Routine Reverse Transcription-polymerase Chain Reaction Research Articles

An Agreement of Antigen Tests on Oral Pharyngeal Swabs or Less Invasive Testing With Reverse Transcription Polymerase Chain Reaction for Detecting SARS-CoV-2 in Adults: Protocol for a Prospective Nationwide Observational Study.

BackgroundThe SARS-CoV-2 pandemic has resulted in an unprecedented level of worldwide testing for epidemiologic and diagnostic purposes, and due to the extreme need for tests, the gold-standard Reverse Transcription Polymerase Chain Reaction (RT-PCR) testing capacity has been unable to meet the overall worldwide testing demand. Consequently, although the current literature has shown the sensitivity of rapid antigen tests (RATs) to be inferior to RT-PCR, RATs have been implemented on a large scale without solid data on performance.ObjectiveThis study will compare analytical and clinical sensitivities and specificities of 50 lateral flow– or laboratory-based RATs and 3 strand invasion–based amplification (SIBA)-RT-PCR tests from 30 manufacturers to RT-PCR testing of samples obtained from the deep oropharynx. In addition, the study will compare sensitivities and specificities of the included RATs as well as RT-PCR on clinical samples obtained from the deep oropharynx, the anterior nasal cavity, saliva, the deep nasopharynx, and expired air to RT-PCR on deep oropharyngeal samples.MethodsIn the prospective part of the study, 200 individuals found SARS-CoV-2 positive and 200 individuals found SARS-CoV-2 negative by routine RT-PCR testing will be retested with each RAT, applying RT-PCR as the reference method. In the retrospective part of the study, 304 deep oropharyngeal cavity swabs divided into 4 groups based on RT-PCR quantification cycle (Cq) levels will be tested with each RAT.ResultsThe results will be reported in several papers with different aims. The first paper will report retrospective (analytical sensitivity, overall and stratified into different Cq range groups) and prospective (clinical sensitivity) data for RATs, with RT-PCR as the reference method. The second paper will report results for RAT based on anatomical sampling location. The third paper will compare different anatomical sampling locations by RT-PCR testing. The fourth paper will focus on RATs that rely on central laboratory testing. Tests from 4 different manufacturers will be compared for analytical performance data on retrospective deep oropharyngeal swab samples. The fifth paper will report the results of 4 RATs applied both as professional use and as self-tests. The last paper will report the results from 2 breath tests in the study. A comparison of sensitivity and specificity between RATs will be conducted using the McNemar test for paired samples and the chi-squared test for unpaired samples. Comparison of the positive predictive value (PPV) and negative predictive value (NPV) between RATs will be performed by the bootstrap test, and 95% CIs for sensitivity, specificity, PPV, and NPV will be calculated as bootstrap CIs.ConclusionsThe study will compare the sensitivities of a large number of RATs for SARS-CoV-2 to with those of RT-PCR and will address whether lateral flow–based RATs differ significantly from laboratory-based RATs. The anatomical test locations for both RATs and RT-PCR will also be compared.Trial RegistrationClinicalTrials.gov NCT04913116; https://clinicaltrials.gov/ct2/show/NCT04913116International Registered Report Identifier (IRRID)DERR1-10.2196/35706

Diagnosis of COVID-19 by Serology in Admitted Patients with Negative RT-PCR Assay

Considering the increasing prevalence and burden of coronavirus disease 2019 (COVID-19) disease and false-negative results in routine reverse transcription-polymerase chain reaction (RT-PCR) tests, additional diagnostic methods are needed to diagnose active cases of this disease. This prospective study was conducted on patients, in whom clinical and radiological symptoms/signs were in favor of COVID-19 while their first PCR test was negative. Later on, a second RT-PCR was performed and serological evaluation was carried out and results were compared with each other. Out of 707 patients who had been referred to the hospital and were clinically and radiologically suspicious of disease, 137 patients with negative RT-PCR tests entered the study. RT-PCR assay became positive for the second time in 45 (32.8%). Anti-COVID-19 IgM and IgG antibodies were positive in 83 (60.6%) and 86 (62.8%) patients, respectively. Finally, it was determined that serological test was diagnostic in 73% of patients and the diagnostic yield of serology was significantly higher after the first week of illness (54.8% in the first week and 88% after that). Taking advantage of both serological tests and RT-PCR helps in diagnosing 83.9% of cases. Based on the present study, the serology may be useful as a complementary test and in parallel to RT-PCR assay for diagnosis of COVID-19 among admitted symptomatic cases.

Optimizing SARS-CoV-2 Surveillance in the United States: Insights From the National Football League Occupational Health Program.

Evidence to understand effective strategies for surveillance and early detection of SARS-CoV-2 is limited. To describe the results of a rigorous, large-scale COVID-19 testing and monitoring program. The U.S. National Football League (NFL) and the NFL Players Association (NFLPA) instituted a large-scale COVID-19 monitoring program involving daily testing using 2 reverse transcription polymerase chain reaction (RT-PCR) platforms (Roche cobas and Thermo Fisher QuantStudio), a transcription-mediated amplification platform (Hologic Panther), and an antigen point-of-care (aPOC) test (Quidel Sofia). 32 NFL clubs in 24 states during the 2020 NFL season. NFL players and staff. SARS-CoV-2 test results were described in the context of medically adjudicated status. Cycle threshold (Ct) values are reported when available. A total of 632370 tests administered across 11668 persons identified 270 (2.4%) COVID-19 cases from 1 August to 14 November 2020. Positive predictive values ranged from 73.0% to 82.0% across the RT-PCR platforms. High Ct values (33 to 37) often indicated early infection. For the first positive result, the median Ct value was 32.77 (interquartile range, 30.02 to 34.72) and 22% of Ct values were above 35. Among adjudicated COVID-19 cases tested with aPOC, 42.3% had a negative result. Positive concordance between aPOC test result and adjudicated case status increased as viral load increased. Platforms varied by laboratory, and test variability may reflect procedural differences. Routine RT-PCR testing allowed early detection of infection. Cycle threshold values provided a useful guidepost for understanding results, with high values often indicating early infection. Antigen POC testing was unable to reliably rule out COVID-19 early in infection. The NFL and the NFLPA.

Rapid and sensitive detection of potato virus X by one-step reverse transcription-recombinase polymerase amplification method in potato leaves and dormant tubers

Potato virus X (PVX), is a serious threat to global potato production. A simple and rapid detection method is imperative for PVX diagnosis and early management. In this study, an isothermal one-step reverse transcription-recombinase polymerase amplification (RT-RPA) method was optimized for the quick and convenient detection of PVX in potato leaves and tubers. Our results revealed that this one-step RT-RPA method was highly efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR). The amplification reaction was free from cross-reactivity with other common potato viruses and completed within 30 min. Moreover, this RT-RPA assay did not require a thermocycler based specific temperature phase amplification and can be easily performed using a simple heating block or water bath at a temperature range of 39–42 °C. The sensitivity assay demonstrated that the developed one-step RT-RPA method was 100 times more sensitive than a routine one-step RT-PCR. Initially, the purified total RNA as the template isolated from infected leaves of potato was used for the detection of PVX. One-step RT-RPA was later performed using cellular disc paper-based simple RNA extract as a template that could detect the virus more efficiently than purified total RNA. The performance of the one-step RT-RPA assay was further evaluated using 500 field samples of leaves and tubers representing different cultivars and geographical regions. To our knowledge, this is the first report of rapid, sensitive, and reliable detection of PVX infection by one-step RT-RPA using cellular disc paper-based simple RNA extract from leaves and dormant tubers of potato. It is superior to the common RT-PCR assay in terms of its versatility, quickness, and independence of highly purified RNA template and can be adopted as a substitute to RT-PCR as an effective technique for seed potato certification, quarantine, breeding, and field surveys.

Implementation and Evolution of Mitigation Measures, Testing, and Contact Tracing in the National Football League, August 9-November 21, 2020.

The National Football League (NFL) and the NFL Players Association (NFLPA) began the 2020 football season in July, implementing extensive mitigation and surveillance measures in facilities and during travel and gameplay. Mitigation protocols* were evaluated and modified based on data from routine reverse transcription-polymerase chain reaction (RT-PCR) tests for SARS-CoV-2, the virus that causes coronavirus 2019 (COVID-19); proximity tracking devices; and detailed interviews. Midseason, transmission was observed in persons who had cumulative interactions of <15 minutes' duration, leading to a revised definition of high-risk contacts that required consideration of mask use, setting and room ventilation in addition to proximity and duration of interaction. The NFL also developed an intensive protocol that imposed stricter infection prevention precautions when a case was identified at an NFL club. The intensive protocol effectively prevented the occurrence of high-risk interactions, with no high-risk contacts identified for 71% of traced cases at clubs under the intensive protocol. The incorporation of the nature and location of the interaction, including mask use, indoor versus outdoor setting, and ventilation, in addition to proximity and duration, likely improved identification of exposed persons at higher risk for SARS-CoV-2 infection. Quarantine of these persons, along with testing and intensive protocols, can reduce spread of infection.

C jak COVID-19 — powikłania układu sercowo-naczyniowego u młodego pacjenta

The authors present the case of a 27-year-old patient with cardiovascular complications after undergoing severe acute respiratory syndrome-related coronavirus 2 infection. The first symptoms included muscle aches and somnolence, severe sore throat, and enlargement of submandibular and cervical lymph nodes, followed by dyspnoea and chest pain with body temperature 37.5°C. Because of the medical profession, the patient underwent a routine reverse-transcription polymerase chain reaction test for coronavirus infection, which was positive, before returning to work. In an electrocardiogram, it was observed pathological Q waves in II, III, aVF, V6, and 1 mm of ST elevation in V1–V3. The laboratory tests showed normal levels of cardiac troponin T, creatine kinase MB fraction, and N-terminal pro-B-type natriuretic peptide. Transthoracic echocardiography revealed the hypokinesis of the middle and apical segment of the anterior and lateral wall with preserved left ventricular ejection fraction (LVEF) — 54%. The magnetic resonance imaging (MRI) revealed the normal volume and global left ventricular systolic function (LVEF 52%) with segmental wall motion abnormalities. Subepicardial banded foci of late gadolinium enhancement (LGE), located in the lower right and left ventricular segments of the basal, central and apical segments of the inferior wall suggested inflammatory aetiology of changes found. It may be assumed that the suspected myocarditis was caused by coronavirus disease 2019 (COVID-19) infection. Since LGE was present in the inferior wall, the patient may be considered having a low risk of major cardiac events and heart failure hospitalizations, referring to the ITAMY study. In patients without a history of cardiovascular diseases with COVID-19, cardiac MRI may reveal preserved, mid-range, or reduced LVEF. The patient requires further follow-up. The coronavirus is the most dangerous for the elderly, but it can also affect the hearts of otherwise healthy people, including young adolescents.

"Test and Standard Precautions"-Is It Enough to Protect Us From False-Negative Severe Acute Respiratory Syndrome Coronavirus 2 Test Results?

"Test and Standard Precautions"-Is It Enough to Protect Us From False-Negative Severe Acute Respiratory Syndrome Coronavirus 2 Test Results?

KHK-A promotes the proliferation of oesophageal squamous cell carcinoma through the up-regulation of PRPS1

Background and study aimsThe metabolism of dietary fructose by ketohexokinase (KHK) is an important step in glucose metabolism in various tumour types. However, the expression, function and underlying mechanisms of KHK in oesophageal squamous cell carcinoma (ESCC) remain largely unclear. The objective of this study was to investigate the effects of KHK-A, a peripheral isoform of KHK, on the proliferation of ESCC cell lines. Material and methodsThe function and mechanism of KHK-A in ESCC cells were investigated by constructing stable KHK-A-knockdown and -overexpressing ESCC cell lines (KYSE410 and KYSE150, respectively). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and colony formation assays were used to analyse the effects of KHK-A on cell proliferation, cell cycle and colony formation, respectively. KHK-A and phosphoribosyl pyrophosphate synthetase isoform 1 (PRPS1) mRNA and protein expressions in several ESCC cell lines were determined using routine reverse transcription-polymerase chain reaction and immunoblotting, respectively. KHK and PRPS1 expressions in ESCC tumour tissues and corresponding adjacent non-tumour tissues were evaluated according to the gene expression omnibus (GEO) database (GSE20347). ResultsIn vitro experiments showed that KHK-A significantly promoted cell proliferation by modulating the G1/S phase transition in the cell cycle, which was probably regulated by PRPS1 expression. GEO database-based analysis showed that KHK levels were significantly higher in the ESCC tissues than in the corresponding adjacent non-tumour tissues. Pearson’s correlation coefficient analysis showed that KHK expression in ESCC cell lines and tissues was significantly positively associated with the up-regulation of PRPS1, suggesting that KHK-A levels regulate PRPS1 expression in ESCC. ConclusionKHK-A may serve as a driving gene in ESCC for the activation of PRPS1, resulting in the up-regulation of PRPS1. This could lead to enhanced nucleic acid synthesis for tumourigenesis. Our study showed that KHK-A is a potential target for ESCC diagnosis and therapy.

First Report of Apple Hammerhead Viroid in the United States, Japan, Italy, Spain, and New Zealand

HomePlant DiseaseVol. 102, No. 12First Report of Apple Hammerhead Viroid in the United States, Japan, Italy, Spain, and New Zealand PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of Apple Hammerhead Viroid in the United States, Japan, Italy, Spain, and New ZealandS. A. Szostek, A. A. Wright, and S. J. HarperS. A. Szostek†Corresponding author: S. A. Szostek; E-mail: E-mail Address: [email protected]http://orcid.org/0000-0002-6185-4558Search for more papers by this author, A. A. WrightSearch for more papers by this author, and S. J. HarperSearch for more papers by this authorAffiliationsAuthors and Affiliations S. A. Szostek † A. A. Wright S. J. Harper , Department of Plant Pathology, Washington State University, Prosser, 99350. Published Online:12 Oct 2018https://doi.org/10.1094/PDIS-04-18-0557-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat In June of 2017, a ‘Honeycrisp’ apple (Malus pumila Mill.) cultivar from central Washington exhibiting shoot decline and dieback symptoms was submitted for diagnosis to the Washington State University Clean Plant Center Northwest (WSU-CPCNW). Total RNA was extracted (RNeasy Plant Mini Kit) from blade, midrib, and petiole tissue of leaves removed from the symptomatic plant. High-throughput sequencing (HTS) library preparation (Illumina TruSeq Stranded Total RNA Kit with Ribo-Zero Plant) and sequencing (Illumina HiSeq 50 Cycle Single Read Sequencing version 4 platform) was performed by Huntsman Cancer Institute (University of Utah, Salt Lake City). Sequence data analysis (CLC Genomics Workbench version 10.1.1) showed that the plant was infected with apple stem grooving virus, apple stem pitting virus, apple chlorotic leaf spot virus, apple green crinkle-associated virus, and apple hammerhead viroid (AHVd), a circular RNA containing hammerhead ribozymes first identified in China (Zhang et al. 2014) and recently shown to be a bona fide viroid (Serra et al. 2018). Approximately 23.5 million 50 nt raw reads were trimmed, filtered, and mapped against the Malus domestica genome to remove host sequences. Contigs were constructed by de novo assembly of the remaining 775,909 reads then subjected to BLAST analysis against the NCBI nucleotide, viroid, and virus reference databases. A single 451-nt contig covering the entire AHVd genome was assembled from 42,925 reads with a mean read coverage of 4,729. This isolate shared 90.2% identity with the AHVd reference sequence KR605506 from China (Zhang et al. 2014) and 88.4% with the Pacific Gala isolate MF402929 from Canada (Messmer et al. 2017). AHVd presence in this plant was confirmed by one-step reverse transcription polymerase chain reaction (RT-PCR) using the primers AHVd-13F_PG and AHVd-12R_PG (Messmer et al. 2017). The sequence of the Washington isolate was deposited in NCBI GenBank under accession number MG662372. On the basis of routine HTS and RT-PCR results, we screened 24 additional apple plants held in isolation and under quarantine that had been submitted by breeding programs and nurseries during 2016 to 2017 to the WSU-CPCNW from the United States and other countries. Screening by RT-PCR, as described above, indicated AHVd presence in 10 additional samples. HTS data were available for 8 of the 10 positive samples and confirmed AHVd presence in an additional sample from Washington, one from Ohio (U.S.A.), one sample each from Japan and New Zealand, and two samples each from Italy and Spain. Full-length or partial sequences of these eight isolates were deposited in NCBI GenBank under accession numbers MG662373 to MG662376, MH049330 to MH049332, and MH049334. Taken with the earlier reports (Messmer et al. 2017; Zhang et al. 2014), these results indicate that AHVd has a worldwide distribution. The aligned region spanning 30 to 427 nt of these additional isolates was compared with the reference sequences. These eight isolates shared 82 to 90.5 and 86.2 to 93.9% identities, respectively, with the AHVd reference sequences KR605506 from China and isolate Pacific Gala (MF402929) from Canada. AHVd has thus far been detected only in apple, indicating a single species, despite some sequence identities below the arbitrary cutoff of 90% (Serra et al. 2018). Although AHVd has been detected in apple trees with trunk splitting (Messmer et al. 2017) and shoot decline and dieback (this study), none of the other apples surveyed in this study or others (Serra et al. 2018) exhibited disease symptoms. Further work is needed before this recently described and apparently widespread viroid can be definitively associated with a disease.

Molecular Detection of Sentinel Node Micrometastases in Patients with Clinical N0 Gastric Carcinoma with Real-time Multiplex Reverse Transcription-Polymerase Chain Reaction Assay

Described is a novel real-time multiplex reverse transcription-polymerase chain reaction (RT-PCR) assay suitable for intraoperative detection of micrometastasis (MM) in sentinel nodes (SNs) dissected from patients with clinical N0 (cN0) gastric carcinoma. One hundred three patients with gastric cancer, who were preoperatively diagnosed with cN0 and clinical T1 or T2, were enrolled. The patients underwent SN mapping followed by standard radical gastrectomy with lymph node dissection. In addition to all SNs, non-SNs (NSNs) within the SN lymphatic basin and NSN from a different lymphatic basin were randomly sampled. All SNs and NSNs were examined by routine histologic diagnosis and RT-PCR for the expression of cytokeratin (CK) 19, CK20, and carcinoembryonic antigen (CEA). The RT-PCR assay and histologic examination were performed in 512 SNs and 299 NSNs from 103 patients. Pathologic l lymph node metastasis was revealed in 13 (12.6%) of 103 patients. All metastatic lymph nodes were identified within SNs. SNs of these 13 patients had positive findings on RT-PCR. Twenty-eight (27.2%) of 103 patients had negative histopathology but positive findings on RT-PCR. In 7 patients (6.8%), SNs were negative but NSNs were positive on RT-PCR. RT-PCR-positive NSNs were present in the same station as corresponding SNs in 3 of these 7 patients and in the same basin as SNs in 4 patients. The real-time multiplex RT-PCR assay is a useful tool for the detection of MM in SNs and NSNs in patients with gastric cancer.

Correlation Between Histology and PAX/FKHR Fusion Status in Alveolar Rhabdomyosarcoma

At the molecular level, alveolar rhabdomyosarcomas (ARMS) are characterized by 3 mutually exclusive PAX/FKHR conditions: PAX3/FKHR fusion (present in 60% of cases), PAX7/FKHR fusion (present in 20%), and PAX/FKHR fusion-negativity (present in 20%). The possibility of morphologic variation among these molecular subtypes has not been investigated. We undertook a blinded retrospective study of 65 cases of ARMS (16 PAX/FKHR fusion-negative, 36 PAX3/FKHR-positive, and 13 PAX7/FKHR-positive by routine reverse transcription-polymerase chain reaction). We evaluated cytohistologic parameters such as microcyst formation, solid foci, differentiation, giant cell formation, anaplasia, nuclear grade, mitosis/karyorrhexis index, rosette formation, geographic necrosis, presence and amount of rhabdomyoblastic differentiation, and the presence of foci resembling embryonal rhabdomyosarcoma. We analyzed the results using a simple chi formula. Of these features, only totally solid alveolar architecture reached significance (P=0.00014), with 7 of 16 PAX/FKHR-negative cases lacking this feature, compared with 0 of 36 PAX3/FKHR cases and 2/13 PAX7/FKHR cases. These preliminary results indicate that in general, only totally solid alveolar architecture in ARMS may predict the absence of a PAX/FKHR fusion. No features seemed to predict the presence of a particular fusion type. Our results suggest that histologic assessment of ARMS has limited correlation with PAX/FKHR fusion status.

Platelet-derived growth factor-A mRNA expression in fetal, normal adult, and atherosclerotic human aortas. Analysis by competitive polymerase chain reaction.

To understand which growth factors are important for growth of atherosclerotic plaques, it is necessary to know the factor's relative abundance and how its gene is regulated in relation to cell proliferation. We tested whether platelet-derived growth factor-A (PDGF-A) mRNA levels correlated with cell proliferation in developing aorta, normal adult aorta, and atherosclerotic plaques. We developed a competitive reverse transcription-polymerase chain reaction (RT-PCR) assay to measure human PDGF-A mRNA levels in small tissue samples. A mutated PDGF-A synthetic RNA was used as an internal standard to compete with endogenous PDGF-A mRNA for amplification. The assay is highly sensitive and much more precise than routine RT-PCR. Correction for heteroduplex pairing between the endogenous and mutant PCR products correlates precisely with synthetic RNA standards and quantitative Northern blotting. Immunostaining with the proliferation marker (proliferating cell nuclear antigen) showed the following rank order of proliferation: fetal aorta >> atherosclerotic plaque > normal aortic media. PDGF-A mRNA levels, however, did not correlate with proliferation. Normal adult aorta contained the most PDGF-A mRNa (34.0+/-7.6 amol/microgram total RNA). Fetal aortas were intermediate (10.2+/-1.6 amol/microgram total RNA); advanced atherosclerotic plaques contained the least PDGF-A mRNA (0.3+/-0.1 amol/microgram total RNA). PDGF-A protein was readily detectable in normal media by immunostaining. Advanced plaques generally had less cell-associated PDGF-A protein, although A-chain was also detected in plaque matrix. PDGF-A mRNA and protein do not correlate with proliferation among these three groups. The significance of high levels of PDGF-A mRNA in the "quiescent" aortic media is unknown but it clearly does not promote cell replication.