Real-time Real-time Reverse Transcriptase-polymerase Chain Reaction Research Articles

Optimization of an allelic discrimination real-time RT-PCR assay for detection of H275Y oseltamivir resistance gene mutation among influenza A(H1N1)pdm09 patients from 2020 to 2022.

Influenza virus is known to cause mild to severe respiratory infections and is also prone to genetic mutations. Of all the mutations, neuraminidase (NA) gene mutations are a matter of concern, as most approved antivirals target this protein. During the 2020 influenza season, an emergence of mutation in the NA gene, affecting the binding of the World Health Organization (WHO)-recommended probes to the specific site of the NA gene, was reported by our group. As a result of this mutation, the WHO-recommended allelic discrimination real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was unable to detect wild-type (H275) or mutant oseltamivir-resistant (Y275) strains of influenza A(H1N1)pmd09 viruses. In the current study, the WHO-recommended probes were redesigned according to the mutation in the probe binding site. Fifty undetermined samples (2020-2021) from the previous study were retested with the newly designed probes and found to be positive for H275 and/or Y275. The results obtained were similar to the Sanger sequencing results from the previous study, suggesting that the redesigned probes were efficient in discriminating between wild-type and mutant-type viruses. Furthermore, 133 samples from 2022, making a total of 183 samples (2020-2022), were tested using improved allelic discrimination real-time RT-PCR, and the overall prevalence rate of oseltamivir resistance in 2020-2022 was found to be 0.54%.

Epidemiological and Genetic Characteristics of Mycoplasma pneumoniae Pneumonia after the Outbreak of COVID-19

Abstract Objective Mycoplasma pneumoniae pneumonia (MPP) accounts for a major part of community-acquired pneumonia in children, and we performed this study to investigate the epidemiological and genetic characteristics of MPP after the outbreak of COVID-19. Methods A total of 15,538 throat swab samples were collected from inpatients with respiratory tract infections from January 2021 to December 2021. All specimens were detected by real-time reverse transcriptase polymerase chain reaction (RT-PCR). The P1 gene of Mycoplasma pneumoniae (MP) in positive samples was amplified and sequenced. Results From January 2021 to December 2021, a total of 15,538 children with acute respiratory tract infection were tested by real-time RT-PCR in our study, including 9,056 boys and 6,482 girls. Overall, 469 (3.0%, 469/15,538) tested positive for MP, with 266 (2.9%, 266/9,056) males and 203 (3.1%, 203/6,482) females (p = 0.48). The positive rates of MP infection in < 1 year old, 1 to 3 years old, 3 to 5 years old, 5 to 7 years old, and >7 years old groups were 1.31% (85/6,474), 1.87% (64/3,423), 3.65% (95/2,601), 8.02% (127/1,583), and 6.72% (98/1,458), respectively. The homology analysis of the P1 gene of 24 MP positive samples showed that the nucleotide sequence consistency was up to 91.2 to 99.3%. Conclusion After the outbreak of COVID-19, positive detection rate was the highest from 5 to 7 years old among children with MPP, and the genotype of MP in Hangzhou, China area was focused on P1 subtype, type I.

Performance of MassARRAY system for the detection of SARS-CoV-2 compared to real-time PCR.

Early identification of COVID-19 (coronavirus disease of 2019) by diagnostic tests played an important role in the isolation of infectious patients and management of this pandemic. Various methodologies and diagnostic platforms are available. The current "gold standard" for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) diagnosis is real-time reverse transcriptase-polymerase chain reaction (RT-PCR). To overcome the limitations posed by the short supply experienced early during the pandemic and to increase our capacity, we assessed the performance of the MassARRAY System (Agena Bioscience). MassARRAY System (Agena Bioscience) combines RT-PCR (reverse transcription-polymerase chain reaction) with high-throughput mass spectrometry processing. We compared the MassARRAY performance to a research-use-only E-gene/EAV (Equine Arteritis Virus) assay and RNA Virus Master PCR. Discordant results were tested with a laboratory-developed assay using the Corman etal. E-gene primers and probes. 186 patient specimens were analyzed using the MassARRAY SARS-CoV-2 Panel. The performance characteristics were as follows: the positive agreement was 85.71%, 95% CI (78.12 - 91.45), and the negative agreement was 96.67%, 95% CI (88.47 - 99.59). 19/186 (10.2%) results were found to be discordant and assessed by a different assay with the exception of 1, where the sample was not available for repeat testing. 14 out of 18 agreed with the MassARRAY after testing with the secondary assay. The overall performance after discordance testing was as follows: the positive agreement was 97.3%, 95% CI (90.58 - 99.67), and the negative agreement was 97.14%, 95% CI (91.88 - 99.41). Our study demonstrates that the MassARRAYSystem is an accurate and sensitive method for SARS-CoV-2 detection. Following the discordant agreement with an alternate RT-PCR test, the performance was found to have sensitivity, specificity, and accuracy exceeding 97%, making it a viable diagnostic tool. It can be used as an alternative method during periods when real-time RT-PCR reagent supply chains are disrupted.

The Impact of Childhood and Parental Vaccination on SARS-CoV-2 Infection Rates in Children.

Background:The data on the indirect protection of children via the coronavirus disease 2019 (COVID-19) vaccination of household members are insufficient, and analyses to evaluate the efficacy of COVID-19 vaccines are limited.Methods:We gathered data on 12,442 patients under the age of 18 regarding the vaccination status of their household members, their vaccine preferences and doses, and their previous history of COVID-19 infection immediately before the patients were administered a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) between September 1, 2021 and December 5, 2021.Results:A total of 18.4% (2289) were vaccinated, 91.4% with BNT162b2mRNA vaccine, 8.6% with inactivated COVID-19 vaccine; 48.7% received a single dose, and 51.3% had 2 doses. Real-time RT-PCR positivity proportions were much higher in older children (P < 0.001) and were higher in children 12 years of age and older [odds ratio (OR), 1.34; 95% confidence interval (CI): 1.21–1.47] compared with others. SARS-CoV-2 infection was significantly lower in the vaccinated group (fully and incompletely) (P < 0.001). Unvaccinated (OR, 4.88; 95% CI: 3.77–6.13) and incompletely vaccinated children (OR, 1.83; 95% CI: 1.52–2.12) had a higher risk of COVID-19 infection compared with fully vaccinated patients No significant association was found between the COVID-19 real-time RT-PCR positivity rates of patients and the vaccination status or vaccine preferences of household members (P > 0.05 each).Conclusions:SARS-CoV-2 infection rates were significantly lower in vaccinated children, especially with mRNA vaccines. The indirect protection of unvaccinated children via the vaccination of household members against COVID-19 seems inadequate. The individual vaccination of children remains crucial.

Association of SARS-CoV-2 Seropositivity and Symptomatic Reinfection in Children in Nicaragua

The impact of the SARS-CoV-2 pandemic on children remains unclear. Better understanding of the burden of COVID-19 among children and their risk of reinfection is crucial, as they will be among the last groups vaccinated. To characterize the burden of COVID-19 and assess how risk of symptomatic reinfection may vary by age among children. In this prospective, community-based pediatric cohort study conducted from March 1, 2020, to October 15, 2021, 1964 nonimmunocompromised children aged 0 to 14 years were enrolled by random selection from the Nicaraguan Pediatric Influenza Cohort, a community-based cohort in District 2 of Managua, Nicaragua. Additional newborn infants aged 4 weeks or younger were randomly selected and enrolled monthly via home visits. Prior COVID-19 infection as confirmed by positive anti-SARS-CoV-2 antibodies (receptor binding domain and spike protein) or real-time reverse transcriptase-polymerase chain reaction (RT-PCR)-confirmed COVID-19 infection at least 60 days before current COVID-19 infection. Symptomatic COVID-19 cases confirmed by real-time RT-PCR and hospitalization within 28 days of symptom onset of a confirmed COVID-19 case. This cohort study assessed 1964 children (mean [SD] age, 6.9 [4.4] years; 985 [50.2%] male). Of 1824 children who were tested, 908 (49.8%; 95% CI, 47.5%-52.1%) were seropositive during the study. There were also 207 PCR-confirmed COVID-19 cases, 12 (5.8%) of which were severe enough to require hospitalization. Incidence of COVID-19 was highest among children younger than 2 years (16.1 cases per 100 person-years; 95% CI, 12.5-20.5 cases per 100 person-years), which was approximately 3 times the incidence rate in any other child age group assessed. In addition, 41 symptomatic SARS-CoV-2 episodes (19.8%; 95% CI, 14.4%-25.2%) were reinfections. In this prospective, community-based pediatric cohort study, rates of symptomatic and severe COVID-19 were highest among the youngest participants, with rates stabilizing at approximately 5 years of age. In addition, symptomatic reinfections represented a large proportion of symptomatic COVID-19 cases.

DirectDetect SARS-CoV-2 Direct Real-Time RT-PCR Study Using Patient Samples.

COVID-19 is an infectious disease that caused a global pandemic affecting people worldwide. As disease detection and vaccine rollout continue to progress, there is still a need for efficient diagnostic tools to satisfy continued testing needs. This preliminary study evaluated a novel SARS-CoV-2 diagnostic test called DirectDetect SARS-CoV-2 Direct Real-time reverse transcriptase polymerase chain reaction (RT-PCR) based on a limited sample size of 24 respiratory samples from 14 SARS-CoV-2-positive patients. The test is advantageous compared to others on the market since it does not require viral transport medium or viral RNA extraction prior to nucleic acid amplification and detection. This capability transforms the hours-long sample preparation time into a minutes-long procedure while also eliminating the need for many costly reagents which may be difficult to obtain during the surge in nucleic acid-based testing during the pandemic. The results show a positive agreement of 94.7, 100, and 94.7% between dry sample swabs, treated samples, and untreated samples tested using the DirectDetect SARS-CoV-2 Direct Real-time RT-PCR compared to tests used in a clinical laboratory, respectively. The findings indicate that DirectDetect can be used for multiple different sample types while reducing the number of reagents and time needed for diagnosis. Although this study shows promising results using the DirectDetect results, further validation of this test using a larger sample set is required to assess the true performance of this test.

Predictive Value of Chest CT Score in Assessing Disease Severity and Short-term Mortality in COVID-19 Pneumonia at a Tertiary Care Centre in Northern India: A Prospective Observational Study

Introduction: Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV-2) infection, also known as Coronavirus Disease2019 (COVID-19) is the global pandemic, first described in Wuhan city of China in December of 2019. Its diagnosis depends upon real-time Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). On chest Computerised Tomography (CT), it is almost similar to other viral pneumonia with extensive parenchymal involvement. Semiquantitative scores depicting this extensiveness of involvement could correlate with disease severity, laboratory parameters, mortality, like Intensive Care Unit (ICU) admission, requirements of ventilatory support and longer hospital stay. Aim: To define role of chest CT score in determining disease severity, predicting poor prognosis and mortality of COVID-19 pneumonia in short-term follow-up. Materials and Methods: This prospective study enrolled all admitted real-time RT-PCR positive patients for COVID19 at All India Institute of Medical Sciences, Rishikesh, India between 15th April and 31st May 2021. All patients were assigned semiquantitative CT scores based on the extent of lung parenchymal involvement of 20 lung regions in chest CT. Clinical severity was matched with chest CT scoring and laboratory findings. Survival curves along with univariate and multivariate analysis were applied to define the role of CT scoring in predicting short term prognosis. Results: Total 547 subjects were included in the study, of which the chest CT score showed a significant association with clinical severities (p-value &lt;0.001). CT score were correlating significantly with increased serum C-Reactive Protein (CRP) (p-value=0.001) and D-dimer (p-value=0.01), and decreased lymphocyte count (p-value=0.003). A CT score ≥31 was found to be associated with an increased risk of mortality in both univariate and multivariate analysis {Odd Ratio (OR)=276.8; 95% Confidence Interval (CI)= 45.21-1695.43; p-value &lt;0.001}. Conclusion: Chest CT score can be imaging measure of disease severity and predict a higher probability of mortality in score ≥31. It can also predict other defined variables of short-term prognosis. So, it has an advantage in speedy diagnostic workflow of symptomatic cases, timely referral of patients to higher centre, and better management of critical care resources.

Assessment of SARS-CoV-2 RNA shedding in semen of 36 males with symptomatic, asymptomatic, and convalescent infection during the first and second wave of COVID-19 pandemic in Italy.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) receptor, angiotensin-converting enzyme 2 (ACE2), has been identified in the human testis, but the risk of transmission of SARS-CoV-2 through sexual intercourse still needs to be defined. The goal of our study was to determine if SARS-CoV-2 is detectable in the semen of patients suffering or recovering from coronavirus disease-19 (COVID-19), still testing positive at nasopharyngeal swabs but showing mild or no symptoms at the time of sampling. Detection of SARS-CoV-2 RNA in semen was performed by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR targeting open reading frame (ORF) 1ab. Medical history of the enrolled patients was taken, including COVID-19-correlated symptoms, both at the time of diagnosis and at the time of interview. Results of real-time RT-PCR and nested PCR in semen showed no evidence of SARS-CoV-2 RNA in the 36 patients suffering or recovering from COVID-19 but still positive in a nasopharyngeal swab, from over 116 patients enrolled in the study. SARS-CoV-2 detection and persistence in semen would have an impact on both clinical practice and public health strategies, but our results would suggest that SARS-CoV-2 is not present in the semen of men recovering from COVID-19.

149. Extraction-free RT-PCR to Detect SARS-CoV-2 Variants of Concern

BackgroundSARS-CoV-2 variants of concern (VOC) have challenged real-time reverse transcriptase polymerase chain reaction (RT-PCR) methods for the diagnosis of COVID-19. MethodsThe CDC 2019-Novel Coronavirus real-time RT-PCR panel was modified to create a single-plex extraction-free proxy RT-PCR assay, VOCFast™. This assay uses the nucleocapsid N1 as well as novel primer/probe pairs to target VOC mutations in the Orf1a and spike (S) genes. For analytical validation of VOCFast, synthetic controls for the Wuhan, alpha/B.1.1.7, beta/B.1.351, and gamma/P.1 strains were tested at various concentrations. Clinical validation was performed using patient anterior nares swab and saliva specimens collected in the Denver, CO area between Nov 2020 and Feb 2021 or in March 2021. Orthogonal next-generation sequencing (NGS) was also performed. ResultsSimilar N1 quantification cycle (Cq) values corresponding to viral load were observed for all strains, suggesting that VOC mutations do not affect performance of the N1 primer/probe. Orf1a-mut and S1-mut primer/probes generated a stable high Cq value for the Wuhan strain. Conversely, Orf1a-mut Cq values were inversely correlated with viral load for all VOC. The S1-mut Cq was inversely correlated with viral load of the alpha strain, but did not reliably amplify beta/gamma VOC. The limit of detection was 8 copies/uL. The first set of COVID-19 patient specimens revealed no amplification using Orf1a-mut whereas 53% of specimens collected in Mar 2021 demonstrated amplification by Orf-1a. Orthogonal testing by the SARS-CoV-2 NGS Assay and COVID-DX software demonstrated that 12/12 alpha strains, 2/2 beta/gamma strains, and 33/33 Wuhan strains were correctly identified by VOCFast. Detection of VOC in clinical specimens and validation by NGS ConclusionThe combination of the N1, Orf1a-mut, and S1-mut primers/probes in VOCFast can distinguish the Wuhan, alpha, and beta/gamma strains and it consistent with NGS results. Testing of clinical samples revealed that VOC emerged in Denver, CO in March 2021. Future work to discriminate beta, gamma, and emerging VOC is ongoing. In summary, VOCFast is an extraction-free RT-PCR assay for nasal swab and saliva specimens that can identify VOC with a turnaround time suitable for clinical testing. Disclosures Brian L. Harry, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder) Mara Couto-Rodriguez, MS, Biotia (Employee) Dorottya Nagy-Szakal, MD PhD, Biotia Inc (Employee, Shareholder) Niamh B. O’Hara, PhD, Biotia (Board Member, Employee, Shareholder) Shi-Long Lu, MD PhD, Summit Biolabs Inc. (Grant/Research Support, Shareholder)

An Insight Into Detection Pathways/Biosensors of Highly Infectious Coronaviruses.

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.

Performance of the Diasorin SARS-CoV-2 antigen detection assay on the LIAISON XL.

BackgroundThe current reference standard to diagnose a SARS-CoV-2 infection is real-time reverse transcriptase polymerase chain reaction (RT-PCR). This test poses substantial challenges for large-scale community testing, especially with respect to the long turnaround times. SARS-CoV-2 antigen tests are an alternative, but typically use a lateral flow assay format rendering them less suitable for analysis of large numbers of samples.MethodsWe conducted an evaluation of the Diasorin SARS-CoV-2 antigen detection assay (DAA) compared to real-time RT-PCR (Abbott). The study was performed on 248 (74 qRT-PCR positive, 174 qRT-PCR negative) clinical combined oro-nasopharyngeal samples of individuals with COVID-19-like symptoms obtained at a Municipal Health Service test centre. In addition, we evaluated the analytical performance of DAA with a 10-fold dilution series of SARS-CoV-2 containing culture supernatant and compared it with the lateral flow assay SARS-CoV-2 Roche/SD Biosensor Rapid Antigen test (RRA).ResultsThe DAA had an overall specificity of 100% (95%CI 97.9%–100%) and sensitivity of 73% (95%CI 61.3%–82.7%) for the clinical samples. Sensitivity was 86% (CI95% 74.6%–93.3%) for samples with Ct-value below 30. Both the DAA and RRA detected SARS-CoV-2 up to a dilution containing 5.2 × 102 fifty-percent-tissue-culture-infective-dose (TCID50)/ml.DiscussionThe DAA performed adequately for clinical samples with a Ct-value below 30. Test performance may be further optimised by lowering the relative light unit (RLU) threshold for positivity assuming the in this study used pre-analytical protocol . The test has potential for use as a diagnostic assay for symptomatic community-dwelling individuals early after disease onset in the context of disease control.

Human Saliva as a Diagnostic Specimen for Early Detection of Inflammatory Biomarkers by Real-Time RT-PCR.

Nowadays human saliva is more frequently studied as a non-invasive, stress-free, and preferable diagnostic material than blood. Supporting evidences acknowledge saliva as a mirror that reflects the body’s physical state. Numerous studies have also demonstrated the presence and use of RNA derived from saliva in the early diagnosis of disease by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Assessing the host inflammatory response in patients and its resolution at an early stage can serve as a prognostic and predictive method in determining therapeutic response or disease progression. In this context, the potential of saliva as a specimen to diagnose early inflammatory biomarkers using RT-PCR seems fascinating and useful. Here, we review inflammatory biomarkers within the saliva, focusing on early detection of these biomarkers using RT-PCR and the factors influencing the quality of saliva specimen.

Saliva Samples for Detection of SARS-CoV-2 in Mildly Symptomatic and Asymptomatic Patients

Background: The ongoing Covid-19 pandemic has been rapidly spreading throughout the world with confirmed case numbers already exceeding 10 millions. Implementation of infection control measures and public health interventions through timely diagnosis of suspected cases seems the most efficient way to control the outbreak.The reference standard for Covid-19 diagnosis is real-time reverse transcriptase polymerase chain reaction (RT-PCR) based SARS-CoV-2 RNA detection in respiratory samples. Although nasopharyngeal swabs are the most commonly utilized samples for diagnosis, collecting these specimens requires healthcare workers, causes discomfort for patients and necessitates the use of personal protective equipment since it presents a nosocomial transmission risk.We aimed to assess the diagnostic value of saliva samples in mildly symptomatic and asymptomatic patients with confirmed Covid-19 disease.Methods: We did a cohort study to validate the use of saliva for SARS-CoV-2 detection in mildly symptomatic and asymptomatic patients with confirmed diagnosis of Covid-19. We obtained self-collected saliva samples from patients within 12 hours after the diagnosis of Covid-19 provided by RT-PCR on nasopharyngeal swabs. Saliva samples of the patients were analyzed by RT-PCR.Findings: In May 2020, 28 asymptomatic and 25 mildly symptomatic patients were enrolled in the study. The median age was 37 years (range 4-70). None of the patients had fever on presentation. Nine (%17) patients had patchy ground-glass opacities on chest tomography.Among 53 patients with SARS-CoV-2 detected in the nasopharyngeal sample, the real-time RT-PCR was positive in the saliva specimens in 48 (90·56%) patients. The mean cycle threshold (CT) values for nasopharyngeal and saliva specimens (27·80±3·44 and 30·64±2·83, respectively) were significantly correlated between the two sample types (p=0·016).The mean CT values of nasopharyngeal and saliva samples in mildly symptomatic and asymptomatic patients (27·18±3·53 and 30·24±3·29 vs. 28·36±3·31 and 30·98±2·39, respectively) were not significantly different (p=0·236 and p=0·733, respectively).The mean CT values for nasopharyngeal and saliva specimens in patients with evidence of pneumonia on tomography were lower than those without pneumonia (26·23±3·56 and 29·15±3·90 vs. 28·12±3·36 and 30·94±2·52, respectively) although the difference was not significant (p=0·077 and p=0·154, respectively).Interpretation: The sensitivity of SARS-CoV-2 detection from saliva is comparable to nasopharyngeal swabs in mildly symptomatic and asymptomatic patients with Covid-19. Saliva specimens can be considered as a reliable and less resource intensive alternative to nasopharyngeal specimens for screening asymptomatic SARS-CoV-2 infections.Funding Statement: This study was supported by donation of qPCR Detection Kit from Bioeksen (Bioeksen R&D, Istanbul, Turkey).Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: This study was approved by both the Republic of Turkey Ministry of Health COVID-19 Scientific Research Evaluation Commission (Approval date: 02/05/2020; number: 2020-05- 02T16-07-46) and the Local Ethics Committee of Kafkas University Faculty of Medicine (Approval date: 06/05/2020 number: 80576354-050-99/131). The informed consent form had been obtained from each patient before the saliva specimens were collected.

Pre-Operative SARS CoV-2 Rapid Antigen Test and Reverse Transcription Polymerase Chain Reaction: A conundrum in surgical decision making.

Purpose:Since the eye is in close proximity to the oro-nasal cavity, transmission of SARS CoV-2 is higher during ophthalmic surgical procedures, it is vital to ensure the safety of the healthcare team by pre-operative Rapid Antigen Test (RAT) and real-time Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for SARS CoV-2 viral RNA of all patients planned for elective or emergency surgery. Hence, understanding the profile of pre-operative RAT and real-time RT-PCR among patients planned for various ophthalmic procedures, will help us make better decisions for future guidelines, for optimal planning of ophthalmic elective or emergency surgeries, keeping in mind the current scenario.Methods:This is a prospective descriptive study conducted on patients planned for elective or emergency ophthalmic surgeries, who had no COVID-19-related symptoms pre-operatively and were subjected to preoperative RAT and real-time RT-PCR of nasopharyngeal and throat swabs for SARS CoV-2 viral RNA. Data was compiled and statistically analysed.Results:204 patients underwent pre-operative RAT and RT-PCR testing; of which, 85 were females and 119 were males. Mean age of the study population was 51.44 ± 16.501 years. Among the 204, 2 tested positive for SARS CoV-2 with Rapid Antigen testing as well as RT-PCR and 10 patients tested positive via RT-PCR after a negative RAT result of the same sample.Conclusion:Pre-operative testing for COVID-19 disease is indispensable as there is a high chance of transmission from patient to healthcare workers. RAT has the advantage of quick results and may play a role in case of emergency procedures. However, it is prudent to perform the more sensitive real-time RT-PCR before any elective, non-emergency procedures or surgeries to avoid any undue risk to the healthcare team.

Self-Sampling of Oropharyngeal Swabs Among Healthcare Workers for Molecular Detection of Respiratory Viruses: A Valuable Approach for Epidemiological Studies and Surveillance Programs.

This study aimed at assessing the validity of self-collected (self-sampled) oropharyngeal (OP) swabs among healthcare workers compared to those collected by trained sentinel general practitioners (GP-sampled) from individuals with influenza-like illness (ILI), to be implemented in epidemiological studies and/or surveillance programs of viral pathogens involved in community respiratory infections. In our study, OP swabs were collected from adults (>18 years) with ILI during the 2018–2019 influenza season. Two groups of samples were considered: group 1−131 self-sampled OP swabs collected by healthcare workers after being trained on the sampling procedure; group 2−131 GP-sampled OP swabs collected from outpatients by sentinel GPs operating within the Italian Influenza Surveillance Network. To assess swabbing quality, following RNA extraction, each sample was tested for the presence of the human ribonuclease P gene (RNP) by in-house real-time reverse transcriptase–polymerase chain reaction (RT-PCR). Samples with a cycle threshold (Ct) <35 were considered adequate for further virological analysis. Influenza viruses (IVs), respiratory syncytial virus (RSV), and rhinovirus (RV) genomes were detected by in-house real-time RT-PCR. All samples were positive to RNP detection with Ct <35. The mean Ct value was similar in the two groups (group 1 vs. group 2: 25.93 ± 2.22 vs. 25.46 ± 2.40; p = 0.10). IVs, RSV, and RV positivity rates were 26.7 vs. 52.7% (p < 0.01), 7.6 vs. 9.9% (p = 0.52), and 21.4 vs. 19.9% (p = 0.76), respectively. Self-sampled OP swabs resulted as valid as GP-sampled OP swabs for molecular detection of respiratory viruses. Self-swabbing can thus be a worthwhile strategy for sample collection to implement molecular surveillance of respiratory pathogens and carry out epidemiological studies, easily reaching a larger population size.

Detection of anti-HEV antibodies and RNA of HEV in pigs from a hyperendemic Italian region with high human seroprevalence.

Pigs are considered the main reservoir of genotypes 3 and 4 of hepatitis E virus (HEV), which is the major cause of acute hepatitis of viral origin in humans worldwide. An increasing number of autochthonous HEV infections have been observed in recent years in industrialized countries, most likely as a result of zoonotic transmission through the consumption of raw or undercooked meat products. Two hundred and thirty-three blood and liver samples were collected at four different local slaughterhouses from domestic pigs bred in Abruzzo, a region of south-central Italy, where there is the highest human seroprevalence to HEV compared with the rest of Italy. An indirect enzyme-linked immunosorbent assay kit was used for detecting anti-HEV IgG in the sera, while the presence of HEV RNA was investigated by performing a real-time reverse transcriptase-polymerase chain reaction (RT-PCR). Between 87.3% and 100% of swine serum samples collected in different slaughterhouses of Abruzzo were positive for anti-HEV antibodies. Conversely, none of the liver samples collected from the same animals were positive for HEV by real-time RT-PCR. The hypothesis of foodborne zoonotic transmission from local pigs as responsible for the hyperendemic status of Abruzzo cannot be corroborated. However, the high seroprevalence observed in pigs indicates that HEV is highly circulating in these territories. We propose to further investigate the role of wild fauna and trade in carrier pigs, and the maintenance of HEV virulence in the environment and meat supply chain to shed light on the possible sources of human infection and the degree of occupational risk.

A TaqMan-MGB real-time RT-PCR assay with an internal amplification control for rapid detection of Muscovy duck reovirus

A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for the detection of Muscovy duck reovirus (MDRV) RNA in clinical samples is described. The assay is based on TaqMan-MGB technology, consisting of two primers and one probe labeled with the reporter dye 6-carboxyfluorescein that binds selectively to the sigma B-protein gene of MDRV. This technique also includes an Internal Positive Control (IPC). The real-time RT-PCR assay was able to detect MDRVs, whereas other common waterfowl-origin viral pathogens were not recognised by the established oligonucleotide set, thus showing that the test was specific for MDRV. The sensitivity of the assay was 2.83 × 101 copies/μL and was 100 times higher than that of the conventional RT-PCR. The variation coefficients of intra-assay and inter-assay were less than 1.5% which verified sufficient repeatability of this assay.The use of β-actin mRNA as an IPC in order not to reduce the efficiency of the assay was adopted. The detection for 100 clinical samples showed that the positive rate of the established TaqMan-MGB real-time RT-PCR method was 87% (87/100), while the positive rate of the conventional RT-PCR was 83% (83/100), with the coincidence rate was 97.14%. Sensitivity and positive rate for clinical samples of TaqMan fluorescent quantitative RT-PCR were higher than conventional RT-PCR. The high specificity, sensitivity, and rapidity TaqMan-MGB real-time RT-PCR assay with the use of IPC to monitor for false negative results can make this method suitable for the pathogenic surveillance and epidemiological investigation of MDRV infection.

Multifocal outbreak of equine influenza in vaccinated horses in Argentina in 2018: Epidemiological aspects and molecular characterisation of the involved virus strains.

Equine influenza is an important cause of respiratory disease of horses worldwide. The equine influenza virus (EIV) undergoes antigenic drift through the accumulation of amino acid substitutions in the viral proteins, which may lead to vaccine breakdown. To describe the epidemiological findings and the molecular characteristics of the EIV detected during the multifocal outbreak that occurred in Argentina between March and July 2018 and evidence a vaccine breakdown. Observational, descriptive study. Virus was detected in nasopharyngeal swabs using real-time reverse transcriptase PCR (RT-PCR). Nucleotide and deduced amino acid sequences of the haemagglutinin (HA) and neuraminidase (NA) genes were obtained from EIV positive nasopharyngeal swabs, and phylogenetic analysis was undertaken. Amino acid sequences were compared against the current World Organisation for Animal Health (OIE)-recommended Florida clade 1 vaccine strain and strain components of vaccines used in Argentina. Serum samples were tested using haemagglutination inhibition test. Equine influenza virus infection was confirmed using real-time RT-PCR and serological testing. The phylogenetic analysis of the HA and NA genes revealed that all the EIV identified during the outbreak belong to the H3N8 subtype, Florida clade 1. Multiple amino acid changes, some of them at antigenic sites, were observed in the circulating virus when compared with the strains included in the most commonly used vaccine in Argentina. Seventy-six percent of the affected horses had been vaccinated with this vaccine, suggesting the occurrence of vaccine breakdown. The study does not include antigenic characterisation and full genome sequencing of Argentinian strains, that could provide additional information. The occurrence of this multifocal equine influenza outbreak in regularly vaccinated horses is a field evidence of vaccine breakdown, reinforcing the necessity of keeping vaccine strains updated according to OIE recommendations. It also underlines the importance of the implementation of appropriate quarantine measures and restriction of horse movement in the face of disease.

Immune Response during Influenza Virus Infection among the Population of Assam, Northeast India

AbstractIntroduction: The pathogenicity of influenza virus infection is modulated by the cytokine expressions in patients. The present study was aimed to measure some important pro- and anti-inflammatory cytokines in influenza-infected population of Assam, Northeast India. Materials and Methods: Influenza viruses consisting of subtypes influenza A(H1N1)pdm09, H3N2 and influenza-B were detected in patients with symptoms of influenza-like-illness by Real-time reverse transcriptase polymerase chain reaction (RT-PCR) method. Relative messenger ribonucleic acid (mRNA) quantification of four pro-inflammatory cytokines (interleukin [IL]-6, IL-8, interferon-gamma [IFN-γ] and tumour necrosis factor-alpha [TNF-α]) and one anti-inflammatory cytokine (IL-10) were measured in influenza-positive cases and non-influenza controls, by real-time RT-PCR. The plasma concentration of the cytokines was determined using cytometric-bead-array with flow cytometry. Results: Influenza viruses were detected in 14.28% (50/350) of 350 patients screened. The expression of IL-6 was significantly raised in cases compared to controls (P = 0.018). IL-8 and IL-10 were also raised in cases, compared to controls (P = 0.284 and P = 0.018). An increased plasma TNF-α was observed in cases (1.36-fold and P = 0.289). The mRNA expression of IFN-γ was also increased in cases compared to controls (0.87-fold). However, the plasma level of IFN-γ was higher in the non-influenza controls compared to cases. Conclusions: The study revealed a differential cytokine profile during influenza virus infection in the population, which may influence disease severity. An extended study on host immune response may provide better insights for the use of cytokine antagonists in therapeutic treatments among severe cases of influenza virus infection.

Validation of the Seegene RV15 multiplex PCR for the detection of influenza A subtypes and influenza B lineages during national influenza surveillance in hospitalized adults.

Background The Serious Outcomes Surveillance Network of the Canadian Immunization Research Network (CIRN SOS) has been performing active influenza surveillance since 2009 (ClinicalTrials.gov identifier: NCT01517191). Influenza A and B viruses are identified and characterized using real-time reverse-transcriptase polymerase chain reaction (RT-PCR), and multiplex testing has been performed on a subset of patients to identify other respiratory virus aetiologies. Since both methods can identify influenza A and B, a direct comparison was performed.Methods Validated real-time RT-PCRs from the World Health Organization (WHO) to identify influenza A and B viruses, characterize influenza A viruses into the H1N1 or H3N2 subtypes and describe influenza B viruses belonging to the Yamagata or Victoria lineages. In a subset of patients, the Seeplex RV15 One-Step ACE Detection assay (RV15) kit was also used for the detection of other respiratory viruses.Results In total, 1111 nasopharyngeal swabs were tested by RV15 and real-time RT-PCRs for influenza A and B identification and characterization. For influenza A, RV15 showed 98.0 % sensitivity, 100 % specificity and 99.7 % accuracy. The performance characteristics of RV15 were similar for influenza A subtypes H1N1 and H3N2. For influenza B, RV15 had 99.2 % sensitivity, 100 % specificity and 99.8 % accuracy, with similar assay performance being shown for both the Yamagata and Victoria lineages.Conclusions Overall, the detection of circulating subtypes of influenza A and lineages of influenza B by RV15 was similar to detection by real-time RT-PCR. Multiplex testing with RV15 allows for a more comprehensive respiratory virus surveillance in hospitalized adults, without significantly compromising the reliability of influenza A or B virus detection.