Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These?

ABSTRACT Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. HIGHLIGHTS

Download Full-text

The 501Y.V2 SARS-CoV-2 variant has an intermediate viral load between the 501Y.V1 and the historical variants in nasopharyngeal samples from newly diagnosed COVID-19 patients

10.1101/2021.03.21.21253498 ◽

2021 ◽

Author(s):

Elisa Teyssou ◽

Cathia Soulie ◽

Benoit Visseaux ◽

Sidonie Lambert-Niclot ◽

Valentine Ferre ◽

...

Keyword(s):

Viral Load ◽

Newly Diagnosed ◽

Threshold Values ◽

Rt Pcr ◽

Cycle Threshold ◽

The World

The 501Y.V2 and the 501Y.V1 SARS-CoV-2 variants emerged and spread rapidly into the world. We analysed the RT-PCR cycle threshold values of 643 nasopharyngeal samples of COVID-19 patients at diagnosis and found that the 501Y.V2 variant presented an intermediate viral load between the 501Y.V1 and the historical variants.

Download Full-text

Cycle threshold values in RT‐PCR to determine dynamics of SARS‐CoV‐2 viral load: An approach to reduce isolation period for COVID‐19 patients

Journal of Medical Virology ◽

10.1002/jmv.27206 ◽

2021 ◽

Author(s):

Clara Aranha ◽

Vainav Patel ◽

Vikrant Bhor ◽

Dimpu Gogoi

Keyword(s):

Viral Load ◽

Threshold Values ◽

Rt Pcr ◽

Cycle Threshold ◽

Isolation Period

Download Full-text

Antigen-based testing but not real-time PCR correlates with SARS-CoV-2 virus culture

10.1101/2020.10.02.20205708 ◽

2020 ◽

Cited By ~ 7

Author(s):

Andrew Pekosz ◽

Charles K. Cooper ◽

Valentin Parvu ◽

Maggie Li ◽

Jeffrey C. Andrews ◽

...

Keyword(s):

Real Time ◽

Infectious Virus ◽

Virus Isolation ◽

Low Cost ◽

Test Results ◽

Rt Pcr ◽

Chain Reaction ◽

Effective Implementation ◽

Virus Culture ◽

Polymerase Chain

SUMMARYIndividuals can test positive for SARS-CoV-2 by real-time polymerase chain reaction (RT-PCR) after no longer being infectious.1-8 Positive SARS-CoV-2 antigen-based testing exhibits a temporal pattern that corresponds with active, replicating virus and could therefore be a more accurate predictor of an individual’s potential to transmit SARS-CoV-2.2,3,9 Using the BD Veritor System for Rapid Detection of SARS-CoV-2 later flow antigen detection test, we demonstrate a higher concordance of antigen-positive test results with the presence of cultured, infectious virus when compared to RT-PCR. When compared to infectious virus isolation, the sensitivity of antigen-based testing is similar to RT-PCR. The correlation between SARS-CoV-2 antigen and SARS-CoV-2 culture represents a significant advancement in determining the risk for potential transmissibility beyond that which can be achieved by detection of SARS-CoV-2 genomic RNA. Coupled with a rapid time-to-result, low cost, and scalability, antigen-based testing should facilitate effective implementation of testing and public health interventions that will better contain COVID-19.

Download Full-text

Incorporating temporal distribution of population-level viral load enables real-time estimation of COVID-19 transmissibility

10.21203/rs.3.rs-841953/v1 ◽

2021 ◽

Author(s):

Yun Lin ◽

Bingyi Yang ◽

Sarah Cobey ◽

Eric Lau ◽

Dillon Adam ◽

...

Keyword(s):

Viral Load ◽

Real Time ◽

Temporal Distribution ◽

Time Estimation ◽

Population Level ◽

Reproductive Number ◽

Threshold Values ◽

Illness Onset ◽

Cycle Threshold ◽

Viral Loads

Abstract Many locations around the world have used real-time estimates of the time-varying effective reproductive number (\({R}_{t}\)) of COVID-19 to provide evidence of transmission intensity to inform control strategies. Estimates of \({R}_{t}\) are typically based on statistical models applied to case counts and typically suffer lags of more than a week because of the incubation period and reporting delays. Noting that viral loads tend to decline over time since illness onset, analysis of the distribution of viral loads among confirmed cases can provide insights into epidemic trajectory. Here, we analyzed viral load data on confirmed cases during two local epidemics in Hong Kong, identifying a strong correlation between temporal changes in the distribution of viral loads (measured by cycle threshold values) and estimates of \({R}_{t}\) based on case counts. We demonstrate that cycle threshold values could be used to improve real-time \({R}_{t}\) estimation, enabling more timely tracking of epidemic dynamics.

Download Full-text

Sampling site for SARS-CoV-2 RT-PCR—An intrapatient four-site comparison from Tampere, Finland

PLoS ONE ◽

10.1371/journal.pone.0260184 ◽

2021 ◽

Vol 16 (11) ◽

pp. e0260184

Author(s):

Dominik Kerimov ◽

Pekka Tamminen ◽

Hanna Viskari ◽

Lauri Lehtimäki ◽

Janne Aittoniemi

Keyword(s):

Informed Consent ◽

Study Design ◽

Nasal Swab ◽

Sampling Site ◽

Threshold Values ◽

Rt Pcr ◽

Cycle Threshold ◽

Significant Difference ◽

Nasal Swabs ◽

Written Informed Consent

Background SARS-CoV-2 diagnosis relies on the performance of nasopharyngeal swabs. Alternative sample sites have been assessed but the heterogeneity of the studies have made comparing different sites difficult. Objectives Our aim was to compare the performance of four different sampling sites for SARS-CoV-2 samples with nasopharynx being the benchmark. Study design COVID-19 positive patients were recruited prospectively, and samples were collected and analysed for SARS-CoV-2 with RT-PCR from all four anatomical sites in 43 patients, who provided written informed consent. Results All anterior nasal and saliva samples were positive, while two oropharyngeal samples were negative. There was no significant difference in the cycle threshold values of nasopharyngeal and anterior nasal samples while saliva and oropharynx had higher cycle threshold values. Conclusions Anterior nasal swab performs as good as nasopharynx swab with saliva also finding all the positives but with higher cycle threshold values. Thus, we can conclude that anterior nasal swabs can be used for SARS-CoV-2 detection instead of nasopharyngeal swabs if the situation would require so.

Download Full-text