scholarly journals Quantitative measurement of infectious virus in SARS-CoV-2 Alpha, Delta and Epsilon variants reveals higher infectivity (viral titer:RNA ratio) in clinical samples containing the Delta and Epsilon variants.

2021 ◽  
Author(s):  
Hannah W Despres ◽  
Margaret G Mills ◽  
David J Shirley ◽  
Madaline M Schmidt ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Quantitative Measurement ◽  
Infectious Virus ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Genome Copy ◽  
Live Virus ◽  
High Degree ◽  
The Relationship ◽  
Rna Levels

ABSTRACT Background Novel SARS-CoV-2 Variants of Concern (VoC) pose a challenge to controlling the COVID-19 pandemic. Previous studies indicate that clinical samples collected from individuals infected with the Delta variant may contain higher levels of RNA than previous variants, but the relationship between viral RNA and infectious virus for individual variants is unknown. Methods We measured infectious viral titer (using a micro-focus forming assay) as well as total and subgenomic viral RNA levels (using RT-PCR) in a set of 165 clinical samples containing SARS-CoV-2 Alpha, Delta and Epsilon variants that were processed within two days of collection from the patient. Results We observed a high degree of variation in the relationship between viral titers and RNA levels. Despite the variability we observed for individual samples the overall infectivity differed among the three variants. Both Delta and Epsilon had significantly higher infectivity than Alpha, as measured by the number of infectious units per quantity of viral E gene RNA (6 and 4 times as much, p=0.0002 and 0.009 respectively) or subgenomic E RNA (11 and 7 times as much, p<0.0001 and 0.006 respectively). Conclusion In addition to higher viral RNA levels reported for the Delta variant, the infectivity (amount of replication competent virus per viral genome copy) may also be increased compared to Alpha. Measuring the relationship between live virus and viral RNA is an important step in assessing the infectivity of novel SARS-CoV-2 variants. An increase in the infectivity of the Delta variant may further explain increased spread and suggests a need for increased measures to prevent viral transmission.

scholarly journals Predicting Infectivity: Comparing Four PCR-based Assays to Detect Culturable SARS-CoV-2 in Clinical Samples

2021 ◽  
Author(s):  
Emily A Bruce ◽  
Margaret G A Mills ◽  
Reigran Sampoleo ◽  
Garrett A. Perchetti ◽  
Meeili Huang ◽  
...  
Keyword(s):  
Infectious Virus ◽  
Diagnostic Testing ◽  
Viral Rna ◽  
Superior Performance ◽  
Clinical Samples ◽  
Analytical Sensitivity ◽  
Live Virus ◽  
Negative Strand ◽  
Replicative Intermediates ◽  
Probe Set

With the COVID-19 pandemic caused by SARS-CoV-2 now in its second year, there remains an urgent need for diagnostic testing that can identify infected individuals, particularly those who harbor infectious virus. Various RT-PCR strategies have been proposed to identify specific viral RNA species that may predict the presence of infectious virus, including detection of transcriptional intermediates (e.g. subgenomic RNA [sgRNA]) and replicative intermediates (e.g. negative-strand RNA species). Using a novel primer/probe set for detection of subgenomic (sg)E transcripts, we successfully identified 100% of specimens containing culturable SARS-CoV-2 from a set of 126 clinical samples (total sgE CT values ranging from 12.3-37.5). This assay showed superior performance compared to a previously published sgRNA assay and to a negative-strand RNA assay, both of which failed to detect target RNA in a subset of samples from which we isolated live virus. In addition, total levels of viral RNA (genome, negative-strand, and sgE) detected with the WHO/Charite primer-probe set correlated closely with levels of infectious virus. Specifically, infectious virus was not detected in samples with a CT above 31.0. Clinical samples with higher levels of viral RNA also displayed cytopathic effect (CPE) more quickly than those with lower levels of viral RNA. Finally, we found that the infectivity of SARS-CoV-2 samples is significantly dependent on the cell type used for viral isolation, as Vero E6 cells expressing TMRPSS2 extended the analytical sensitivity of isolation by more than 3 CT compared to parental Vero E6 cells and resulted in faster isolation. Our work shows that using a total viral RNA Ct cut-off of >31 or specifically testing for sgRNA can serve as an effective rule-out test for viral infectivity.

scholarly journals Inactivation of SARS-CoV-2 virus in saliva using a guanidium based transport medium suitable for RT-PCR diagnostic assays

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252687
Author(s):  
Sukalyani Banik ◽  
Kaheerman Saibire ◽  
Shraddha Suryavanshi ◽  
Glenn Johns ◽  
Soumitesh Chakravorty ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Viral Rna ◽  
Clinical Samples ◽  
Sample Collection ◽  
Rt Pcr ◽  
Diagnostic Assays ◽  
Guanidinium Thiocyanate ◽  
Upper Respiratory ◽  
Polymerase Chain ◽  
The Stability

Background Upper respiratory samples used to test for SARS-CoV-2 virus may be infectious and present a hazard during transport and testing. A buffer with the ability to inactivate SARS-CoV-2 at the time of sample collection could simplify and expand testing for COVID-19 to non-conventional settings. Methods We evaluated a guanidium thiocyanate-based buffer, eNAT™ (Copan) as a possible transport and inactivation medium for downstream Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) testing to detect SARS-CoV-2. Inactivation of SARS-CoV-2 USA-WA1/2020 in eNAT and in diluted saliva was studied at different incubation times. The stability of viral RNA in eNAT was also evaluated for up to 7 days at room temperature (28°C), refrigerated conditions (4°C) and at 35°C. Results SARS-COV-2 virus spiked directly in eNAT could be inactivated at >5.6 log10 PFU/ml within a minute of incubation. When saliva was diluted 1:1 in eNAT, no cytopathic effect (CPE) on VeroE6 cells was observed, although SARS-CoV-2 RNA could be detected even after 30 min incubation and after two cell culture passages. A 1:2 (saliva:eNAT) dilution abrogated both CPE and detectable viral RNA after as little as 5 min incubation in eNAT. SARS-CoV-2 RNA from virus spiked at 5X the limit of detection remained positive up to 7 days of incubation in all tested conditions. Conclusion eNAT and similar guanidinium thiocyanate-based media may be of value for transport, stabilization, and processing of clinical samples for RT-PCR based SARS-CoV-2 detection.

Concordance of local and central laboratory hormone and HER2 receptor status in ECOG 2197

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 21022-21022 ◽  
Author(s):  
S. S. Badve ◽  
F. L. Baehner ◽  
R. Gray ◽  
B. Childs ◽  
T. Maddala ◽  
...  
Keyword(s):  
Hormone Receptor ◽  
Pcr Analysis ◽  
Central Laboratory ◽  
Rt Pcr ◽  
Her2 Receptor ◽  
High Incidence ◽  
High Degree ◽  
The Relationship ◽  
Very High ◽  
Local Laboratory

21022 Background: Central and local laboratory concordance for hormone and HER2 receptor measurement is of national interest. This study compares ER/PR/HER2 by local laboratories using immunohistochemistry (IHC) and central laboratories (IHC & quantitative RT-PCR). Methods: Of 2952 patients in E2197, a case-cohort sample of 776 patients who either did (N=179) or did not recur was studied. Central IHC for ER/PR/HER2 was performed using single 0.6 mm microarrays; Allred score (AS) was used for ER/PR (AS>2 = positive). Positive HER2 was 3+ staining in >10% cells for Central IHC and 2+ or 3+ for Local IHC. RT-PCR analysis by Oncotype DX™ for ER/PR/HER2 was performed using pre-defined cutoffs of 6.5, 5.5 and 11.5 units, respectively. Hormone receptor (HR) pos was defined as ER &/or PR pos. Results: Results from Local IHC (ER/PR in 776 & HER2 in 517 pts) were compared with Central IHC (760 pts) and RT-PCR results (776 pts). The discordance between HR positivity by Local IHC and RT-PCR was very low. However, 12% of HR neg pts by Local IHC (38/321) & Central IHC (39/326) were HR pos by RT-PCR. The relationship between ER and recurrence as a function of AS was examined. Patients with AS of 3–4 were found to be closer to the AS=2 group than to the AS>4 group Patients with AS of 3–4 were found to be closer to the AS ÿ 2 group than to the AS > 4 group (Est.HR for ER 0.97 for AS 3–4 vs. 0–2 and 0.46 for AS 5–8 vs. 0–2, and for PR were 0.84 for AS 3–4 vs. 0–2 and 0.41 for AS 5–8 vs. 0–2). Conclusions: There is a high degree of overall concordance among Local IHC, Central IHC, and Central RT-PCR for ER and PR. The degree of concordance is even greater for HR compared to ER or PR alone. Although the concordance with local labs for HER2 testing was poor, the concordance between Central IHC and RT-PCR was very high. The relatively high incidence (12%) of IHC HR neg pts who are HR pos by RT- PCR is notable. [Table: see text] [Table: see text]

scholarly journals Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR

1998 ◽  
Vol 36 (9) ◽  
pp. 2634-2639 ◽  
Author(s):  
Eva Harris ◽  
T. Guy Roberts ◽  
Leila Smith ◽  
John Selle ◽  
Laura D. Kramer ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dengue Fever ◽  
Internal Control ◽  
Extraction Procedure ◽  
Rna Degradation ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Single Tube ◽  
Dengue Viruses

In recent years, dengue viruses (serotypes 1 to 4) have spread throughout tropical regions worldwide. In many places, multiple dengue virus serotypes are circulating concurrently, which may increase the risk for the more severe form of the disease, dengue hemorrhagic fever. For the control and prevention of dengue fever, it is important to rapidly detect and type the virus in clinical samples and mosquitoes. Assays based on reverse transcriptase (RT) PCR (RT-PCR) amplification of dengue viral RNA can offer a rapid, sensitive, and specific approach to the typing of dengue viruses. We have reduced a two-step nested RT-PCR protocol to a single-tube reaction with sensitivity equivalent to that of the two-step protocol (1 to 50 PFU) in order to maximize simplicity and minimize the risk of sample cross-contamination. This assay was also optimized for use with a thermostable RT-polymerase. We designed a plasmid-based internal control that produces a uniquely sized product and can be used to control for both reverse transcription or amplification steps without the risk of generating false-positive results. This single-tube RT-PCR procedure was used to type dengue viruses during the 1995 and 1997-1998 outbreaks in Nicaragua. In addition, an extraction procedure that permits the sensitive detection of viral RNA in pools of up to 50 mosquitoes without PCR inhibition or RNA degradation was developed. This assay should serve as a practical tool for use in countries where dengue fever is endemic, in conjunction with classical methods for surveillance and epidemiology of dengue viruses.

Diagnosis of foot-and-mouth disease by RT-PCR: use of phylogenetic data to evaluate primers for the typing of viral RNA in clinical samples

2001 ◽  
Vol 146 (12) ◽  
pp. 2421-2434 ◽  
Author(s):  
S. M. Reid ◽  
N. P. Ferris ◽  
G. H. Hutchings ◽  
K. De Clercq ◽  
B. J. Newman ◽  
...  
Keyword(s):  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Phylogenetic Data ◽  
Foot And Mouth

scholarly journals Molecular and Virological Investigation of a Focal Chikungunya Outbreak in Northern India

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Manisha Soni ◽  
Anil Kumar Singh ◽  
Shashi Sharma ◽  
Ankita Agarwal ◽  
Natarajan Gopalan ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Chikungunya Virus ◽  
Viral Rna ◽  
Clinical Samples ◽  
Nucleotide Sequencing ◽  
The Novel ◽  
Rt Pcr ◽  
Northern India ◽  
Genotype Identification ◽  
Mosquito Pool

Chikungunya (CHIK) fever is one of the most important arboviral infections of medical significance. The objective of the present study is to identify and characterize the etiology of a focal febrile arthritis outbreak from Gwalior, northern India, during October-November 2010. A detailed virological (isolation) and molecular (end-point RT-PCR, quantitative RT-PCR, and nucleotide sequencing) investigation of this outbreak was carried out by collecting and studying 52 clinical samples and 15 mosquito pools from the affected region. The investigation revealed the presence of CHIK viral RNA in 29% of clinical samples and 13% mosquito pool by RT-PCR. The quantification of CHIK viral RNA in samples varied from 102.50to 106.67 copies/mL, as demonstrated through quantitative RT-PCR. In addition, six CHIK viruses were isolated from RT-PCR positive samples. The nucleotide sequences of partial E1 gene of five representative CHIK viruses were deciphered, which revealed that all the viral strains from this outbreak belong to the recently emerging ECS African genotype. Identification of Chikungunya virus ECSA African genotype as the etiology of the present outbreak confirms the continued circulation of the novel genotype, since 2006, in India. The identification of CHIK virus inAedes aegyptialso confirmed it as the major vector in northern India.

A refined long RT-PCR technique to amplify complete viral RNA genome sequences from clinical samples: Application to a novel hepatitis C virus variant of genotype 6

2005 ◽  
Vol 126 (1-2) ◽  
pp. 139-148 ◽  
Author(s):  
Ling Lu ◽  
Tatsunori Nakano ◽  
Gregory A. Smallwood ◽  
Thomas G. Heffron ◽  
Betty H. Robertson ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Rna ◽  
Clinical Samples ◽  
Rt Pcr ◽  
Genome Sequences ◽  
Virus Variant ◽  
Rna Genome

scholarly journals Role of Nucleocapsid Protein Antigen Detection for Safe End of Isolation of SARS-CoV-2 Infected Patients with Long Persistence of Viral RNA in Respiratory Samples

2021 ◽  
Vol 10 (18) ◽  
pp. 4037
Author(s):  
Antonella Mencacci ◽  
Alessio Gili ◽  
Anna Gidari ◽  
Elisabetta Schiaroli ◽  
Carla Russo ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Infectious Virus ◽  
Viral Rna ◽  
Concomitant Disease ◽  
Rt Pcr ◽  
Antigen Assay ◽  
Diagnosis Of Infection ◽  
Better Than

Background. In SARS-CoV-2 infection, viral RNA may persist in respiratory samples for several weeks after the resolution of symptoms. Criteria to assess the end of infectivity are not unequivocally defined. In some countries, time from diagnosis is the unique criterion used, in addition to symptom cessation. This study evaluates the role of the Lumipulse® Antigen Assay (LAA) for the safe end of isolation of patients ≥21 days after the diagnosis of infection. Methods. A total of 671 nasopharyngeal swabs from patients diagnosed with infection at least 21 days before were assessed by RT-PCR and LAA, and the role of LAA in predicting the absence of infectivity was evaluated by virus cell culture. Results. Viable virus was present in 10/138 cultured samples. Eight out of ten infective patients suffered from a concomitant disease, predisposing them to long-term shedding of infective virus. In particular, infectious virus was isolated from 10/20 RT-PCR+/LAA+ cultured samples, whereas no viable virus was found in all 118 RT-PCR+/LAA– cultured swabs. LLA and RT-PCR agreed in 484/671 (72.1%) samples, with 100% and 26.7% concordance in RT-PCR negative and positive samples, respectively. Conclusions. Viable virus can be found ≥21 days after diagnosis in immunocompromised or severely ill patients. LAA better than RT-PCR predicts non-infectivity of patients and can be safely used to end isolation in cases with long persistence of viral RNA in the respiratory tract.

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