymerase (RdRp) and nucleocapsid (N) genes, respectively. ScriptTaq COVID PCR assay was able to detect up to 100 copies per reaction of the viral RdRP and N genes. The test demonstrated an overall agreement of 95.4%, a positive percent agreement (PPA) of 90.2%, and a negative percent agreement (NPA) of 100.0% when compared with two commercially available kits. ScriptTaq COVID PCR diagnostic test is a specific, sensitive, and low-cost alternative for low-resource settings.

Development of a multiplex RT-PCR method for the detection of four porcine enteric coronaviruses.

Porcine enteric coronaviruses are pathogens that cause viral diarrhea in pigs and are widely prevalent worldwide. Moreover, studies have shown that some porcine enteric coronaviruses can infect humans and poultry. In order to effectively monitor these viruses, it is necessary to establish a multiple detection method to understand their prevalence and conduct in-depth research. Common porcine enteric coronaviruses include Porcine epidemic diarrhea virus (PEDV), Porcine transmissible gastroenteritis virus (TGEV), Porcine delta coronavirus (PDCoV), and Swine acute diarrhea syndrome coronavirus (SADS-CoV). Pigs infected with these viruses have the common clinical symptoms that are difficult to distinguish. A quadruplex RT-PCR (reverse transcription-polymerase chain reaction) method for the simultaneous detection of PEDV, PDCoV, TGEV and SADS-CoV was developed. Four pairs of specific primers were designed for the PEDV M gene, PDCoV N gene, TGEV S gene and SADS-CoV RdRp gene. Multiplex RT-PCR results showed that the target fragments of PDCoV, SADS-CoV, PEDV and TGEV could be amplified by this method. and the specific fragments with sizes of 250 bp, 368 bp, 616 bp and 801 bp were amplified, respectively. This method cannot amplify any fragment of nucleic acids of Seneca Valley virus (SVV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Atypical Porcine Pestivirus (APPV), and has good specificity. The lowest detection limits of PDCoV, PEDV, TGEV and SADS-CoV were 5.66 × 105 copies/µL, 6.48 × 105 copies/µL, 8.54 × 105 copies/µL and 7.79 × 106 copies/µL, respectively. A total of 94 samples were collected from pig farms were analyzed using this method. There were 15 positive samples for PEDV, 3 positive samples for mixed infection of PEDV and PDCoV, 2 positive samples for mixed infection of PEDV and TGEV, and 1 positive sample for mixed infection of PEDV, TGEV, and PDCoV. Multiplex RT-PCR method could detect four intestinal coronaviruses (PEDV, PDCoV, TGEV, and SADS-CoV) in pigs efficiently, cheaply and accurately, which can be used for clinical large-scale epidemiological investigation and diagnosis.

SARS-CoV-2 coinfections with variant genomic lineages identified by multiplex fragment analysis.

Immunocompromised patients can experience prolonged SARS-CoV-2 infections in the setting of a lack of protectivity immunity despite vaccination. As circulating SARS-CoV-2 strains become more heterogeneous, concomitant infection with multiple SARS-CoV-2 variants has become an increasing concern. Immunocompromised patient populations represent potential reservoirs for the emergence of novel SARS-CoV-2 variants through mutagenic change or coinfection followed by recombinatory events. Identification of SARS-CoV-2 coinfections is challenging using traditional next generation sequencing pipelines; however, targeted genotyping approaches can facilitate detection. Here we describe five COVID-19 cases caused by coinfection with different SARS-CoV-2 variants (Delta/Omicron BA.1 and Omicron BA.1/BA.2) as identified by multiplex fragment analysis.

Understanding Host-Virus Interactions: Assessment of Innate Immune Responses in Mastomys natalensis Cells after Arenavirus Infection.

Mastomys natalensis is the natural host of various arenaviruses, including the human-pathogenic Lassa virus. Homologous arenaviruses, defined here as those having M. natalensis as a natural host, can establish long-lasting infection in M. natalensis, while these animals rapidly clear arenaviruses having another rodent species as a natural host (heterologous viruses). Little is known about the mechanisms behind the underlying arenavirus-host barriers. The innate immune system, particularly the type I interferon (IFN) response, might play a role. In this study, we developed and validated RT-PCR assays to analyse the expression of M. natalensis interferon-stimulated genes (ISGs). We then used these assays to study if homologous and heterologous viruses induce different IFN responses in M. natalensis cells. Infection experiments were performed with the homologous Lassa and Morogoro viruses and the related but heterologous Mobala virus. Compared to the direct induction with IFN or Poly(I:C), arenaviruses generally induced a weak IFN response. However, the ISG-expression profiles of homologous and heterologous viruses were similar. Our data indicate that, at least in M. natalensis cells, the IFN system is not a major factor in the virus-host barrier for arenaviruses. Our system provides a valuable tool for future in vivo investigation of arenavirus host restrictions at the level of the innate immune response.

Development of multiplex S-gene-targeted RT-PCR for rapid identification of SARS-CoV-2 variants by extended S-gene target failure.

BACKGROUND: Tracking SARS-CoV-2 variants of concern (VOC) by genomic sequencing is time-consuming. The rapid screening of VOCs is necessary for clinical laboratories. In this study, we developed a rapid screening method based on multiplex RT-PCR by extended S-gene target failure (eSGTF), a false negative result caused by S-gene mutations. METHODS: Three S-gene target (SGT) regions (SGT1, codons 65-72; SGT2, codons 152-159; and SGT3, codons 370-377) and an N-gene region (for internal control) were detected in single-tube. Four types of VOC (Alpha, Delta, Omicron BA.1, and Omicron BA.2) are classified by positive/negative patterns of 3 S-gene regions (eSGTF pattern). RESULTS: The eSGTF patterns of VOCs were as follows (SGT1, SGT2, SGT3; P, positive; N, negative): Alpha, NPP; Delta, PNP; Omicron BA.1, NPN pattern; and Omicron BA.2, PPN. As compared with the S-gene sequencing, eSGTF patterns were identical to the specific VOCs (concordance rate = 96.7%, N = 206/213). Seven samples with discordant results had a minor mutation in the probe binding region. The epidemics of VOCs estimated by eSGTF patterns were similar to those in Japan. CONCLUSIONS: Multiplex RT-PCR and eSGTF patterns enable high-throughput screening of VOCs. It will be useful for the rapid determination of VOCs in clinical laboratories.

Etiological Analysis of Viral Encephalitis in Children in Zhejiang Province from 2018 to 2019.

OBJECTIVE: To investigate the common pathogens of viral encephalitis (VE) in children, and to provide guidance for the empirical diagnosis and treatment of patients with VE. METHODS: A total of 227 cerebrospinal fluid (CSF) samples were collected from pediatric patients with VE in Zhejiang province from January 2018 to December 2019. The samples were tested using multiplex and singleplex Reverse Transcription-Polymerase Chain Reaction (RT-PCR) with primers specific to enterovirus (EV), varicella-zoster virus (VZV), mumps virus (MuV), cytomegalovirus (CMV), herpes simplex virus type 1 (HSV-1)/type 2 (HSV-2), Epstein-Barr virus (EBV), and human herpesvirus 6 (HHV-6). The data of the two analyses were compared and then verified using Sanger sequencing. RESULTS: Of the 227 CSF samples, 90 were shown to be positive for multiplex RT-PCR with a positivity rate of 39.65% and a 95% confidence interval (33.2%, 46.1%). EV was the most common cause of VE, followed by EBV, HHV-6, MuV, CMV, VZV, and HSV-1. Most included cases occurred in summer, accounting for 49.78% of all cases. For EV, EBV, and HSV-2, multiplex RT-PCR showed a positivity rate of 34.36%, which was not statistically different from that of 30.4% shown by singleplex RT-PCR. The sequences of EV, EBV, VZV, MuV, CMV, HSV-1, HHV-6, and HSV-2 were confirmed by sequencing the PCR products obtained from multiplex and singleplex PCR. CONCLUSIONS: In children, VE is more prevalent in the summer than in other seasons in Zhejiang province, and EV may be the most common causative pathogen.

SARS-CoV-2 Continuous Genetic Divergence and Changes in Multiplex RT-PCR Detection Pattern on Positive Retesting Median 150 Days after Initial Infection.

Being in the epicenter of the COVID-19 pandemic, our lab tested 193,054 specimens for SARS-CoV-2 RNA by diagnostic multiplex reverse transcription polymerase chain reaction (mRT-PCR) starting in March 2020, of which 17,196 specimens resulted positive. To investigate the dynamics of virus molecular evolution and epidemiology, whole genome amplification (WGA) and Next Generation Sequencing (NGS) were performed on 9516 isolates. 7586 isolates with a high quality were further analyzed for the mutation frequency and spectrum. Lastly, we evaluated the utility of the mRT-PCR detection pattern among 26 reinfected patients with repeat positive testing three months after testing negative from the initial infection. Our results show a continuation of the genetic divergence in viral genomes. Furthermore, our results indicate that independent mutations in the primer and probe regions of the nucleocapsid gene amplicon and envelope gene amplicon accumulate over time. Some of these mutations correlate with the changes of detection pattern of viral targets of mRT-PCR. Our data highlight the significance of a continuous genetic divergence on a gene amplification-based assay, the value of the mRT-PCR detection pattern for complementing the clinical diagnosis of reinfection, and the potential for WGA and NGS to identify mutation hotspots throughout the entire viral genome to optimize the design of the PCR-based gene amplification assay.

Clinical Evaluation of Conventional Human Coronavirus Infection in Adults.

Human coronaviruses (HCoVs) are distributed globally and they cause a range of respiratory symptoms. Since HCoV infection usually causes mild upper respiratory tract disease and currently has no specific therapy, there are limited reports on its features, especially in adults. We aimed to evaluate the features of HCoV infections in clinical settings. Adult patients with respiratory symptoms from October 2014 to September 2019 at Nagasaki Genbaku Isahaya Hospital were enrolled. Multiplex reverse transcription-polymerase chain reaction as performed for 15 viruses, including HCoVs, and eight bacterial species on the patients' respiratory specimens. A total of 121 cases were recruited with HKU1, OC43, 229E, and NL63 strains in 80, 21, 12, and 11 cases, respectively. The percentage of HCoV-infected patients peaked in winter (47.5%). Symptoms of fever (69.4%), cough (47.9%), and comorbidities of asthma/cough variant asthma (34.7%) were frequently observed. Lymphocytopenia and increased C-reactive protein levels were observed in laboratory tests. Co-infection with other viruses was identified in 38.8% of the cases. In the repeat-positive cases, 42% were repeat positive within 100 days. HCoV-infected patients showed winter seasonality with a high frequency of comorbidity with asthma and co-infections. Re-infection within an early period was suspected, but further consideration is required.

Clinical evaluation of the molecular-based BD SARS-CoV-2/Flu for the BD MAX™ system.

BACKGROUND: COVID-19 and influenza (flu) share similar clinical symptoms. Therefore, differential detection of these viruses during the respiratory virus season will be an important component for proper patient triage, management, and treatment. OBJECTIVES: Establish the diagnostic performance related to SARS-CoV-2 and Flu A/B detection for the BD SARS-CoV-2/Flu for BD MAX™ System ("MAX SARS-CoV-2/Flu") multiplex assay. MATERIALS AND METHODS: Two hundred and thirty-five (235) retrospective nasopharyngeal specimens were obtained from external vendors. The BD BioGx SARS-CoV-2 Reagents for BD MAX™ System ("BioGx SARS-CoV-2″) and the Cepheid Xpert® Xpress Flu/RSV ("Xpert Flu/RSV") were utilized as reference methods. RESULTS: By reference methods, 52 specimens were SARS-CoV-2-positive, 59 were Flu A-positive, and 60 were Flu B-positive. MAX SARS-CoV-2/Flu had positive percent agreement (PPA) and negative percent agreement (NPA) values for SARS-CoV-2 detection of 96.2% ([95%CI]:87.0-98.9) and 100% [95%CI:88.7-100], respectively; PPA values for Flu A and Flu B of 100% [95%CI:93.9-100] and 98.3% [95%CI:91.1-99.7], respectively, and NPA values for Flu A and Flu B of 98.9% [95%CI:94.0-99.8] and 100% [95%CI:95.9-100], respectively. CONCLUSIONS: The MAX SARS-CoV-2/Flu assay met FDA-EUA performance criteria for SARS-CoV-2 (≥95% for PPA and NPA) and FDA clearance criteria for Flu A/B (PPA ≥90%; lower bound of the 95%CI ≥80% and NPA ≥95%; lower bound of the 95%CI ≥90%).

The clinical impact of PCR-based point-of-care diagnostic in respiratory tract infections in children.

BACKGROUND: Children are commonly affected by respiratory tract infections. Based on clinical symptoms, laboratory evaluation, and imaging, the causative pathogen often cannot be delineated. Point-of-care-testing systems that provide an opportunity for fast detection of common viruses and some bacteria can therefore influence treatment's options. We aimed to examine whether the Biofire® FilmArray® has an effect on antibiotic treatment, duration of antibiotic therapy, and length of hospital stay within a pediatric cohort. METHODS: We included children who were admitted to inpatient treatment with an acute respiratory tract infection from 02/2017 to 04/2018 using the FA respiratory panel for pathogen detection. The study group data were compared to the retrospective data of children admitted from 02/2016 to 02/2017, using a proprietary multiplex RT-PCR. RESULTS: A total of 322 children of the study group and 464 children of the control group were analyzed for clinical symptoms, laboratory findings, antibiotic treatment, and length of hospital stay. There was no significant reduction (P < .05) of antibiotic treatment and length of hospital stay. CRP, prehospital antibiotic treatment, antibiotic treatment, past medical history, age, and further pathogen detection showed a significant impact on antibiotic therapy, duration of antibiotic treatment, and length of hospital stay. CONCLUSION: The use of the FA did not result in a significant reduction of antibiotic treatment or in length of hospital stay. Other parameters had a more significant impact. Therefore, we suggest that standard operation procedures with therapy guidelines are necessary to provide an effective application of POCT systems.

No SARS-CoV-2 detection in the German CAPNETZ cohort of community acquired pneumonia before COVID-19 peak in March 2020.

PURPOSE: The first SARS-CoV-2 cases in Europe were reported in January 2020. Recently, concern arose on unrecognized infections before this date. For a better understanding of the pandemic, we retrospectively analyzed patient samples for SARS-CoV-2 from the prospective CAPNETZ study cohort. METHODS: We used nasopharyngeal swab samples from a cohort of well characterized patients with community acquired pneumonia of the CAPNETZ study group, recruited from different geographic regions across Germany, Austria, the Netherlands, and Switzerland between 02nd December 2019 and 28th April 2020. Multiplex real-time RT-PCR for a broad range of respiratory pathogens and SARS-CoV-2 real-time RT-PCR were performed on all samples. RESULTS: In our cohort, respiratory pathogens other than SARS-CoV-2 were detected in 21.5% (42/195) of patients with rhinovirus as the most frequently detected pathogen. The detection rate increased to 29.7% (58/195) when SARS-CoV-2 was included. No SARS-CoV-2 positive sample was detected before end of March 2020. CONCLUSIONS: Respiratory viral pathogens accounted for a considerable number of positive results but no SARS-CoV-2 case was identified before the end of March 2020.