A Viral Fragmentation Signature for SARS-CoV-2 in Clinical Samples Correlating with Contagiousness

AbstractThe viral load of SARS-CoV-2 in clinical samples as measured by the primary diagnostic tool of RT-PCR is an imperfect readout for infection potential as most targeted assays designed for sensitivity, indiscriminately detect short and long RNA fragments, although infectivity is embodied only in the whole virus and its intact genome. Here, we used next-generation sequencing (NGS) to characterize 155 clinical samples and show sensitive and quantitative detection of viral RNA which confirmed subgenomic RNA in 57.6% of samples and provided a novel method to determine relative integrity of viral RNA in samples. The relative abundance of long fragments quantified as a viral fragmentation score was positively associated with viral load and inversely related to time from disease onset. An empirically determined score cut-off for presence of substantially fragmented RNA was able to identify 100% of samples collected after 8 days of illness with poor infection potential in line with current clinical understanding of infectiousness of SARS-CoV-2. The quantification of longer fragments in addition to existing short targets in an NGS or RT-PCR-based assay could provide a valuable readout of infection potential simultaneous to the detection of any fragments of SARS-CoV-2 RNA in test samples.

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Performance of Self-Collected Saliva Testing Compared with Nasopharyngeal Swab Testing for the Detection of SARS-CoV-2

Viruses ◽

10.3390/v13050895 ◽

2021 ◽

Vol 13 (5) ◽

pp. 895

Author(s):

Florence Carrouel ◽

Martine Valette ◽

Hervé Perrier ◽

Maude Bouscambert-Duchamp ◽

Claude Dussart ◽

...

Keyword(s):

Viral Load ◽

Quantitative Pcr ◽

Statistical Analyses ◽

Fair Agreement ◽

Quantitative Detection ◽

Nasopharyngeal Swab ◽

Rt Pcr ◽

Noninvasive Techniques ◽

Concordance Probability ◽

Salivary Test

The aim of this study was to determine whether self-collected pure saliva (SCPS) is comparable to nasopharyngeal (NP) swabs in the quantitative detection of SARS-CoV-2 by RT-PCR in asymptomatic, mild patients with confirmed COVID-19. Thirty-one patients aged from 18 to 85 years were included between 9 June and 11 December 2020. A SCPS sample and a NP sample were taken for each patient. Quantitative PCR was performed to detect SARS-CoV-2 viral load. Results of SCPS vs NP samples testing were compared. Statistical analyses were performed. Viral load was significantly correlated (r = 0.72). The concordance probability was estimated at 73.3%. In symptomatic adults, SCPS performance was similar to that of NP swabs (Percent Agreement = 74.1%; p = 0.11). Thus, the salivary test based on pure oral saliva samples easily obtained by noninvasive techniques has a fair agreement with the nasopharyngeal one in asymptomatic, mild patients with a confirmed diagnosis of COVID-19.

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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.

10.1101/2021.09.07.21263229 ◽

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.

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

PLoS ONE ◽

10.1371/journal.pone.0252687 ◽

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.

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Detecting SARS-CoV-2 by Realtime RT-PCR in clinical samples collected in the North of Vietnam

Tạp chí Y học Dự phòng ◽

10.51403/0868-2836/2020/114 ◽

2021 ◽

Vol 30 (9) ◽

pp. 11-17

Author(s):

Hoang Vu Mai Phuong ◽

Ung Thi Hong Trang ◽

Nguyen Vu Son ◽

Le Thi Thanh ◽

Nguyen Le Khanh Hang ◽

...

Keyword(s):

Viral Load ◽

Average Rate ◽

Clinical Samples ◽

Viet Nam ◽

Rt Pcr ◽

Ct Value ◽

The North ◽

Viral Load Data ◽

The Mean ◽

High Level

From January to August 2020, Northern Viet Nam faced a COVID-19 outbreak, up to September 2020, there were 1122 confrmed cases of SARS-CoV-2, of which 465 cases were imported from Europe, America and Asia, 657 cases were identifed domestically. A total of 30,686 samples were collected during the SARS-CoV-2 outbreak in Northern Viet Nam and examined by Real-time RT-PCR using primers and probe from Charite - Berlin protocol. This study showed the initial results of SARS-CoV-2 detection and RNA quantitative in positive samples. The positive rate was 0.8%, ranging from 0.4 to 3.5% according to collection sites. Out of 251 positive samples, the mean Ct value was 28 (IQR: 22.3-32; range 14 - 38). The positive samples had a Ct value below 30 was 68.5%, there was no signifcant difference between the Ct value of the group ≤ 30 and > 30. The mean of the RNA copies/µl was 8.4.107, (IQR: 2.29.106 - 1.83.109 RNA copies/µl, range: 1.95.103 – 4.95.1011). In the group of imported COVID-19 cases, the rate of virus at low level was 29%, an average was 56% and at high level was 15%. In the community groups, the viral load data showed that the average rate at low, intermediate and high level were 20%, 63% and 17% respectively. The proportion of high-level viral load may raise an alert to start the quarantine process to reduce the transmission of SARS-CoV-2

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Microfluidic Nano-Scale qPCR Enables Ultra-Sensitive and Quantitative Detection of SARS-CoV-2

Processes ◽

10.3390/pr8111425 ◽

2020 ◽

Vol 8 (11) ◽

pp. 1425

Author(s):

Xin Xie ◽

Tamara Gjorgjieva ◽

Zaynoun Attieh ◽

Mame Massar Dieng ◽

Marc Arnoux ◽

...

Keyword(s):

Viral Infections ◽

Limit Of Detection ◽

False Negative ◽

False Negative Rate ◽

Clinical Samples ◽

Quantitative Detection ◽

Rt Pcr ◽

Viral Loads ◽

Nano Scale ◽

Negative Rate

A major challenge in controlling the COVID-19 pandemic is the high false-negative rate of the commonly used RT-PCR methods for SARS-CoV-2 detection in clinical samples. Accurate detection is particularly challenging in samples with low viral loads that are below the limit of detection (LoD) of standard one- or two-step RT-PCR methods. In this study, we implemented a three-step approach for SARS-CoV-2 detection and quantification that employs reverse transcription, targeted cDNA preamplification, and nano-scale qPCR based on a commercially available microfluidic chip. Using SARS-CoV-2 synthetic RNA and plasmid controls, we demonstrate that the addition of a preamplification step enhances the LoD of this microfluidic RT-qPCR by 1000-fold, enabling detection below 1 copy/µL. We applied this method to analyze 182 clinical NP swab samples previously diagnosed using a standard RT-qPCR protocol (91 positive, 91 negative) and demonstrate reproducible and quantitative detection of SARS-CoV-2 over five orders of magnitude (<1 to 106 viral copies/µL). Crucially, we detect SARS-CoV-2 with relatively low viral load estimates (<1 to 40 viral copies/µL) in 17 samples with negative clinical diagnosis, indicating a potential false-negative rate of 18.7% by clinical diagnostic procedures. In summary, this three-step nano-scale RT-qPCR method can robustly detect SARS-CoV-2 in samples with relatively low viral loads (<1 viral copy/µL) and has the potential to reduce the false-negative rate of standard RT-PCR-based diagnostic tests for SARS-CoV-2 and other viral infections.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.36.9.2634-2639.1998 ◽

1998 ◽

Vol 36 (9) ◽

pp. 2634-2639 ◽

Cited By ~ 142

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.

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Sensitive quantitative detection of SARS-CoV-2 in clinical samples using digital warm-start CRISPR assay

10.1101/2020.11.21.20236109 ◽

2020 ◽

Author(s):

Xiong Ding ◽

Kun Yin ◽

Ziyue Li ◽

Maroun M. Sfeir ◽

Changchun Liu

Keyword(s):

Room Temperature ◽

Rna Extraction ◽

Clinical Samples ◽

Quantitative Detection ◽

High Tolerance ◽

Rt Pcr ◽

One Pot ◽

Nucleoprotein Gene ◽

Warm Start ◽

Heat Treated

AbstractQuantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in clinical samples is crucial for assessing the infectivity of coronavirus disease 2019 and the efficacy of antiviral drugs. Here, we describe a digital warm-start CRISPR (WS-CRISPR) assay for sensitive quantitative detection of SARS-CoV-2 in clinical samples. The WS-CRISPR assay combines low-temperature reverse transcription dual-priming mediated isothermal amplification (RT-DAMP) and CRISPR-Cas12a-based detection in one-pot, attributed to the mediation role by pyrophosphatase and phosphorothioated primers. The WS-CRISPR assay is initiated at above 50 °C and overcomes undesired premature target amplification at room temperature, enabling accurate digital nucleic acid quantification. By targeting SARS-CoV-2’s nucleoprotein gene, digital WS-CRISPR assay is able to detect down to 5 copies/μl SARS-CoV-2 RNA in the chip within 90 minutes. It is clinically validated by quantitatively determining 32 clinical swab samples and three clinical saliva samples, showing 100% agreement with RT-PCR results. Moreover, the digital WS-CRISPR assay has been demonstrated to directly detect SARS-CoV-2 in heat-treated saliva samples without RNA extraction, showing high tolerance to inhibitors. Thus, the digital WS-CRISPR method, as a sensitive and reliable CRISPR assay, facilitates accurate SARS-CoV-2 detection toward digitized quantification.

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Development of a nanoparticle-assisted PCR assay to distinguish canine coronaviruses I and II

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638720974114 ◽

2020 ◽

Vol 33 (1) ◽

pp. 104-107

Author(s):

Tong Qin ◽

Jing Wang ◽

Shang-Jin Cui

Keyword(s):

Mixed Infection ◽

Annealing Temperature ◽

Clinical Samples ◽

Rt Pcr ◽

M Gene ◽

Specific Detection ◽

Pcr Assay ◽

Reaction Conditions ◽

Novel Method ◽

Beijing China

Nanoparticle-assisted PCR (nanoPCR) is a novel method for the simple, rapid, and specific detection of viruses. We developed a nanoPCR method to detect and differentiate canine coronavirus I (CCoV I) and II (CCoV II). Primer pairs were designed against the M gene conserved region of CCoV I and CCoV II, producing specific fragments of 239 bp (CCoV I) and 105 bp (CCoV II). We optimized the annealing temperature and primer concentrations for the CCoV nanoPCR assay and assessed its sensitivity and specificity. Under optimized nanoPCR reaction conditions, the detection limits were 6.47 × 101 copies/μL for CCoV I and 6.91 × 102 copies/μL for CCoV II. No fragments were amplified using other canine viruses as templates. The sensitivity of the nanoPCR assay was 100-fold higher than that of a conventional RT-PCR assay. Among 60 clinical samples collected from Beijing, China, the assay detected 12% positive for CCoV I and 48% positive for CCoV II. Our nanoPCR method is an effective method to rapidly detect CCoV I and CCoV II alone, or as a mixed infection, in dogs.

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Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients

Clinical Infectious Diseases ◽

10.1093/cid/ciaa345 ◽

2020 ◽

Vol 71 (15) ◽

pp. 793-798 ◽

Cited By ~ 184

Author(s):

Fengting Yu ◽

Liting Yan ◽

Nan Wang ◽

Siyuan Yang ◽

Linghang Wang ◽

...

Keyword(s):

Viral Load ◽

Target Genes ◽

Single Gene ◽

Clinical Staging ◽

N Gene ◽

Quantitative Detection ◽

Imaging Data ◽

Rt Pcr ◽

Viral Loads ◽

Nasal Swabs

Abstract Background Coronavirus disease 2019 (COVID-19) has become a public health emergency. The widely used reverse transcription–polymerase chain reaction (RT-PCR) method has limitations for clinical diagnosis and treatment. Methods A total of 323 samples from 76 COVID-19–confirmed patients were analyzed by droplet digital PCR (ddPCR) and RT-PCR based 2 target genes (ORF1ab and N). Nasal swabs, throat swabs, sputum, blood, and urine were collected. Clinical and imaging data were obtained for clinical staging. Results In 95 samples that tested positive by both methods, the cycle threshold (Ct) of RT-PCR was highly correlated with the copy number of ddPCR (ORF1ab gene, R2 = 0.83; N gene, R2 = 0.87). Four (4/161) negative and 41 (41/67) single-gene positive samples tested by RT-PCR were positive according to ddPCR with viral loads ranging from 11.1 to 123.2 copies/test. The viral load of respiratory samples was then compared and the average viral load in sputum (17 429 ± 6920 copies/test) was found to be significantly higher than in throat swabs (2552 ± 1965 copies/test, P < .001) and nasal swabs (651 ± 501 copies/test, P < .001). Furthermore, the viral loads in the early and progressive stages were significantly higher than that in the recovery stage (46 800 ± 17 272 vs 1252 ± 1027, P < .001) analyzed by sputum samples. Conclusions Quantitative monitoring of viral load in lower respiratory tract samples helps to evaluate disease progression, especially in cases of low viral load.

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