Delta Variant SARS-CoV-2 infections in pediatric cases during the second wave in India

AbstractThe aim of this study was to identify the SARS-CoV-2 lineages circulating in the pediatric population of India during the second wave of the pandemic. Clinical and demographic details linked with the nasopharyngeal/oropharyngeal swabs (NPS/OPS) collected from SARS-CoV-2 cases (n=583) aged 0-18 year and tested positive by real-time RT-PCR were retrieved from March to June 2021.Symptoms were reported among 37.2% of patients and 14.8% reported to be hospitalized. The E gene CT value had significant statistical difference at the point of sample collection when compared to that observed in the sequencing laboratory. Out of these 512 sequences 372 were VOCs, 51 were VOIs. Most common lineages observed were Delta, followed by Kappa, Alpha and B.1.36, seen in 65.82%, 9.96%, 6.83% and 4.68%, respectively in the study population. Overall, it was observed that Delta strain was the leading cause of SARS-CoV-2 infection in Indian children during the second wave of the pandemic. We emphasize on the need of continuous genomic surveillance in SARS-CoV-2 infection even amongst children.

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Lyophilized Matrix Containing Ready-to-Use Primers and Probe Solutions for Standardization of Real-Time PCR and RT-qPCR Diagnostics

Proceedings ◽

10.3390/proceedings2020050007 ◽

2020 ◽

Vol 50 (1) ◽

pp. 7

Author(s):

Remi N Charrel ◽

Laurence Thirion

Keyword(s):

Real Time ◽

Molecular Techniques ◽

Cold Chain ◽

Rt Pcr ◽

Emerging Viruses ◽

Valley Fever ◽

Term Stability ◽

E Gene ◽

Long Term Stability

Real-time molecular techniques have become the reference methods for the direct diagnosis of pathogens. The reduction of steps is a key factor in order to decrease the risk of human errors resulting in invalid series and delayed results. We describe here a process involving the preparation of oligonucleotide primers and a hydrolysis probe in a single tube at predefined optimized concentrations that are stabilized via lyophilization (Lyoph-P&P). Lyoph-P&P was compared to the classic protocol using extemporaneously prepared liquid reagents, assaying (i) sensitivity, (ii) long-term stability at 4 °C, and (iii) long-term stability at 37 °C, mimicking transportation without a cold chain. Two previously published molecular assays were selected for this study. They target two emerging viruses that are listed on the blueprint of the WHO to be considered for preparedness and response actions: chikungunya virus (CHIKV) and Rift Valley fever phlebovirus (RVFV). The results of our study demonstrate that (i) Lyoph-P&P is stable for at least four days at 37 °C, supporting shipping without the need of a cold chain, (ii) Lyoph-P&P rehydrated solution is stable at 4 °C for at least two weeks, (iii) the sensitivity observed with Lyoph-P&P is at least equal to, and often better than, that observed with liquid formulation, and (iv) the validation of results observed with low-copy specimens is rendered easier by higher fluorescence levels. In conclusion, Lyoph-P&P holds several advantages over extemporaneously prepared liquid formulations and merits consideration as a novel real-time molecular assay for implementation into a laboratory with routine diagnostic activity. Since the meeting, this concept has been applied to the COVID-19 situation: two diagnostic assays (E gene and RdRp) have been developed and can be ordered on the European Virus Archive catalog (https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-2019-ncov-e-gene; https://www.european-virus-archive.com/detection-kit/lyophilized-primers-and-probe-rt-pcr-sars-cov-2-rdrp-gene).

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Development of a Real-Time Loop-Mediated Isothermal Amplification Method for the Detection of West Nile Virus

Jundishapur Journal of Microbiology ◽

10.5812/jjm.105443 ◽

2020 ◽

Vol 13 (10) ◽

Author(s):

Jae Woong Lee ◽

Yu-Jung Won ◽

Sung-Geun Lee ◽

Soon-Young Paik

Keyword(s):

West Nile Virus ◽

Real Time ◽

Lamp Assay ◽

Isothermal Amplification ◽

Genomic Sequences ◽

The Novel ◽

Rt Pcr ◽

West Nile ◽

The Real ◽

E Gene

Background: The West Nile Virus (WNV), discovered in New York, USA in 1999 after it was first isolated in Uganda in 1937, has since spread not only in the United States but also around the world. Africa, Eurasia, Australia, and the Middle East have sporadic cases of the disease. Objectives: We aimed to find real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay to be more sensitive than conventional RT-PCR, and more rapid and efficient than conventional RT-PCR and real-time RT-PCR for WNV detection. Methods: A total of 32 genomic sequences from different strains of WNV were analyzed to identify conserved nucleotide sequence regions. Six WNV specific RT-LAMP primers targeting the E gene were designed. Results: The novel primer for the real-time RT-LAMP assay can detect WNV with high specificity. The efficiency of the real-time RT-LAMP assay is higher than the conventional RT-PCR and real-time RT-PCR. Real-time RT-PCR and conventional PCR require at least 30 – 40 min and 2 h, respectively, to yield results, whereas real-time RT-LAMP provides positive results in only 10 – 20 min. Conclusions: The novel primers were developed by analyzing of 32 genomic sequences of WNV strains. The primers were designed from the most conserved region of the E gene for real-time RT-LAMP. The LAMP assay is a rapid, efficient, highly sensitive, and specific tool for the identification of WNV.

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CORRELATION OF PROGNOSIS OF COVID POSITIVE PATIENTS BETWEEN REAL TIME RT-PCR AND CT-VALUES OF E. GENE, N.GENE, RDRP GENE

10.36106/ijsr/4608909 ◽

2021 ◽

pp. 1-3

Author(s):

Sreyoshi Som ◽

Indraneel Dasgupta

Keyword(s):

Real Time ◽

Virus Disease ◽

The Other ◽

Rdrp Gene ◽

N Gene ◽

Absolute Amount ◽

Target Sequence ◽

Rt Pcr ◽

E Gene ◽

Corona Virus

INTRODUCTION: COVID-19 – Corona virus disease-19 – A respiratory illness caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2), colloquially referred to as coronavirus. Real-time PCR is conducted to quantify the absolute amount of target sequence or to compare relative amount of a target sequence among samples. This technique monitors amplication of the target in real time via a target specic uorescent signal emitted during amplication. During most real times PCR, a considerable amount of background uorescence occurs. Inspite of this fact PCR uorescent dyes and probes should be sequence specic. This CT value or cycle threshold value in a real time RT- PCR is dened as the number of cycles required for the uorescent signal to cross the threshold i.e., exceeds background level). This is inversely proportional to the amount of target nuclei in the sample. i.e., lower the CTlevel the greater the amount of target nuclei in the sample. AIM: To explore the association of the mutation pattern of covid-19 genetic variation with the severity and fatality of patients with covid-19 viral illness. Factors considered are Age, Sex, and Duration of hospital stay and outcome of patient. MATERIALS AND METHODS: The study was a comparative, correlation, retrospective, lab values based study. All covid+ve patients tested at Peerless hospital, Kolkata by RT-PCR on admission. It is imperative to choose reference genes whose expression levels are not expected to change during our experiment. Common housekeeping genes include actin, alpha-tubuliun, gapdh and ubiquitin. it is wise to use at least two reference gene for one study may not be suitable for another. Total 370 participants were present in this study. RESULT: In our study, 114(30.8%) COVID positive patients were in E GENE GR 11-20, 147(39.7%) COVID positive patients were in E GENE GR 21-30 and 109(29.5%) COVID positive patients were in E GENE GR 31-40. 163(44.1%) COVID positive patients were in N GENE GR 11-20, 132(35.7%) COVID positive patients were in N GENE GR 21-30 and 75(20.3%) COVID positive patients were in N GENE GR 31-40. 228(61.6%) COVID positive patients were in RdRp GENE GR 11-20, 112(30.3%) COVID positive patients were in RdRp GENE GR 21-30 and 30(8.1%) COVID positive patients were in RdRp GENE GR 31-40. We found that, 30(8.1%) COVID positive patients died and 340 (91.9%) patients have survived. CONCLUSION: The patients who died had signicantly lower RT-PCR CT-values OF E. GENE, N.GENE, RdRp GENE than patients who survived. We concluded that the time of onset of symptoms were earlier for patients with lower CTvalue, than the other group who survived. So we concluded after correlating CT-values of real time RT-PCR and E. GENE, N.GENE, RdRp GENE, that the patients with lower CTvalues had more severity and had poor prognosis than the other group with higher RT-PCR CT-values of E.GENE, N, GENE, RdRp GENE

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Rapid Adaptation and Continuous Performance Evaluation of SARS-CoV-2 Envelope Gene (E-gene) Real-Time RT-PCR Assays to Support the Hospital Surge in Test Demand

10.21203/rs.3.rs-38621/v1 ◽

2020 ◽

Author(s):

Yolanda Iok Ieng Ho ◽

Ann Han Wong ◽

Eddie Chi Man Leung ◽

River Chun Wai Wong ◽

Raymond Wai Man Lai

Keyword(s):

Real Time ◽

Tertiary Hospital ◽

Envelope Gene ◽

Rt Pcr ◽

Continuous Training ◽

E Gene ◽

Gene Assay ◽

Test Platform ◽

Time Analyzer ◽

One Step

Abstract We describe timely adaption of both published WHO E-gene protocol and commercially available LightMix Modular E-gene assay to the test platform (ABI 7900 Fast real-time analyzer and TaqMan Fast One-step Virus Master Mix) available in an accredited tertiary hospital laboratory with on-going evaluation to ensure provision of quality service within time constraint. The LightMix Modular E-gene was slightly more sensitive when compared to the WHO E-gene, both analytically and diagnostically. The assay was recommended for screening of SARS-CoV-2 infection. With the availability of technically competent staff through continuous training, provision of round the clock service is feasible despite the test is of high complexity. The shorten turn-around-time of about 4 hours per batch could support surges in testing demand, which is essential to control the current SARS-CoV-2 pandemic, to prevent potential overwhelming of the healthcare system and to optimize utilization of the isolation beds.

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Clinical Evaluation of a Combo Rapid Antigen Test QuickNavi-Flu+COVID19 Ag for Simultaneous Detection of SARS-CoV-2 and Influenza Viruses

10.1101/2021.12.05.21267215 ◽

2021 ◽

Author(s):

Yuto Takeuchi ◽

Yusaku Akashi ◽

Yoshihiko Kiyasu ◽

Norihiko Terada ◽

Yoko Kurihara ◽

...

Keyword(s):

Real Time ◽

Sensitivity And Specificity ◽

Clinical Performance ◽

Simultaneous Detection ◽

Influenza Viruses ◽

Antigen Test ◽

Sample Collection ◽

Rt Pcr ◽

The Real ◽

Reverse Transcription Pcr

AbstractIntroductionSince respiratory sample collection is an uncomfortable experience, simultaneous detection of pathogens with a single swab is preferable. We prospectively evaluated the clinical performance of a newly developed antigen test QuickNavi-Flu+COVID19 Ag (Denka Co., Ltd., Tokyo, Japan) which can detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses at the same time with a single testing device.MethodsIncluded were those who were suspected of contracting coronavirus disease 2019 (COVID-19) and referred to a PCR center at Ibaraki prefecture in Japan, between August 2, 2021 to September 13, 2021, when the L452R mutant strains of SARS-CoV-2 were prevalent. Additional nasopharyngeal samples and anterior nasal samples were obtained for the antigen test and were compared with a reference reverse transcription PCR (RT-PCR) using nasopharyngeal samples.ResultsIn total, 1510 nasopharyngeal samples and 862 anterior nasal samples were evaluated. For SARS-CoV-2 detection in nasopharyngeal samples, the sensitivity and specificity of the antigen test were 80.9% and 99.8%, respectively. The sensitivity and specificity using anterior nasal samples were 67.8% and 100%, respectively. In symptomatic cases, the sensitivities increased to 88.3% with nasopharyngeal samples and 73.7% with anterior nasal samples. There were three cases of discrepant results between the antigen test and the real-time RT-PCR. All of them were positive with the antigen test but negative with the real-time RT-PCR in SARS-CoV-2 detection. During the study period, influenza viruses were not detected.ConclusionA combo kit, QuickNavi-Flu+COVID19 Ag, showed an acceptable sensitivity and sufficient specificity for SARS-CoV-2 detection, especially using nasopharyngeal sample collected from symptomatic patients.

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