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Preanalytical Issues and Cycle Threshold Values in SARS-CoV-2 Real-Time RT-PCR Testing: Should Test Results Include These?
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  • Ilka Engelmann*
    Ilka Engelmann
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    *Email: [email protected]. Tel: 00 33 (0)3 20 44 48 80/57 95.
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  • Enagnon Kazali Alidjinou
    Enagnon Kazali Alidjinou
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
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  • Judith Ogiez
    Judith Ogiez
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
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  • Quentin Pagneux
    Quentin Pagneux
    Univ. Lille, CNRS, Centrale Lille, University Polytechnique Hauts-de-France, UMR 8520−IEMN, F-59000 Lille, France
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  • Sana Miloudi
    Sana Miloudi
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
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  • Ilyes Benhalima
    Ilyes Benhalima
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
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  • Mahdi Ouafi
    Mahdi Ouafi
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
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  • Famara Sane
    Famara Sane
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
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  • Didier Hober
    Didier Hober
    Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
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  • Alain Roussel
    Alain Roussel
    Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Campus de Luminy, CEDEX 20, 13020 Marseille, France
    Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, CEDEX 20, 13020 Marseille, France
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  • Christian Cambillau
    Christian Cambillau
    Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Campus de Luminy, CEDEX 20, 13020 Marseille, France
    Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, CEDEX 20, 13020 Marseille, France
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  • David Devos
    David Devos
    Univ. Lille, CHU-Lille, Inserm, U1172, Lille Neuroscience & Cognition, LICEND, F-59000 Lille, France
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  • Rabah Boukherroub
    Rabah Boukherroub
    Univ. Lille, CNRS, Centrale Lille, University Polytechnique Hauts-de-France, UMR 8520−IEMN, F-59000 Lille, France
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  • Sabine Szunerits*
    Sabine Szunerits
    Univ. Lille, CNRS, Centrale Lille, University Polytechnique Hauts-de-France, UMR 8520−IEMN, F-59000 Lille, France
    *Email: [email protected]. Tel: 0033-3 62 53 17 25.
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    Orcidhttp://orcid.org/0000-0002-1567-4943
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ACS Omega

Cite this: ACS Omega 2021, 6, 10, 6528–6536
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https://doi.org/10.1021/acsomega.1c00166
Published March 6, 2021

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Abstract

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Since the emergence of SARS-CoV-2 pandemic, clinical laboratories worldwide are overwhelmed with SARS-CoV-2 testing using the current gold standard: real-time reverse-transcription polymerase chain reaction (RT-PCR) assays. The large numbers of suspected cases led to shortages in numerous reagents such as specimen transport and RNA extraction buffers. We try to provide some answers on how strongly preanalytical issues affect RT-PCR results by reviewing the utility of different transport buffer media and virus inactivation procedures and comparing the literature data with our own recent findings. We show that various viral inactivation procedures and transport buffers are available and are less of a bottleneck for PCR-based methods. However, efficient alternative lysis buffers remain more difficult to find, and several fast RT-PCR assays are not compatible with guanidine-containing media, making this aspect more of a challenge in the current crisis. Furthermore, the availability of different SARS-CoV-2-specific RT-PCR kits with different sensitivities makes the definition of a general cutoff level for the cycle threshold (Ct) value challenging. Only a few studies have considered how Ct values relate to viral infectivity and how preanalytical issues might affect viral infectivity and RNA detection. We review the current data on the correlation between Ct values and viral infectivity. The presence of the SARS-CoV-2 viral genome in its own is not sufficient proof of infectivity and caution is needed in evaluation of the infectivity of samples. The correlation between Ct values and viral infectivity revealed an RT-PCR cutoff value of 34 cycles for SARS-CoV-2 infectivity using a laboratory-developed RT-PCR assay targeting the RdRp gene. While ideally each clinical laboratory should perform its own correlation, we believe this perspective article could be a reference point for others, in particular medical doctors and researchers interested in COVID-19 diagnostics, and a first step toward harmonization.

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1. From COVID-19 Symptoms to SARS-CoV-2 RT-PCR

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It is now more than a year ago, in December 2019, when health officials in Wuhan, China, reported on a disease outbreak with pneumonia syndromes. Since then, the highly pathogenic 2019 coronavirus disease (COVID-19) linked to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus particles spread worldwide, causing more than 2.19 million fatalities and more than 101 million confirmed cases worldwide (as of January 29, 2021). With a lack of approved antiviral agents, only recent vaccines (1,2) and passive immunotherapy approaches using COVID-19 convalescent plasma (3,4) at hand, testing has proven to be critical in the management of the pandemic. Target amplification and its subsequent detection is the most widely used approach for viral diagnosis and also employed for the detection of coronaviruses. (5) These methods, and, in particular, the real-time reverse-transcription polymerase chain reaction (abbreviated as RT-PCR throughout the article), are based on the use of SARS-CoV-2-specific primers and probes, designed according to the SARS-CoV-2 genome (Figure 1a). SARS-CoV-2 is an enveloped, positive-sense, single-stranded RNA virus and possesses a genome approaching 30 kB. It is arranged in the order of a 5′ untranslated region (UTR)–replicase complex [open reading frame(ORF)1ab]–structural proteins [spike (S)–envelope (E)–membrane (M)–nucleocapsid (N)]–3′UTR and nonstructural accessory proteins. (6) The RNA-dependent RNA polymerase (RdRp; nsp12) positioned on ORF1ab plays a crucial role in RNA synthesis with features of rapid mutation and recombination. (7) Theoretically, the amplification of the target genes using assay-specific primers and probes should have not only high specificity but also high sensitivity. In addition, multiple mutations were detected over the entire genomes of SARS-CoV-2, speculated to influence the rate of disease transmission and mortality but also the false-negative rate, if the mutation occurs at the position of primers or probes of the currently used target genes. (8,9)

Figure 1

Figure 1. (a) Genome of SARS-CoV-2 with the most common RT-PCR targets highlighted. (b) Essential steps of the RT-PCR diagnostic workflow including sample collection, storage in a transport medium, lysis and RNA extraction, reverse transcription, amplification, and detection. (c) Example of a real-time RT-PCR amplification curve for SARS-CoV-2 and postrun analysis to interpret results.

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Two of the first real-time RT-PCR assays receiving emergency use authorization (EUA) in February 2020 were the COVID-19 RT-PCR panel assay (IDT) as well as the RealStar SARS-CoV-2 RT-PCR (Altona Diagnostics). Since then, a large panel of COVID-19 RT-PCR assays are available (a nonexhaustive selection in Table 1 and reviewed in ref (10)) and allowed not only better management of the crisis but helped in understanding both the disease and its epidemiology. (11,12) These assays are all based on some of the essential steps of RT-PCR (Figure 1b), including (i) lysis of SARS-CoV-2 virus particles and purification of nucleic acids from collected samples (oropharyngeal, nasopharyngeal swabs, and/or lower respiratory tract samples, e.g., sputum, tracheal aspirates, or bronchoalveolar lavage specimens) dispersed in a universal or viral transport medium (UTM/VTM), (ii) reverse transcription to complementary DNA (cDNA), (iii) amplification of the specific region of the cDNA, and (iv) optical detection. The assays differ in the primers/probes used to amplify/detect the different target regions of the SARS-CoV-2 genome (Table 1), which consequently affects their sensitivity. (13−15) The difference is generally more pronounced for samples with low viral load, with a median cycle threshold (Ct) of >33, as recommended by the “Société Française de Microbiologie”. (16)
Table 1. Some Selected SARS-CoV-2 RNA Detection Assays
developerkitextractionamplificationcycler
Thermofisher ScientificTaqPath COVID-19 CE-IVD RT-PCR KitMGI Nucleic Acid Extraction Kit on the MGI SP-960 instrumentORF1ab, S, NQuantStudio 5 Real-Time PCR System (Applied Biosystems)
Altona DiagnosticsRealStar SARS-CoV-2 RT-PCR Kit 1.0AltoStar Purification Kit 1.5 extraction kit on AltoStar Automation System AM16E, SCFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad Laboratories)
Beijing Genomics InstituteReal-Time Fluorescent RT-PCR Kit for Detecting SARS-CoV-2MGI Nucleic Acid Extraction Kit on the MGI SP-960 instrumentORF1QuantStudio 5 Real-Time PCR System (Applied Biosystems)
DiaSorin MolecularSimplexa COVID-19 directNoneS, ORF1abLIAISON MDX
BDBD SARS-CoV-2 reagents for BD MAX systemBD MAX ExK TNA-3N1, N2BD MAX
SeegeneAllPlex 2019-nCoV assayStarMag 96 Extraction Kit on Microlab NimbusE, N, RdRpCFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories)
QiagenQIAstat-Dx respiratory panelIncluded automated RNA extractionE, ORF1bQIAstat
CepheidXpert Xpress SARS-CoV-2Included automated RNA extractionN2, EGeneXpert Infinity
Institut Pasteur Parislaboratory developed (17)QIAamp Viral RNA Mini Kit (Qiagen, France)RdRp (two target regions, named IP2, IP4)7500 Real-Time PCR System (Applied Biosystems)
If SARS-CoV-2 RNA is present, then each round of amplification results in a doubling of the amount of the target. As a result, amplification occurs exponentially, producing an exponential curve of amplification (Figure 1c) visualized by the use of a fluorescent nucleic acid probe. The Ct value is defined as the number of cycles of amplification required for the fluorescence of a PCR product to be detected crossing a threshold, which is above the background signal (a low-level signal in the assay regardless of whether the target is present or not) (Figure 1c).
This rather complex process is based on the use of correct preanalytical procedures, namely, appropriate transport media, storage conditions, and lysis buffers without causing RNA damage as well as on a correct interpretation of the Ct values. The risk of false-positive laboratory tests increases as the probability of COVID-19 infection decreases. One key parameter with infected patients harboring low virus loads (either at the very beginning or at the end of the infection) is high Ct values near the detection limit of the assay (Figure 1c). The risk of false-negative results is correlated with a lack of sensitivity of the analytical method, when the infectious dose is too low but is most strongly affected by the preanalytical steps (storage and transport) and the sampling approach itself (i.e., type and quality of the specimen). Indeed, nasopharyngeal swabs are recommended for screening and need to be correctly performed to contain a sufficient number of infected cells in order to give a right picture of the infection state of the patient. What guidance can be given to enable the discernment of true positives from false positives? How much does sample preparation and the use of lysis buffers and heat inactivation affect the results? Are Ct values correlated to viral infectivity or does a positive result only indicate that the person has come into contact with the genomic sequence at some time in the recent past?

2. Does the Success of RT-PCR Depend on the Sample Quality and Preparation?

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Diagnostics of viral infection using PCR or RT-PCR is now well-established for many viruses. Most of the time, these techniques are performed on blood-derived specimens, i.e., whole blood, serum, or plasma. In these cases, there is no need for transport buffers and virus inactivation is not performed. Other preanalytical issues, like storage or lysis, also apply to these specimen types. However, in SARS-CoV-2 diagnostics, two additional challenges apply. First, the specimen type is not blood-derived but, in general, a respiratory swab specimen. Therefore, there is a need to place this specimen in some kind of a transport medium. Second, the pathogen is classified biosafety level 3 (BSL-3) and diagnostic procedures except virus culture can be performed in BSL-2 level facilities with appropriate protective measures. (18) Because of high specimen numbers, automatization of diagnostic assays is necessary, and due to the severity of the illness and high infectiousness of the virus, many laboratories, including ours, perform virus inactivation before analysis. The success of the diagnosis of SARS-CoV-2 depends largely on the quality of the collected specimens and the conditions under which they are transported and stored before being processed in the laboratory. The most important point is that the specimen is of good quality, i.e., it contains virus/virus-infected cells. This is especially critical for nasopharyngeal swab specimens. Amplification of a cellular gene, e.g., G6PDH, enables monitoring of the cellular content as a surrogate marker of the specimen quality. However, in routine practice, most commercial assays include instead an internal control, e.g., an artificial RNA that is added to the specimen prior to RNA extraction, which only monitors extraction and amplification steps. In the case of SARS-CoV-2, specimens are mostly dispersed in nutrient solutions such as a viral transport medium (VTM) or a universal transport medium (UTM). The constituents of an appropriate viral transport medium are selected to create an isotonic solution containing proteins to protect the viral structure, antibiotics to inhibit microbial contamination, and one or more buffers to control the pH. Often, VTM/UTM are based on Hanks balanced salt solutions, calcium and magnesium, proteins (fetal bovine serum and albumin), as well as antibiotics such as gentamycin and amphotericin. Viral transport media are prepared with the idea of sustaining the viability of the viruses for detection in cell culture. While these complex solutions present a challenge for molecular SARS-CoV-2 diagnostics such as electrochemical-, (19) electrical- (20) and/or optical-based biosensors (11,21,22) due to eventual surface antifouling, (19,20) they are less of a bottleneck for PCR-based methods.
For RT-PCR, effective RNA extraction remains one of the main issues. The key chemical constituent of the lysis buffer is guanidium thiocyanate, a chaotropic agent that denatures proteins, helps the liberation of nuclear RNA and genomic DNA, and protects against RNAse enzyme degradation. In standard formulations, such as those commercialized by, e.g., Qiagen (AVL buffer), up to 6 M guanidium thiocyanate is used. (23) With the epidemic doubling in size every 3–7 days and the increased need of RNA extraction reagents such as guanidium thiocyanate, this chemical became scarce and alternatives were sought after. Scallen and her colleagues validated that a lysis buffer containing only 4 M guanidinium thiocyanate and Triton X-100 (3% v/v) was effective for the extraction of SARS-CoV-2 RNA from respiratory specimens and allowed easing the burden on hospital laboratories. (23) The inclusion of a detergent such as Triton X-100 enhances the disintegration of the viral envelope during extraction. We have recently demonstrated the utility of a specimen transport buffer containing next to Tris-EDTA 10 × (100 mM Tris/10 mM EDTA), only 2.53 M guanidine thiocyanate (30%), and Triton X-100 (2.5% v/v, 34.4 mM). (24) When respiratory specimens were diluted in this buffer, the quantity of viral RNA for two SARS-CoV-2 targets was stable at room temperature until at least day 5. There was a slight increase in Ct values for the SARS-CoV-2 targets after day 5, whereas Ct values for the cellular control gene G6PDH remained stable until day 30. After 30 days of storage at room temperature, SARS-CoV-2 RNA could be detected consistently in the case of high virus loads (i.e., Ct values of up to 30) but not in the case of low virus loads. These results confirm that lower guanidine concentrations had no negative effect on stabilizing viral RNA. This transport buffer could solve two issues: the problems of shortages in a viral transport medium and limited cold room capacities. However, it has to be kept in mind that some rapid RT-PCR assays are not compatible with guanidine-containing media as guanidine thiocyanate is unstable in a sodium hypochlorite solution, commonly known as bleach and a widely used cleaning reagent in molecular testing workflows and is also an inhibitor of the Taq polymerase, which leads to uninterpretable results when using assays without an RNA extraction step. (24−26)

3. Importance of SARS-CoV-2 Inactivation and Its Consequences for RT-PCR

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To allow safe transportation of clinical specimens from collection points to laboratories, as well as for performing experiments involving SARS-CoV-2 in general, inactivation procedures are performed. The efficiency of SARS-CoV-2 inactivation depends on several factors, including next to its concentration, the sample matrix and contact time. Up to date, only limited data on the efficacy of SARS-CoV-2-specific inactivation approaches are reported in the literature (Table 2), (27,28) as risk assessments have largely been based upon virus inactivation information from related coronaviruses such as SARS-CoV-1. (29,30)
Table 2. Inactivation Procedures of SARS-CoV-2
methodconditionsinactivationrefs
heat100 °C (5 min)yes (31)
80 °C (60 min)yes (29)
60 °C (30 min)yes (32)
56 °C (45 min)yes (31)
56 °C (30 min)no (31)
UV irradiation0.01 J cm–2no (29)
0.04 J cm–2yes (29)
chemicalsodium dodecyl sulfate (1 h, 0.5%)yes (28)
Triton X-100 (1 h, 0.5%)yes (28)
NP-40 (1 h, 0.5%)yes (28)
Tween 20 (1 h, 0.5%)no (28)
guanidine thiocyanate (1 day, 1.27 M) Triton X-100 (1 day, 2.5% v/v)no (24)
TRIzol (10 min,10%)yes (31)
formalin (10 min, 0.5–2%)yes (31)
Heat inactivation is the most widely employed method for SARS-CoV-2. (28) Patterson et al. heated SARS-CoV-2 at 80 °C for 1 h and demonstrated complete virus inactivation. The complete inactivation of SARS-CoV-2 viral particles upon heating at 60 °C for 30 min was confirmed by us recently. (32) These results are in line with the data by Jureka et al., (31) where 5 min at 100 °C was sufficient for virus inactivation, while 45 min at 56 °C was required for complete inactivation. We recently confirmed that virus inactivation can be achieved upon heating at 55 °C for 45 min. (32)
It is important to note that heat inactivation at 60 °C for 30 min preserved the antigenic spike protein function (Figure 1a), used currently in several point-of-care antigenic tests. (19,20) This was confirmed by affinity studies between heated and nonheated SARS-CoV-2 receptor-binding domain (RBD) and SARS-CoV-2 surface receptor using surface plasmon resonance (SPR) binding curves (Figure 2a). Heating the RBD at 100 °C for 60 min showed no binding affinity to SPR chips modified with RBD-specific VHH-72 surface ligands. (33) A lower binding efficiency was still observed for RBD heated for 45 min at 60 °C. This contrasts with RBD treatment for 30 or 15 min at 60 °C, where only a slight degradation of the protein seems to have taken place and binding events are comparable to those recorded for non-heat-treated specimens. In addition, heat treatment at 60 °C did not have a negative effect on the RNA stability (Figure 2b–d), making it an ideal and clean inactivation process.

Figure 2

Figure 2. Quantification of SARS-CoV-2 following exposure to different inactivation conditions: (a) surface plasmon resonance (SPR) binding curves recorded on a T200 Biacore for the receptor-binding domain (RBD, 200 nM) and RBD for different time intervals. The interface was modified with SARS-CoV-2-specific VHH-72 nanobodies. (33) (b–d) RNA stability after heat inactivation: SARS-CoV-2 RNA-positive nasopharyngeal swab specimens were pooled and divided into equal volumes and heated for 30 min at 60 °C. The aliquots were either kept at room temperature (RT), 4 °C, or −80 °C for the indicated times before RNA extraction and RT-PCR was performed in triplicate. (32) As a control, untreated specimens were included. Ct values are indicated for the SARS-CoV-2 targets IP2 (b), IP4 (c), and the cellular control G6PDH (d) (Figure 2−2d report mean values of three samples).

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Next to heat, UV irradiation, based on the modification of the nucleic acid structure, proved to be effective in SARS-CoV-2 inactivation. (29) The approach is dependent on the exposed light energy with 0.01 J cm–2, leading to partial viral inactivation, while light energy values higher than 0.04 J cm–2 resulted in complete inactivation.
Chemical inactivation using various detergents (27,28,31) and chemicals, such as formaldehyde (34) and β-propionolactone, (35−37) was also evaluated. In the case of sodium dodecyl sulfate (SDS), Triton 126X-100, and NP-40, a commercially available ethoxylated nonylphenol surfactant, complete inactivation of SARS-CoV-2 was achieved. (28) However, upon treatment with Tween 20 (0.5%) for 1 h, all specimens remained infectious. This was also the case using our proposed transport buffer based on Tris-EDTA 10 × (100 mM Tris/10 mM EDTA), guanidine thiocyanate (30%, 2.53 M), and Triton X-100 (2.5% v/v, 34.4 mM) at a dilution of 1/2 and 9/10. In contrast, SARS-CoV-2 treatment with TRIzol (10%) and a formalin (0.5–2%) solution for 1 h at room temperature proved to be effective in SARS-CoV-2 inactivation. (31) The same was true when SARS-CoV-2 was exposed to β-propiolactone (0.5%) for 16 h at 4 °C, followed by 2 h at 37 °C. (31) While heating of specimens is usually performed in the diagnostic laboratory, chemical inactivation using appropriate specimen transport buffers would enable virus inactivation before transport and thus facilitates transport and preanalytical procedures. (38)

4. Predicting Infectious SARS-CoV-2 from Diagnostic Specimens

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Next to the sampling and specimen preparation, the use of an appropriate cutoff value for the proper interpretation of SARS-CoV-2 PCR results is of paramount importance. However, these values can only be interpreted correctly by having an idea about the health history of the patient, as pointed out lately by communication by Public Health England. (39) Byrne et al. (40) published one of the first overviews of infectious periods of SARS-CoV-2 up to April 1, 2020. Substantial variations were observed with mean infectious period of asymptomatic cases between 6.5 and 9.5 days, shortening in presymptomatic cases to 1–4 days, with 13.4 days estimated from the onset of the symptoms to a negative RT-PCR. (40) This review was complemented more recently by a review and meta-analyses by Weiss and co-workers (41) and Cevik et al. (42) with data on nasopharyngeal swab samples taken in the first week after the onset of COVID-19 symptoms. (41,42) These specimens revealed high viral loads in both studies. (41,42)Figure 3a schematically illustrates the viral load dynamics from the symptom onset to recovery for symptomatic cases. It is believed that infectiousness begins 2–3 days prior to symptoms’ onset and peaks around the symptom onset. (43) The infectious period is thus approximately 9–10 days. In fact, virus load is already high before the symptom onset and almost half of transmissions to secondary cases occur in the presymptomatic period. (44−46) The timeline of SARS-CoV-2 RNA was lately confirmed by some of us (47) using data from 520 COVID-19 patients. The Ct values of nasopharyngeal swab specimens were plotted against the time after the symptom onset (Figure 3b). The lowest Ct values, corresponding to the highest virus loads, were found early after the symptom onset, followed by a decline in virus load with increasing time after the symptom onset.

Figure 3

Figure 3. Clinical significance of SARS-CoV-2 RT-PCR results. (a) Timeline of SARS-CoV-2 infectivity taking into account our own findings and those of others. (39,40) (b) Ct values (target IP2) as a function of time after the symptom onset in nasopharyngeal swab specimens of COVID-19 patients. Ct of specimens with undetectable SARS-CoV-2 RNA were set to 50. (c) Correlation of Ct values with SARS-CoV-2 infectivity. Vero E6 cells were infected with 10-fold dilutions of a SARS-CoV-2 isolate. The plates were incubated for 6 days in 5% CO2 at 37 °C and examined daily using an inverted microscope (ZEISS Primovert) to evaluate the extent of the virus-induced cytopathic effect in cell culture. The calculation of the estimated virus concentration was carried out by the Spearman and Karber method (48,49) and expressed as TCID50/mL (50% tissue culture infectious dose). TCID50/mL values were transformed to PFU mL–1 using the formula PFU mL–1 = TCID50/mL × 0.7. (50) RNA extraction and RT-PCR (target IP4 and target IP2) were performed in duplicate for each dilution. Ct of dilutions with undetectable SARS-CoV-2 RNA were set to 50.

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There is a general agreement that Ct values are strongly associated with SARS-CoV-2 viral load and infectivity. While a threshold cutoff of Ct 33–35 is associated with low infectivity, a Ct value of 20 is linked to a highly infective person. Ct values are semiquantitative, with a 3-point increase in the Ct value being roughly equivalent to a 10-fold decrease in the quantity of viral genetic material. While clinical laboratories often use Ct cutoff values provided by the RT-PCR kit manufacturers, which are between 37 and 40 cycles, to define a specimen with positive SARS-CoV-2 RNA detection, correlation between viral RNA load with patient specimen infectivity can only be determined in BLS-3 settings.
Some studies have been conducted in an attempt to correlate Ct values with SARS-CoV-2 virus infectivity. (46,51−53) While being a laborious process in BLS-3 settings, viability of virus can be determined by inoculating cultured Vero cells and assessing evidence of the cytopathic effect. Data in Figure 3c were obtained by first determining the Ct value of a SARS-CoV-2 clinical isolate, followed by a serial dilution of this specimen in 10-fold steps. As expected, a linear relation between the Ct values and serial dilution was observed for both RT-PCR targets, IP4 and IP2. The same dilutions were further used for infecting Vero cells and determining the infectivity of each dilution. While the initial specimens with a Ct value of 12 correspond to a viral load of 2.2 × 106 pfu mL–1, at a Ct of 33, only 15 pfu mL–1 are detected. Our system correlates with a cutoff value of 34 cycles for SARS-CoV-2 infectivity. The relationship between RT-PCR values and culture positivity was also validated by Singanayagam et al. (54) who found that culturing virus declined to 8% in specimens with Ct > 35.
A study by La Scola and colleagues examined 3790 positive specimens with known Ct values and found that 70% of these specimens could be cultured if having a Ct ≤ 25, but less than 3%, if having Ct > 35. (52) Bullard tested 90 samples from Covid-19-positive specimens through incubation with Vero cells to assess their infectivity. (53) They concluded that specimens with Ct > 24 or taken >8 days after the symptom onset were not able to infect cells. Indeed, positive RT-PCR tests are not correlated to the infection state. Wölfel et al. demonstrated that after 8 days from the symptom onset, virus could not be isolated in cell culture, despite ongoing high viral load in RT-PCR results. (55) Indeed, other studies concluded that SARS-CoV-2 RNA can still be detected 6–8 weeks after the symptom onset and after clearance of symptoms, with no correlated cell culture infectivity. (45,56) So, could Ct values help doctors to indicate if the patient is highly infectious or not and should be isolated and contacts tracked down? Ct values are believed by many to be an imperfect measure and laboratory-dependent. Furthermore, infectivity, measured by infection of cell culture, can only be a surrogate marker for infectivity. A virus might be more effective in infecting its natural host than cell culture. The real answer to the question of infectivity can only be provided by epidemiological studies, correlating the number of secondary cases infected by a person with a given Ct value at a given time. This kind of study would be extremely valuable but would need exact contact tracing to identify contacts that had only contact on the same day when the Ct value was obtained because nobody can predict what Ct the person’s specimen harbored the day before or after.

5. Conclusions and Future Critical Outlook

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With the rapid diagnosis of COVID-19-infected patients using RT-PCR, clinical laboratories played a key role in controlling the spread of SARS-CoV-2 virus. Since the beginning of the pandemic, there are controversial views about the infectiousness of patients as a positive RT-PCR test cannot tell whether the person is infectious or not or when the infection started. In most cases, virus load quickly decreases after the symptom onset, and most patients will no further have SARS-CoV-2 RNA detection in nasopharyngeal swabs in a median of 14–25 days after the symptom onset. However, in elderly, severely ill, or immunosuppressed patients, nasopharyngeal swabs and lower respiratory tract specimens can still be positive for SARS-CoV-2 RNA detection several weeks after the symptom onset. (45,57) In general, these specimens harbor high Ct values corresponding to low virus load. The question arose whether this corresponds to infectious virus or only viral genomic material. The detection of the presence of a viral genome on its own is therefore not sufficient. Ct values are thus believed by many to be an imperfect measure, laboratory-dependent, and not very useful. However, several studies that aimed at quantifying the relationship between Ct and the likelihood of culturing live viruses suggest that the probability to recover a live virus from specimens with Ct > 34 is low. Defining a general Ct cutoff is obviously a real challenge. Infectivity and RT-PCR Ct cutoff values also depend on the time span, which evolved between sampling and analysis. Indeed, samples are transported and stored at 4 °C for several hours prior to analysis. The storage at 4 °C for up to 5 days is recommended by WHO and the use of storage buffers is believed to conserve the sample quality. (58)
However, using the Ct value as a surrogate marker for virus load in a clinical routine will need the harmonization of the numerous SARS-CoV-2 assays. Harmonization of virus load measurements between different laboratories and assays is a general challenge that has not been completely resolved even for virus load assessments that are routinely performed for several years. Development of WHO international standards for quantification is one step to try to harmonize virus load determination. (59) Defining RT-PCR cutoff levels is furthermore necessary for evaluating the performance of other SARS–CoV-2 tests compared to RT-PCR. (60)
The current gold standard for SARS-CoV-2 diagnostics is real-time RT-PCR. Classical assays take 4–6 h while rapid molecular assays have been developed and yield results in less than 2 h. (10) However, these need specific instruments and the price per test is high. While clinical laboratories still play the dominant role in patient specimen screening, integrated point-of-care (POC) platforms are believed to hold a central place in the future. While RT-PCR approaches will remain the gold standard for viral testing and thus for SARS-CoV-2, POC devices will allow overcoming the bottleneck of widespread nucleic acid testing due to their intrinsic characteristics being simple, faster, and cheap with the ability of COVID-19 diagnostic in the doctor’s office as well as at home. Such platforms will ensure reduced turnaround time, unlimited retesting, and a potentially lower cost by reducing reagent consumptions. Currently, such platforms mostly detect SARS-CoV-2 antigens and severely lack sensitivity compared to RT-PCR. (61−63) Recent developments include loop-mediated isothermal amplification (LAMP) POC tests. These are cost-effective, rapid molecular assays with satisfying specificity and sensitivity. However, they generally require a sample preparation step and a specific instrument. (10) Currently available POC tests detect viral components but not the entire virus particle. It would be, however, much more elegant and eventually more efficient to detect the infectivity status of the patient by sensing the presence of the S1 protein on the surface of an intact SARS-CoV-2 particle. We are currently working on the development of such a test. We could indeed discriminate COVID-19-negative from COVID-19-positive patients upon the use of electrodes functionalized with engineered nanobodies in an electrochemical sensing format. (19,64) The developed sensor was tested in a clinical trial on 100 nasal swab specimens and showed a 88% positive percentage agreement (PPA) and a 88% negative percentage agreement (NPA), as compared to RT-PCR with an analysis time of 10 min without any pretreatment step.
The use of POC testing also means treating a diagnostic assay as an integrated unit from specimen collection and processing to final analysis. As these tests are designed to be done on site and immediately, no viral transportation media nor viral inactivation protocols are required, making the assays much easier and faster to perform. This implies moving from nasopharyngeal specimens, which have to be taken by professionals, to alternative specimens such as saliva and exhaled breath condensate (EBC) that can easily be collected by the patient at home. The EasyCov saliva-based screening test, developed jointly by CNRS researchers in Montpellier, France, and two private sector companies (Alcediag and SkillCell), is one of the examples where COVID-19 diagnostics will stand in the future. Easycov enables mass testing via loop-mediated isothermal amplification (LAMP), resulting in a colorimetric readout. The test that is completed in 40 min succeeded in detecting 87% of positive cases among 220 people who had been tested at the Montpellier University Hospital since September 6, 2020.
Breath sampling might be another approach in the future as it does not require trained medical personnel and can be done essentially anywhere in any time frame. Exhaled breath contains tiny aerosols and the collection of these aerosol particles, produced by patients during coughing or breathing, potentially provides a noninvasive method for collection of diagnostic specimens of SARS-CoV-2. It has been indeed postulated lately that the exhaled breath is a significant source of SARS-CoV-2 emission. (65)
In conclusion, the ongoing COVID-19 crisis revealed that complex issues such as viral transportation, viral inactivation, and viral diagnostics require knowledge and expertise in different topics and domains. It is through the tight interplay between biologists, chemists, nanotechnologists, and medical doctors that COVID-19 diagnostics will ultimately allow controlling the worldwide pandemic. The enormous know-how gained with SARS-CoV-2 sensing can often be easily adapted to other viral infection assays and researchers are urged to continue their strong involvement in the actual sanitary crisis. It is timely to put the importance of research and innovation at the forefront to make society understand that money is not wasted while doing research with positive input to society at large.

Author Information

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  • Corresponding Authors
  • Authors
    • Enagnon Kazali Alidjinou - Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    • Judith Ogiez - Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    • Quentin Pagneux - Univ. Lille, CNRS, Centrale Lille, University Polytechnique Hauts-de-France, UMR 8520−IEMN, F-59000 Lille, France
    • Sana Miloudi - Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    • Ilyes Benhalima - Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    • Mahdi Ouafi - Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    • Famara Sane - Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    • Didier Hober - Univ. Lille, CHU Lille, Laboratoire de Virologie ULR3610, F-59000 Lille, France
    • Alain Roussel - Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Campus de Luminy, CEDEX 20, 13020 Marseille, France;  Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, CEDEX 20, 13020 Marseille, France
    • Christian Cambillau - Architecture et Fonction des Macromolécules Biologiques, Aix-Marseille Université, Campus de Luminy, CEDEX 20, 13020 Marseille, France;  Architecture et Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, CEDEX 20, 13020 Marseille, France
    • David Devos - Univ. Lille, CHU-Lille, Inserm, U1172, Lille Neuroscience & Cognition, LICEND, F-59000 Lille, France
    • Rabah Boukherroub - Univ. Lille, CNRS, Centrale Lille, University Polytechnique Hauts-de-France, UMR 8520−IEMN, F-59000 Lille, France
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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This work was financially supported by the Centre National de la Recherche Scientifique (CNRS), the University of Lille, I-SITE via the COVID task force, and the Hauts-de-France region via ANR Resilience (CorDial-FLU). The project is funded by the Horizon 2020 framework programme of the European Union under grant agreement no 101016038. The authors thank all technicians of the virology laboratory of the Lille University Hospital (CHU Lille) for their excellent technical assistance under extremely challenging conditions. They also acknowledge the valuable help of medical and pharmaceutical students.

References

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This article references 65 other publications.

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    A review. Since late Dec. 2019, the coronavirus pandemic (COVID-19; previously known as 2019-nCoV) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world. With more than 1,700,000 confirmed cases, the world faces an unprecedented economic, social, and health impact. The early, rapid, sensitive, and accurate diagnosis of viral infection provides rapid responses for public health surveillance, prevention, and control of contagious diffusion. More than 30% of the confirmed cases are asymptomatic, and the high false-neg. rate (FNR) of a single assay requires the development of novel diagnostic techniques, combinative approaches, sampling from different locations, and consecutive detection. The recurrence of discharged patients indicates the need for long-term monitoring and tracking. Diagnostic and therapeutic methods are evolving with a deeper understanding of virus pathol. and the potential for relapse. In this Review, a comprehensive summary and comparison of different SARS-CoV-2 diagnostic methods are provided for researchers and clinicians to develop appropriate strategies for the timely and effective detection of SARS-CoV-2. The survey of current biosensors and diagnostic devices for viral nucleic acids, proteins, and particles and chest tomog. will provide insight into the development of novel perspective techniques for the diagnosis of COVID-19.
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    Uhteg, K.; Jarrett, J.; Richards, M.; Howard, C.; Morehead, E.; Geahr, M.; Gluck, L.; Hanlon, A.; Ellis, B.; Kaur, H. Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays. J. Clin. Virol. 2020, 127, 104384  DOI: 10.1016/j.jcv.2020.104384
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    Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays
    Uhteg, Katharine; Jarrett, Junko; Richards, Mahmia; Howard, Craig; Morehead, Elizabeth; Geahr, Melissa; Gluck, Linda; Hanlon, Ann; Ellis, Brandon; Kaur, Harsimar; Simner, Patricia; Carroll, Karen C.; Mostafa, Heba H.
    Journal of Clinical Virology (2020), 127 (), 104384CODEN: JCVIFB; ISSN:1386-6532. (Elsevier B.V.)
    In Dec. 2019, a novel coronavirus (SARS-CoV-2) was first isolated from Wuhan city, China and within three months, the global community was challenged with a devastating pandemic. The rapid spread of the virus challenged diagnostic labs. to rapidly develop mol. diagnostic methods. As SARS CoV-2 assays became available for testing on existing mol. platforms, labs. devoted unprecedented energy and resources into evaluating the anal. performance of the new tests and in some cases developed their own diagnostic assays under FDA-EUA guidance. This study compares the validation of three different mol. assays at the Johns Hopkins Mol. Virol. lab.: the RealStar SARS-CoV-2 RT-PCR, ePlex SARS-CoV-2, and the CDC COVID-19 RT-PCR tests. Overall, our studies indicate a comparable anal. performance of the three assays for the detection of SARS-CoV-2.
  14. 14
    Vogels, C. B. F.; Brito, A. F.; Wyllie, A. L.; Fauver, J. R.; Ott, I. M.; Kalinich, C. C.; Petrone, M. E.; Casanovas-Massana, A.; Muenker, M. C.; Moore, A. J. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets. Nat. Microbiol. 2020, 5, 1299– 1305,  DOI: 10.1038/s41564-020-0761-6
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    Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets
    Vogels, Chantal B. F.; Brito, Anderson F.; Wyllie, Anne L.; Fauver, Joseph R.; Ott, Isabel M.; Kalinich, Chaney C.; Petrone, Mary E.; Casanovas-Massana, Arnau; Catherine Muenker, M.; Moore, Adam J.; Klein, Jonathan; Lu, Peiwen; Lu-Culligan, Alice; Jiang, Xiaodong; Kim, Daniel J.; Kudo, Eriko; Mao, Tianyang; Moriyama, Miyu; Oh, Ji Eun; Park, Annsea; Silva, Julio; Song, Eric; Takahashi, Takehiro; Taura, Manabu; Tokuyama, Maria; Venkataraman, Arvind; Weizman, Orr-El; Wong, Patrick; Yang, Yexin; Cheemarla, Nagarjuna R.; White, Elizabeth B.; Lapidus, Sarah; Earnest, Rebecca; Geng, Bertie; Vijayakumar, Pavithra; Odio, Camila; Fournier, John; Bermejo, Santos; Farhadian, Shelli; Dela Cruz, Charles S.; Iwasaki, Akiko; Ko, Albert I.; Landry, Marie L.; Foxman, Ellen F.; Grubaugh, Nathan D.
    Nature Microbiology (2020), 5 (10), 1299-1305CODEN: NMAICH; ISSN:2058-5276. (Nature Research)
    The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the crit. need for accurate and rapid diagnostic assays to prompt clin. and public health interventions. Currently, several quant. reverse transcription-PCR (RT-qPCR) assays are being used by clin., research and public health labs. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR anal. efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charite´) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.
  15. 15
    Nalla, A. K.; Casto, A. M.; Huang, M.-L.; Perchetti, G. A.; Sampoleo, R.; Shrestha, L.; Wei, Y.; Zhu, H.; Jerome, K. R.; Greninger, A. L. Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit. J. Clin. Microbiol. 2022, 58, e00557-20  DOI: 10.1128/JCM.00557-20
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    Avisdu 25 septembre 2020 de la Société Française de Microbiologie (SFM) relatifà l’interprétation de la valeur de Ct (estimation de la charge virale)obtenue en cas de RT-PCR SARS-CoV-2 positive sur les prélèvements cliniquesréalisés à des fins diagnostiques ou de dépistage. https://www.sfm-microbiologie.org/covid-19-fiches-et-documents-sfm/ (Version 3 du 7 octobre, 2020).
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    Institut Pasteur, Paris. Protocol: Real-time RT-PCR Assays for the Detection of SARS CoV-2. [Internet] [cited 2020 Aug 28]. https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2.
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    Arrêté du 18 décembre 2020 relatif à la classification du coronavirus SARS-CoV-2 dans la liste des agents biologiques pathogènes. https://www.legifrance.gouv.fr/jorf/article_jo/JORFARTI000042840497.
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    Szunerits, S.; Pagneux, Q.; Swaidan, A.; Mishyn, V.; Roussel, A.; Cambillau, C.; Devos, D.; Engelmann, I.; Alidjinou, E. K.; Happy, H. The role of the surface ligand on the performance of electrochemical SARS-CoV-2 antigen biosensors. Anal. Bioanal. Chem. 2021. DOI: 10.1007/s00216-020-03137-y .
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    Seo, G.; Lee, G.; Kim, M. J.; Baek, S.-H.; Choi, M.; Ku, K. B.; Lee, C.-S.; Jun, S.; Park, D.; Kim, H. G. Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor. ACS Nano 2020, 14, 5135– 5142,  DOI: 10.1021/acsnano.0c02823
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    20
    Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor
    Seo, Giwan; Lee, Geonhee; Kim, Mi Jeong; Baek, Seung-Hwa; Choi, Minsuk; Ku, Keun Bon; Lee, Chang-Seop; Jun, Sangmi; Park, Daeui; Kim, Hong Gi; Kim, Seong-Jun; Lee, Jeong-O.; Kim, Bum Tae; Park, Edmond Changkyun; Kim, Seung Il
    ACS Nano (2020), 14 (4), 5135-5142CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV). Based on the rapid increase in the rate of human infection, the World Health Organization (WHO) has classified the COVID-19 outbreak as a pandemic. Because no specific drugs or vaccines for COVID-19 are yet available, early diagnosis and management are crucial for contg. the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clin. samples. The sensor was produced by coating graphene sheets of the FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was detd. using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Our FET device could detect the SARS-CoV-2 spike protein at concns. of 1 fg/mL in phosphate-buffered saline and 100 fg/mL clin. transport medium. In addn., the FET sensor successfully detected SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 x 101 pfu/mL) and clin. samples (LOD: 2.42 x 102 copies/mL). Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a highly sensitive immunol. diagnostic method for COVID-19 that requires no sample pretreatment or labeling.
  21. 21
    Kilic, T.; Weissleder, R.; Lee, H. Molecular and Immunological Diagnostic Tests of COVID-19: Current Status and Challenges. iScience 2020, 23, 101406  DOI: 10.1016/j.isci.2020.101406
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    Molecular and Immunological Diagnostic Tests of COVID-19: Current Status and Challenges
    Kilic, Tugba; Weissleder, Ralph; Lee, Hakho
    iScience (2020), 23 (8), 101406CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
    A review. Rapid spread of coronavirus disease 2019 (COVID-19) is ravaging the globe. Since its first report in Dec. 2019, COVID-19 cases have exploded to over 14 million as of July 2020, claiming more than 600,000 lives. Implementing fast and widespread diagnostic tests is paramount to contain COVID-19, given the current lack of an effective therapeutic or vaccine. This focuses on a broad description of currently available diagnostic tests to detect either the virus (SARS-CoV-2) or virus-induced immune responses. We specifically explain the working mechanisms of these tests and compare their anal. performance. These analyses will assist in selecting most effective tests for a given application, for example, epidemiol. or global pandemic research, population screening, hospital-based testing, home-based and point-of-care testing, and therapeutic trials. Finally, we lay out the shortcomings of certain tests and future needs.
  22. 22
    Jayamohan, H.; Lambert, C. J.; Sant, H. J.; Jafek, A.; Patel, D.; Feng, H.; Beeman, M.; Mahmood, T.; Nze, U.; Gale, B. K. SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations. Anal. Bioanal. Chem. 2020, 1– 23,  DOI: 10.1007/s00216-020-02958-1
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    Scallan, M. F.; Dempsey, C.; MacSharry, J.; O’Callaghan, I.; O’Connor, P. M.; Horgan, C. P.; Durack, E.; Cotter, P. D.; Hudson, S.; Moynihan, H. A. Validation of a lysis buffer containing 4 M guanidinium thiocyanate (GITC)/Triton X-100 for extraction of SARS-CoV-2 RNA for COVID-19 testing: comparison of formulated lysis buffers containing 4 to 6 M GITC, Roche external lysis buffer and Qiagen RTL lysis buffer. bioRxiv 2020, 026435  DOI: 10.1101/2020.04.05.026435
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    Engelmann, I.; Benhalima, I.; Ouafi, M.; Pagneux, Q.; Boukherroub, R.; Hober, D.; Alidjinou, E. K.; Szunerits, S. Validation of a specimen transport buffer for stabilization of SARS-CoV-2 RNA at room temperature for molecular diagnostics,2020, unpublished results.
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    Auerswald, H.; Yann, S.; Dul, S.; In, S.; Dussart, P.; Martin, N. J.; Karlsson, E. A.; Garcia-Rivera, J. A. Assessment of Inactivation Procedures for SARS-CoV-2. BioRxiv 2020, 20, 533  DOI: 10.1101/2020.05.28.120444
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    van Bockel, D.; Munier, C. M. L.; Turville, S.; Badman, S. G.; Walker, G.; Stella, A. O.; Anupriya Aggarwal, A.; Yeang, M.; Condylios, A.; Kelleher, A. D. Evaluation of Commercially Available Viral TransportMedium (VTM) for SARS-CoV-2 Inactivation and Usein Point-of-Care (POC) Testing. Viruses 2020, 12, 1208  DOI: 10.3390/v12111208
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    Evaluation of commercially available viral transport medium (VTM) for SARS-CoV-2 inactivation and use in point-of-care (POC) testing
    van Bockel, David; Munier, C. Mee Ling; Turville, Stuart; Badman, Steven G.; Walker, Gregory; Stella, Alberto Ospina; Aggarwal, Anupriya; Yeang, Malinna; Condylios, Anna; Kelleher, Anthony D.; Applegate, Tanya L.; Vallely, Andrew; Whiley, David; Rawlinson, William; Cunningham, Phillip; Kaldor, John; Guy, Rebecca
    Viruses (2020), 12 (11), 1208CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
    Crit. to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted expts. to det. the virucidal activity of com. available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addn. to a buffer exchange method where the virus is phys. sepd. from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titer when incubated with VTM. We demonstrate that VTM formulations, Primestore Mol. Transport Medium (MTM) and COPAN eNAT completely inactivate high-titer SARS-CoV-2 virus (>1 × 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC mol. testing using rapid turnaround GeneXpert platforms or equiv.
  27. 27
    Welch, S. R.; Davies, K. A.; Buczkowski, H.; Hettiarachchi, N.; Green, N.; Arnold, U.; Jones, M.; Hannah, M. J.; Evans, R.; Burton, C. Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives. J. Clin. Microbiol. 20220, e01713– e01720,  DOI: 10.1128/JCM.01713-20
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    Patterson, E.; Prince, T.; Anderson, E. R.; Casas-Sanchez, A.; Smith, S. L.; Cansado-Utrilla, C.; Solomon, T.; Griffith, M.; Acosta-Serrano, A.; Turtle, L. Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays. J. Infect. Dis. 2020, 222, 1462– 1467,  DOI: 10.1093/infdis/jiaa507
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    Methods of inactivation of SARS-CoV-2 for downstream biological assays
    Patterson, Edward I.; Prince, Tessa; Anderson, Enyia R.; Casas-Sanchez, Aitor; Smith, Shirley L.; Cansado-Utrilla, Cintia; Solomon, Tom; Griffiths, Michael J.; Acosta-Serrano, Alvaro; Turtle, Lance; Hughes, Grant L.
    Journal of Infectious Diseases (2020), 222 (9), 1462-1467CODEN: JIDIAQ; ISSN:1537-6613. (Oxford University Press)
    The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment labs.; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment labs., we have tested methods commonly used to inactivate virus and prep. the sample for addnl. expts. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for inhouse inactivation of SARS-CoV-2 in other labs., ensuring the safe use of samples in lower-containment levels.
  29. 29
    Darnell, M. E.; Subbarao, K.; Feinstone, S. M.; Taylor, D. R. Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV. J. Virol. Methods 2004, 121, 85– 91,  DOI: 10.1016/j.jviromet.2004.06.006
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    Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV
    Darnell, Miriam E. R.; Subbarao, Kanta; Feinstone, Stephen M.; Taylor, Deborah R.
    Journal of Virological Methods (2004), 121 (1), 85-91CODEN: JVMEDH; ISSN:0166-0934. (Elsevier B.V.)
    Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment labs. at biosafety level 3 (BSL3). The virus was inactivated by UV light (UV) at 254 nm, heat treatment of 65 or greater, alk. (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV contg. materials, that are rendered non-infectious, to be conducted at reduced safety levels.
  30. 30
    Rabenau, H. F.; Biesert, L.; Schmidt, T.; Bauer, G.; Cinatl, J.; Doerr, H. W. SARS-coronavirus (SARS-CoV) and the safety of a solvent/detergent (S/D) treated immunoglobulin preparation. Biologicals 2005, 33, 95– 99,  DOI: 10.1016/j.biologicals.2005.01.003
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    SARS-coronavirus (SARS-CoV) and the safety of a solvent/detergent (S/D) treated immunoglobulin preparation
    Rabenau, H. F.; Biesert, L.; Schmidt, T.; Bauer, G.; Cinatl, J.; Doerr, H. W.
    Biologicals (2005), 33 (2), 95-99CODEN: BILSEC; ISSN:1045-1056. (Elsevier B.V.)
    SARS-coronavirus (SARS-CoV) is a newly emerged, highly pathogenic agent that caused over 8000 human infections with nearly 800 deaths between Nov. 2002 and Sept. 2003. While direct person-to-person transmission via respiratory droplets accounted for most cases, other modes have not been ruled out. SARS-CoV viremia does not seem to reach high titers, however, it has to be excluded that virus transmission may occur via blood transfusion or application of therapeutic plasma products, e.g. fresh-frozen plasma or single components derived thereof. Manufg. processes of all plasma derivs. are required to comprise dedicated virus inactivation/removal steps. Treatment with a mixt. of solvent and detergent (SD) has successfully been applied to inactivate the most members of the transfusion-relevant viruses without affecting therapeutic properties of the products. The SD treatment irreversibly disrupts the lipid envelope of viruses such as HIV, HBV, HCV, HGV, and CMV. Here, the authors evaluated the manufg. process of an Ig prepn. (OCTAGAM, manufd. by Octapharma Pharmazeutika Produktionsges. m.b.H., Vienna, Austria) for its capacity to inactivate the SARS-CoV. The results demonstrate that SARS-CoV was completely inactivated below the limit of detection. This was found to occur within 1 min of SD treatment.
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    Jureka, A.; Silvas, J. A.; Basler, C. F. Propagation, Inactivation, and Safety Testing of SARS-CoV-2. Viruses 2020, 12, 622  DOI: 10.3390/v12060622
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    Propagation, inactivation, and safety testing of SARS-CoV-2
    Jureka, Alexander S.; Silvas, Jesus A.; Basler, Christopher F.
    Viruses (2020), 12 (6), 622CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
    In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcryst. cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.
  32. 32
    Engelmann, I.; Benhalima, I.; Ouafi, M.; Pagneux, Q.; Boukherroub, R.; Hober, D.; Alidjinou, E. K.; Szunerits, S., Validation of a specimen transport buffer for stabilization of SARS-CoV-2 RNA at room temperature for molecular diagnostics. Diagnostics 2021, submitted.
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    Wrapp, D.; De Vlieger, D. Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 2020, 181, 1004– 1015,  DOI: 10.1016/j.cell.2020.04.031
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    Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies
    Wrapp, Daniel; De Vlieger, Dorien; Corbett, Kizzmekia S.; Torres, Gretel M.; Wang, Nianshuang; Van Breedam, Wander; Roose, Kenny; van Schie, Loes; Hoffmann, Markus; Pohlmann, Stefan; Graham, Barney S.; Callewaert, Nico; Schepens, Bert; Saelens, Xavier; McLellan, Jason S.
    Cell (Cambridge, MA, United States) (2020), 181 (5), 1004-1015.e15CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
    Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, resp. Crystal structures of these VHHs bound to their resp. viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a mol. basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these mols. may serve as useful therapeutics during coronavirus outbreaks.
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    Ke, Z.; Oton, J.; Qu, K.; Cortese, M.; Zila, V.; McKeane, L.; Nakane, T.; Zinanov, J.; Neufeldt, C. J.; Cerikan, B. Structures and distribution of SARS-CoV-2 spike proteins on inatact virions. Nature 2020, 588, 498– 502,  DOI: 10.1038/s41586-020-2665-2
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    Structures and distributions of SARS-CoV-2 spike proteins on intact virions
    Ke, Zunlong; Oton, Joaquin; Qu, Kun; Cortese, Mirko; Zila, Vojtech; McKeane, Lesley; Nakane, Takanori; Zivanov, Jasenko; Neufeldt, Christopher J.; Cerikan, Berati; Lu, John M.; Peukes, Julia; Xiong, Xiaoli; Krausslich, Hans-Georg; Scheres, Sjors H. W.; Bartenschlager, Ralf; Briggs, John A. G.
    Nature (London, United Kingdom) (2020), 588 (7838), 498-502CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
    Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of sol., overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. We applied cryo-electron microscopy and tomog. to image intact SARS-CoV-2 virions and det. the high-resoln. structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
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    Liu, C.; Mendonca, L.; Yang, Y.; Gao, Y.; Shen, G.; Liu, J.; Ni, T.; Liu, C.; Tang, X.; Wei, J. The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and Cryo-ET. Structure 2020, 28, 1218.e4– 1224.e4,  DOI: 10.1016/j.str.2020.10.001
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    Gao, Q.; Bao, L.; Mao, H.; Wang, L.; Xu, K.; Yang, M.; Li, Y.; Zhu, L.; Wang, N.; Lv, Z. Development of an inactivated vaccine candidate for SARS-CoV-2. Science 2020, 369, 77– 81,  DOI: 10.1126/science.abc1932
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    Development of an inactivated vaccine candidate for SARS-CoV-2
    Gao, Qiang; Bao, Linlin; Mao, Haiyan; Wang, Lin; Xu, Kangwei; Yang, Minnan; Li, Yajing; Zhu, Ling; Wang, Nan; Lv, Zhe; Gao, Hong; Ge, Xiaoqin; Kan, Biao; Hu, Yaling; Liu, Jiangning; Cai, Fang; Jiang, Deyu; Yin, Yanhui; Qin, Chengfeng; Li, Jing; Gong, Xuejie; Lou, Xiuyu; Shi, Wen; Wu, Dongdong; Zhang, Hengming; Zhu, Lang; Deng, Wei; Li, Yurong; Lu, Jinxing; Li, Changgui; Wang, Xiangxi; Yin, Weidong; Zhang, Yanjun; Qin, Chuan
    Science (Washington, DC, United States) (2020), 369 (6499), 77-81CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
    The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale prodn. of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6μg per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, resp., without observable antibody-dependent enhancement of infection. These data support the clin. development and testing of PiCoVacc for use in humans.
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    Gao, Q.; Bao, L.; Mao, H.; Wang, L.; Xu, K.; Yang, M.; Li, Y.; Zhu, L.; Wang, N.; Lv, Z. Development of an inactivated vaccine candidate for SARS-CoV-2. Science 2020, 369, eabc1932  DOI: 10.1126/science.abc1932
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    Dewar, R.; Baunoch, D.; Wojno, K.; Parkash, V.; Khosravi-Far, R. Viral transportation in covid-19 pandemic: Inactivated virus transportation should be implemented for safe transportation and handling at diagnostics laboratories. Arch. Pathol. Lab. Med. 2020, 16, 32298137  DOI: 10.5858/arpa.2020-0175-L
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    BACKGROUND: The spatial and temporal dynamics of SARS-CoV-2 have been described in case series and retrospective studies. In this study, we provide a coherent overview of the duration of viral detection and viral RNA load in COVID-19 patients, stratified by specimen type, clinical severity, and age. METHOD: We systematically searched PubMed/MEDLINE and Cochrane review database for studies published between 1.11.2019 and 23.04.2020. We pooled the data of selected studies (22/7226 (650 patients) for meta-analysis) to estimate duration of viral detection and visualized viral load over time. FINDINGS: Our analysis showed consistent viral detection from specimen from the upper respiratory tract (URT), the lower respiratory tract (LRT), and faeces, irrespective of the clinical severity of COVID-19. Our analysis suggests that SARS-CoV-2 persists for a longer duration in the LRT compared to the URT in adult patients (5•7 days in mild; 5•9 days in moderate-severe patients). The differences in the duration of viral detection between mild and moderate-severe patients is limited in the LRT, but an indication of longer duration of viral detection for moderate-severe patients was observed in feces (15 days in mild vs. 21 days in moderate-severe patients) and the URT (12 days in mild vs. 16 days in moderate-severe patients). Further, viral load was demonstrated to peak in earlier stages of infection in the URT compared to LRT. INTERPRETATION: This review may aid mathematical modelling and help in defining appropriate endpoints for clinical trails with antivirals in COVID-19. FUNDING: The project has received funding support from Innovation Fund Denmark.
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    Eurosurveillance (2020), 25 (32), 2001483CODEN: EUROGD; ISSN:1560-7917. (European Centre for Disease Prevention and Control)
    Severe acute respiratory syndrome coronavirus 2 viral load in the upper respiratory tract peaks around symptom onset and infectious virus persists for 10 days in mild-to-moderate coronavirus disease (n = 324 samples analyzed). RT-PCR cycle threshold (Ct) values correlate strongly with cultivable virus. Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset; it is similar in asymptomatic and symptomatic persons. Asymptomatic persons represent a source of transmissible virus.
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    Background: Reverse-transcription polymerase chain reaction (RT-PCR) has become the primary method to diagnose viral diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RT-PCR detects RNA, not infectious virus; thus, its ability to det. duration of infectivity of patients is limited. Infectivity is a crit. determinant in informing public health guidelines/interventions. Our goal was to det. the relationship between E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples, symptom onset to test (STT), and infectivity in cell culture. Methods: In this retrospective cross-sectional study, we took SARS-CoV-2 RT-PCR-confirmed pos. samples and detd. their ability to infect Vero cell lines. Results: Ninety RT-PCR SARS-CoV-2-pos. samples were incubated on Vero cells. Twenty-six samples (28.9%) demonstrated viral growth. Median tissue culture infectious dose/mL was 1780 (interquartile range, 282-8511). There was no growth in samples with a Ct > 24 or STT > 8 days. Multivariate logistic regression using pos. viral culture as a binary predictor variable, STT, and Ct demonstrated an odds ratio (OR) for pos. viral culture of 0.64 (95% confidence interval [CI],.49-.84; P <.001) for every 1-unit increase in Ct. Area under the receiver operating characteristic curve for Ct vs pos. culture was OR, 0.91 (95% CI,.85-.97; P <.001), with 97% specificity obtained at a Ct of > 24. Conclusions: SARS-CoV-2 Vero cell infectivity was only obsd. for RT-PCR Ct < 24 and STT < 8 days. Infectivity of patients with Ct > 24 and duration of symptoms > 8 days may be low. This information can inform public health policy and guide clin., infection control, and occupational health decisions. Further studies of larger size are needed.
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    Singanayagam, A.; Patel, M.; Charlett, A.; Lopez Bernal, J.; Saliba, V.; Ellis, J.; Ladhani, S.; Zambon, M.; Gopal, R. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Eurosurveillance 2020, 25, 2001483  DOI: 10.2807/1560-7917.ES.2020.25.32.2001483
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    Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020
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    Eurosurveillance (2020), 25 (32), 2001483CODEN: EUROGD; ISSN:1560-7917. (European Centre for Disease Prevention and Control)
    Severe acute respiratory syndrome coronavirus 2 viral load in the upper respiratory tract peaks around symptom onset and infectious virus persists for 10 days in mild-to-moderate coronavirus disease (n = 324 samples analyzed). RT-PCR cycle threshold (Ct) values correlate strongly with cultivable virus. Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset; it is similar in asymptomatic and symptomatic persons. Asymptomatic persons represent a source of transmissible virus.
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    Nature (2020), 581 (7809), 465-469 ISSN:.
    Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 2019(1,2). Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses(3). This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung(2,4); the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 2003(5). However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission(6-8). There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 10(8) RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
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    Use of exhaled breath condensate (EBC) in the diagnosis of SARS-COV-2 (COVID-19)
    Ryan Daniel J; Toomey Sinead; Hennessy Bryan T; Ryan Daniel J; Casey Michelle; Branagan Peter; Costello Richard W; Hurley Killian; Gunaratnam Cedric; McElvaney Noel G; OBrien Michael Emmet; Sulaiman Imran; Morgan Ross K; Madden Stephen F; Breathnach Oscar S; Morris Patrick G; Grogan Liam; Hennessy Bryan T; De Barra Eoghan
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    False negatives from nasopharyngeal swabs (NPS) using reverse transcriptase PCR (RT-PCR) in SARS-CoV-2 are high. Exhaled breath condensate (EBC) contains lower respiratory droplets that may improve detection. We performed EBC RT-PCR for SARS-CoV-2 genes (E, S, N, ORF1ab) on NPS-positive (n=16) and NPS-negative/clinically positive COVID-19 patients (n=15) using two commercial assays. EBC detected SARS-CoV-2 in 93.5% (29/31) using the four genes. Pre-SARS-CoV-2 era controls (n=14) were negative. EBC was positive in NPS negative/clinically positive patients in 66.6% (10/15) using the identical E and S (E/S) gene assay used for NPS, 73.3% (11/15) using the N/ORF1ab assay and 14/15 (93.3%) combined.

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  • Abstract

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    Figure 1

    Figure 1. (a) Genome of SARS-CoV-2 with the most common RT-PCR targets highlighted. (b) Essential steps of the RT-PCR diagnostic workflow including sample collection, storage in a transport medium, lysis and RNA extraction, reverse transcription, amplification, and detection. (c) Example of a real-time RT-PCR amplification curve for SARS-CoV-2 and postrun analysis to interpret results.

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    Figure 2

    Figure 2. Quantification of SARS-CoV-2 following exposure to different inactivation conditions: (a) surface plasmon resonance (SPR) binding curves recorded on a T200 Biacore for the receptor-binding domain (RBD, 200 nM) and RBD for different time intervals. The interface was modified with SARS-CoV-2-specific VHH-72 nanobodies. (33) (b–d) RNA stability after heat inactivation: SARS-CoV-2 RNA-positive nasopharyngeal swab specimens were pooled and divided into equal volumes and heated for 30 min at 60 °C. The aliquots were either kept at room temperature (RT), 4 °C, or −80 °C for the indicated times before RNA extraction and RT-PCR was performed in triplicate. (32) As a control, untreated specimens were included. Ct values are indicated for the SARS-CoV-2 targets IP2 (b), IP4 (c), and the cellular control G6PDH (d) (Figure 2−2d report mean values of three samples).

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    Figure 3

    Figure 3. Clinical significance of SARS-CoV-2 RT-PCR results. (a) Timeline of SARS-CoV-2 infectivity taking into account our own findings and those of others. (39,40) (b) Ct values (target IP2) as a function of time after the symptom onset in nasopharyngeal swab specimens of COVID-19 patients. Ct of specimens with undetectable SARS-CoV-2 RNA were set to 50. (c) Correlation of Ct values with SARS-CoV-2 infectivity. Vero E6 cells were infected with 10-fold dilutions of a SARS-CoV-2 isolate. The plates were incubated for 6 days in 5% CO2 at 37 °C and examined daily using an inverted microscope (ZEISS Primovert) to evaluate the extent of the virus-induced cytopathic effect in cell culture. The calculation of the estimated virus concentration was carried out by the Spearman and Karber method (48,49) and expressed as TCID50/mL (50% tissue culture infectious dose). TCID50/mL values were transformed to PFU mL–1 using the formula PFU mL–1 = TCID50/mL × 0.7. (50) RNA extraction and RT-PCR (target IP4 and target IP2) were performed in duplicate for each dilution. Ct of dilutions with undetectable SARS-CoV-2 RNA were set to 50.

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      3
      Early high-titer plasma therapy to prevent severe Covid-19 in older adults
      Libster, R.; Marc, G. Perez; Wappner, D.; Coviello, S.; Bianchi, A.; Braem, V.; Esteban, I.; Caballero, M. T.; Wood, C.; Berrueta, M.; Rondan, A.; Lescano, G.; Cruz, P.; Ritou, Y.; Vina, V. Fernandez; Paggi, D. Alvarez; Esperante, S.; Ferreti, A.; Ofman, G.; Ciganda, A.; Rodriguez, R.; Lantos, J.; Valentini, R.; Itcovici, N.; Hintze, A.; Oyarvide, M. L.; Etchegaray, C.; Neira, A.; Name, I.; Alfonso, J.; Castelo, R. Lopez; Caruso, G.; Rapelius, S.; Alvez, F.; Etchenique, F.; Dimase, F.; Alvarez, D.; Aranda, S. S.; Yanotti, C. Sanchez; De Luca, J.; Baglivo, S. Jares; Laudanno, S.; Nowogrodzki, F.; Larrea, R.; Silveyra, M.; Leberzstein, G.; Debonis, A.; Molinos, J.; Gonzalez, M.; Perez, E.; Kreplak, N.; Arguello, S. Pastor; Gibbons, L.; Althabe, F.; Bergel, E.; Polack, F. P.
      New England Journal of Medicine (2021), 384 (7), 610-618CODEN: NEJMAG; ISSN:1533-4406. (Massachusetts Medical Society)
      Background Therapies to interrupt the progression of early coronavirus disease 2019 (Covid-19) remain elusive. Among them, convalescent plasma administered to hospitalized patients has been unsuccessful, perhaps because antibodies should be administered earlier in the course of illness. methods We conducted a randomized, double-blind, placebo-controlled trial of convalescent plasma with high IgG titers against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in older adult patients within 72 h after the onset of mild Covid-19 symptoms. The primary end point was severe respiratory disease, defined as a respiratory rate of 30 breaths per min or more, an oxygen satn. of less than 93% while the patient was breathing ambient air, or both. The trial was stopped early at 76% of its projected sample size because cases of Covid-19 in the trial region decreased considerably and steady enrollment of trial patients became virtually impossible. results A total of 160 patients underwent randomization. In the intention-to-treat population, severe respiratory disease developed in 13 of 80 patients (16%) who received convalescent plasma and 25 of 80 patients (31%) who received placebo (relative risk, 0.52; 95% confidence interval [CI], 0.29 to 0.94; P = 0.03), with a relative risk redn. of 48%. A modified intention-to-treat anal. that excluded 6 patients who had a primary end-point event before infusion of convalescent plasma or placebo showed a larger effect size (relative risk, 0.40; 95% CI, 0.20 to 0.81). No solicited adverse events were obsd. conclusions Early administration of high-titer convalescent plasma against SARS-CoV-2 to mildly ill infected older adults reduced the progression of Covid-19.
    4. 4
      Joyner, M. J.; Carter, R. E.; Senefeld, J. W.; Klassen, S. A.; Mills, J. R.; Johnson, P. W.; Theel, E. S.; Wiggins, C. C.; Bruni, K. A.; Klompas, A. M. Convalescent Plasma Antibody Levels and the Risk of Death from Covid-19. N. Eng. J. Med. 2021,  DOI: 10.1056/NEJMoa2031893
      There is no corresponding record for this reference.
    5. 5
      Shen, M.; Zhou, Y.; Ye, J.; AL-maskri, A. A. A.; Kang, Y.; Zeng, S.; Cai, S. Recent advances and perspectives of nucleic acid detection for coronavirus. J. Pharm. Anal. 2020, 10, 97– 101,  DOI: 10.1016/j.jpha.2020.02.010
      5
      Recent advances and perspectives of nucleic acid detection for coronavirus
      Shen Minzhe; Zhou Ying; Ye Jiawei; Abdullah Al-Maskri Abdu Ahmed; Kang Yu; Zeng Su; Cai Sheng
      Journal of pharmaceutical analysis (2020), 10 (2), 97-101 ISSN:.
      The recent pneumonia outbreak caused by a novel coronavirus (SARS-CoV-2) is posing a great threat to global public health. Therefore, rapid and accurate identification of pathogenic viruses plays a vital role in selecting appropriate treatments, saving people's lives and preventing epidemics. It is important to establish a quick standard diagnostic test for the detection of the infectious disease (COVID-19) to prevent subsequent secondary spread. Polymerase chain reaction (PCR) is regarded as a gold standard test for the molecular diagnosis of viral and bacterial infections with high sensitivity and specificity. Isothermal nucleic acid amplification is considered to be a highly promising candidate method due to its fundamental advantage in quick procedure time at constant temperature without thermocycler operation. A variety of improved or new approaches also have been developed. This review summarizes the currently available detection methods for coronavirus nucleic acid. It is anticipated that this will assist researchers and clinicians in developing better techniques for timely and effective detection of coronavirus infection.
    6. 6
      Kandeel, M.; Ibrahim, A.; Fayez, M.; Al-Nazawi, M. From SARS and MERS CoVs to SARS-CoV-2: moving toward more biased codon usage in viral structural and non-structural genes. J. Med. Virol. 2020, 92, 660– 666,  DOI: 10.1002/jmv.25754
      6
      From SARS and MERS CoVs to SARS-CoV-2: Moving toward more biased codon usage in viral structural and nonstructural genes
      Kandeel, Mahmoud; Ibrahim, Abdelazim; Fayez, Mahmoud; Al-Nazawi, Mohammed
      Journal of Medical Virology (2020), 92 (6), 660-666CODEN: JMVIDB; ISSN:0146-6615. (Wiley-Blackwell)
      Background : Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an emerging disease with fatal outcomes. In this study, a fundamental knowledge gap question is to be resolved by evaluating the differences in biol. and pathogenic aspects of SARS-CoV-2 and the changes in SARS-CoV-2 in comparison with the two prior major COV epidemics, SARS and Middle East respiratory syndrome (MERS) coronaviruses. Methods : The genome compn., nucleotide anal., codon usage indexes, relative synonymous codons usage, and effective no. of codons (ENc) were analyzed in the four structural genes; Spike (S), Envelope (E), membrane (M), and Nucleocapsid (N) genes, and two of the most important nonstructural genes comprising RNA-dependent RNA polymerase and main protease (Mpro) of SARS-CoV-2, Beta-CoV from pangolins, bat SARS, MERS, and SARS CoVs. Results : SARS-CoV-2 prefers pyrimidine rich codons to purines. Most high-frequency codons were ending with A or T, while the low frequency and rare codons were ending with G or C. SARS-CoV-2 structural proteins showed 5 to 20 lower ENc values, compared with SARS, bat SARS, and MERS CoVs. This implies higher codon bias and higher gene expression efficiency of SARS-CoV-2 structural proteins. SARS-CoV-2 encoded the highest no. of over-biased and neg. biased codons. Pangolin Beta-CoV showed little differences with SARS-CoV-2 ENc values, compared with SARS, bat SARS, and MERS CoV. Conclusion : Extreme bias and lower ENc values of SARS-CoV-2, esp. in Spike, Envelope, and Mpro genes, are suggestive for higher gene expression efficiency, compared with SARS, bat SARS, and MERS CoVs.
    7. 7
      Elfiky, A. A. Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study. Life Sci. 2020, 253, 117592  DOI: 10.1016/j.lfs.2020.117592
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      Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir against SARS-CoV-2 RNA dependent RNA polymerase (RdRp): A molecular docking study
      Elfiky, Abdo A.
      Life Sciences (2020), 253 (), 117592CODEN: LIFSAK; ISSN:0024-3205. (Elsevier B.V.)
      A new human coronavirus (HCoV), which has been designated SARS-CoV-2, began spreading in Dec. 2019 in Wuhan City, China causing pneumonia called COVID-19. The spread of SARS-CoV-2 has been faster than any other coronaviruses that have succeeded in crossing the animal-human barrier. There is concern that this new virus will spread around the world as did the previous two HCoVs-Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS)-each of which caused approx. 800 deaths in the years 2002 and 2012, resp. Thus far, 11,268 deaths have been reported from the 258,842 confirmed infections in 168 countries. In this study, the RNA-dependent RNA polymerase (RdRp) of the newly emerged coronavirus is modeled, validated, and then targeted using different anti-polymerase drugs currently on the market that have been approved for use against various viruses. The results suggest the effectiveness of Ribavirin, Remdesivir, Sofosbuvir, Galidesivir, and Tenofovir as potent drugs against SARS-CoV-2 since they tightly bind to its RdRp. In addn., the results suggest guanosine deriv. (IDX-184), Setrobuvir, and YAK as top seeds for antiviral treatments with high potential to fight the SARS-CoV-2 strain specifically. The availability of FDA-approved anti-RdRp drugs can help treat patients and reduce the danger of the mysterious new viral infection COVID-19. The drugs mentioned above can tightly bind to the RdRp of the SARS-CoV-2 strain and thus may be used to treat the disease. No toxicity measurements are required for these drugs since they were previously tested prior to their approval by the FDA.
    8. 8
      Phan, T. Genetic diversity and evolution of SARS-CoV-2. Infect., Genet. Evol. 2020, 81, 104260  DOI: 10.1016/j.meegid.2020.104260
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      Genetic diversity and evolution of SARS-CoV-2
      Phan, Tung
      Infection, Genetics and Evolution (2020), 81 (), 104260CODEN: IGENCN; ISSN:1567-1348. (Elsevier B.V.)
      COVID-19 is a viral respiratory illness caused by a new coronavirus called SARS-CoV-2. The World Health Organization declared the SARS-CoV-2 outbreak a global public health emergency. We performed genetic analyses of eighty-six complete or near-complete genomes of SARS-CoV-2 and revealed many mutations and deletions on coding and non-coding regions. These observations provided evidence of the genetic diversity and rapid evolution of this novel coronavirus.
    9. 9
      Pachetti, M.; Marini, B.; Benedetti, F.; Giudici, F.; Mauro, E.; Storici, P.; Masciovecchio, C.; Angeletti, S.; Ciccozzi, M.; Gallo, R. C. Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant. J. Transl. Med. 2020, 18, 179,  DOI: 10.1186/s12967-020-02344-6
      9
      Emerging SARS-CoV-2 mutation hot spots include a novel RNA-dependent-RNA polymerase variant
      Pachetti, Maria; Marini, Bruna; Benedetti, Francesca; Giudici, Fabiola; Mauro, Elisabetta; Storici, Paola; Masciovecchio, Claudio; Angeletti, Silvia; Ciccozzi, Massimo; Gallo, Robert C.; Zella, Davide; Ippodrino, Rudy
      Journal of Translational Medicine (2020), 18 (1), 179CODEN: JTMOBV; ISSN:1479-5876. (BioMed Central Ltd.)
      Abstr.: Background: SARS-CoV-2 is a RNA coronavirus responsible for the pandemic of the Severe Acute Respiratory Syndrome (COVID-19). RNA viruses are characterized by a high mutation rate, up to a million times higher than that of their hosts. Virus mutagenic capability depends upon several factors, including the fidelity of viral enzymes that replicate nucleic acids, as SARS-CoV-2 RNA dependent RNA polymerase (RdRp). Mutation rate drives viral evolution and genome variability, thereby enabling viruses to escape host immunity and to develop drug resistance. Methods: We analyzed 220 genomic sequences from the GISAID database derived from patients infected by SARS-CoV-2 worldwide from Dec. 2019 to mid-March 2020. SARS-CoV-2 ref. genome was obtained from the GenBank database. Genomes alignment was performed using Clustal Omega. Mann-Whitney and Fisher-Exact tests were used to assess statistical significance. Results: We characterized 8 novel recurrent mutations of SARS-CoV-2, located at positions 1397, 2891, 14408, 17746, 17857, 18060, 23403 and 28881. Mutations in 2891, 3036, 14408, 23403 and 28881 positions are predominantly obsd. in Europe, whereas those located at positions 17746, 17857 and 18060 are exclusively present in North America. We noticed for the first time a silent mutation in RdRp gene in England (UK) on Feb. 9th, 2020 while a different mutation in RdRp changing its amino acid compn. emerged on Feb. 20th, 2020 in Italy (Lombardy). Viruses with RdRp mutation have a median of 3 point mutations [range: 2-5], otherwise they have a median of 1 mutation [range: 0-3] (p value < 0.001). Conclusions: These findings suggest that the virus is evolving and European, North American and Asian strains might coexist, each of them characterized by a different mutation pattern. The contribution of the mutated RdRp to this phenomenon needs to be investigated. To date, several drugs targeting RdRp enzymes are being employed for SARS-CoV-2 infection treatment. Some of them have a predicted binding moiety in a SARS-CoV-2 RdRp hydrophobic cleft, which is adjacent to the 14408 mutation we identified. Consequently, it is important to study and characterize SARS-CoV-2 RdRp mutation in order to assess possible drug-resistance viral phenotypes. It is also important to recognize whether the presence of some mutations might correlate with different SARS-CoV-2 mortality rates.
    10. 10
      Yu, C. Y.; Chan, K. G.; Yean, C. Y.; Ang, G. Y. Nucleic Acid-Based Diagnostic Tests for the Detection SARS-CoV-2: An Update. Diagnostics 2021, 11, 53  DOI: 10.3390/diagnostics11010053
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      Nucleic acid-based diagnostic tests for the detection SARS-CoV-2: an update
      Yu, Choo Yee; Chan, Kok Gan; Yean, Chan Yean; Ang, Geik Yong
      Diagnostics (2021), 11 (1), 53CODEN: DIAGC9; ISSN:2075-4418. (MDPI AG)
      The coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) began as a cluster of pneumonia cases in Wuhan, China before spreading to over 200 countries and territories on six continents in less than six months. Despite rigorous global containment and quarantine efforts to limit the transmission of the virus, COVID-19 cases and deaths have continued to increase, leaving devastating impacts on the lives of many with farreaching effects on the global society, economy and healthcare system. With over 43 million cases and 1.1 million deaths recorded worldwide, accurate and rapid diagnosis continues to be a cornerstone of pandemic control. In this review, we aim to present an objective overview of the latest nucleic acid-based diagnostic tests for the detection of SARS-CoV-2 that have been authorized by the Food and Drug Administration (FDA) under emergency use authorization (EUA) as of 31 Oct. 2020. We systematically summarize and compare the principles, technologies, protocols and performance characteristics of amplification- and sequencing-based tests that have become alternatives to the CDC 2019-nCoV Real-Time RT-PCR Diagnostic Panel. We highlight the notable features of the tests including authorized settings, along with the advantages and disadvantages of the tests. We conclude with a brief discussion on the current challenges and future perspectives of COVID-19 diagnostics.
    11. 11
      Udugama, B.; Kadhiresan, P.; Kozlowski, H. N.; Malekjahani, A.; Osborne, M.; Li, V. Y. C.; Chen, H.; Mubareka, S.; Gubbay, J. B.; Chan, W. C. W. Diagnosing COVID-19: The Disease and Tools for Detection. ACS Nano 2020, 14, 3822– 3835,  DOI: 10.1021/acsnano.0c02624
      11
      Diagnosing COVID-19: The Disease and Tools for Detection
      Udugama, Buddhisha; Kadhiresan, Pranav; Kozlowski, Hannah N.; Malekjahani, Ayden; Osborne, Matthew; Li, Vanessa Y. C.; Chen, Hongmin; Mubareka, Samira; Gubbay, Jonathan B.; Chan, Warren C. W.
      ACS Nano (2020), 14 (4), 3822-3835CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      A review. COVID-19 has spread globally since its discovery in Hubei province, China in Dec. 2019. A combination of computed tomog. imaging, whole genome sequencing, and electron microscopy were initially used to screen and identify SARS-CoV-2, the viral etiol. of COVID-19. The aim of this review article is to inform the audience of diagnostic and surveillance technologies for SARS-CoV-2 and their performance characteristics. We describe point-of-care diagnostics that are on the horizon and encourage academics to advance their technologies beyond conception. Developing plug-and-play diagnostics to manage the SARS-CoV-2 outbreak would be useful in preventing future epidemics.
    12. 12
      Yuan, X.; Yang, C.; He, Q.; Chen, J.; Yu, D.; Li, J.; Zhai, S.; Qin, Z.; Du, K.; Chu, Z. Current and Perspective Diagnostic Techniques for COVId-19. ACS Infect. Dis. 2020, 6, 1998– 2016,  DOI: 10.1021/acsinfecdis.0c00365
      12
      Current and Perspective Diagnostic Techniques for COVID-19
      Yuan, Xi; Yang, Chengming; He, Qian; Chen, Junhu; Yu, Dongmei; Li, Jie; Zhai, Shiyao; Qin, Zhifeng; Du, Ke; Chu, Zhenhai; Qin, Peiwu
      ACS Infectious Diseases (2020), 6 (8), 1998-2016CODEN: AIDCBC; ISSN:2373-8227. (American Chemical Society)
      A review. Since late Dec. 2019, the coronavirus pandemic (COVID-19; previously known as 2019-nCoV) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been surging rapidly around the world. With more than 1,700,000 confirmed cases, the world faces an unprecedented economic, social, and health impact. The early, rapid, sensitive, and accurate diagnosis of viral infection provides rapid responses for public health surveillance, prevention, and control of contagious diffusion. More than 30% of the confirmed cases are asymptomatic, and the high false-neg. rate (FNR) of a single assay requires the development of novel diagnostic techniques, combinative approaches, sampling from different locations, and consecutive detection. The recurrence of discharged patients indicates the need for long-term monitoring and tracking. Diagnostic and therapeutic methods are evolving with a deeper understanding of virus pathol. and the potential for relapse. In this Review, a comprehensive summary and comparison of different SARS-CoV-2 diagnostic methods are provided for researchers and clinicians to develop appropriate strategies for the timely and effective detection of SARS-CoV-2. The survey of current biosensors and diagnostic devices for viral nucleic acids, proteins, and particles and chest tomog. will provide insight into the development of novel perspective techniques for the diagnosis of COVID-19.
    13. 13
      Uhteg, K.; Jarrett, J.; Richards, M.; Howard, C.; Morehead, E.; Geahr, M.; Gluck, L.; Hanlon, A.; Ellis, B.; Kaur, H. Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays. J. Clin. Virol. 2020, 127, 104384  DOI: 10.1016/j.jcv.2020.104384
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      Comparing the analytical performance of three SARS-CoV-2 molecular diagnostic assays
      Uhteg, Katharine; Jarrett, Junko; Richards, Mahmia; Howard, Craig; Morehead, Elizabeth; Geahr, Melissa; Gluck, Linda; Hanlon, Ann; Ellis, Brandon; Kaur, Harsimar; Simner, Patricia; Carroll, Karen C.; Mostafa, Heba H.
      Journal of Clinical Virology (2020), 127 (), 104384CODEN: JCVIFB; ISSN:1386-6532. (Elsevier B.V.)
      In Dec. 2019, a novel coronavirus (SARS-CoV-2) was first isolated from Wuhan city, China and within three months, the global community was challenged with a devastating pandemic. The rapid spread of the virus challenged diagnostic labs. to rapidly develop mol. diagnostic methods. As SARS CoV-2 assays became available for testing on existing mol. platforms, labs. devoted unprecedented energy and resources into evaluating the anal. performance of the new tests and in some cases developed their own diagnostic assays under FDA-EUA guidance. This study compares the validation of three different mol. assays at the Johns Hopkins Mol. Virol. lab.: the RealStar SARS-CoV-2 RT-PCR, ePlex SARS-CoV-2, and the CDC COVID-19 RT-PCR tests. Overall, our studies indicate a comparable anal. performance of the three assays for the detection of SARS-CoV-2.
    14. 14
      Vogels, C. B. F.; Brito, A. F.; Wyllie, A. L.; Fauver, J. R.; Ott, I. M.; Kalinich, C. C.; Petrone, M. E.; Casanovas-Massana, A.; Muenker, M. C.; Moore, A. J. Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT–qPCR primer–probe sets. Nat. Microbiol. 2020, 5, 1299– 1305,  DOI: 10.1038/s41564-020-0761-6
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      Analytical sensitivity and efficiency comparisons of SARS-CoV-2 RT-qPCR primer-probe sets
      Vogels, Chantal B. F.; Brito, Anderson F.; Wyllie, Anne L.; Fauver, Joseph R.; Ott, Isabel M.; Kalinich, Chaney C.; Petrone, Mary E.; Casanovas-Massana, Arnau; Catherine Muenker, M.; Moore, Adam J.; Klein, Jonathan; Lu, Peiwen; Lu-Culligan, Alice; Jiang, Xiaodong; Kim, Daniel J.; Kudo, Eriko; Mao, Tianyang; Moriyama, Miyu; Oh, Ji Eun; Park, Annsea; Silva, Julio; Song, Eric; Takahashi, Takehiro; Taura, Manabu; Tokuyama, Maria; Venkataraman, Arvind; Weizman, Orr-El; Wong, Patrick; Yang, Yexin; Cheemarla, Nagarjuna R.; White, Elizabeth B.; Lapidus, Sarah; Earnest, Rebecca; Geng, Bertie; Vijayakumar, Pavithra; Odio, Camila; Fournier, John; Bermejo, Santos; Farhadian, Shelli; Dela Cruz, Charles S.; Iwasaki, Akiko; Ko, Albert I.; Landry, Marie L.; Foxman, Ellen F.; Grubaugh, Nathan D.
      Nature Microbiology (2020), 5 (10), 1299-1305CODEN: NMAICH; ISSN:2058-5276. (Nature Research)
      The recent spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exemplifies the crit. need for accurate and rapid diagnostic assays to prompt clin. and public health interventions. Currently, several quant. reverse transcription-PCR (RT-qPCR) assays are being used by clin., research and public health labs. However, it is currently unclear whether results from different tests are comparable. Our goal was to make independent evaluations of primer-probe sets used in four common SARS-CoV-2 diagnostic assays. From our comparisons of RT-qPCR anal. efficiency and sensitivity, we show that all primer-probe sets can be used to detect SARS-CoV-2 at 500 viral RNA copies per reaction. The exception for this is the RdRp-SARSr (Charite´) confirmatory primer-probe set which has low sensitivity, probably due to a mismatch to circulating SARS-CoV-2 in the reverse primer. We did not find evidence for background amplification with pre-COVID-19 samples or recent SARS-CoV-2 evolution decreasing sensitivity. Our recommendation for SARS-CoV-2 diagnostic testing is to select an assay with high sensitivity and that is regionally used, to ease comparability between outcomes.
    15. 15
      Nalla, A. K.; Casto, A. M.; Huang, M.-L.; Perchetti, G. A.; Sampoleo, R.; Shrestha, L.; Wei, Y.; Zhu, H.; Jerome, K. R.; Greninger, A. L. Comparative Performance of SARS-CoV-2 Detection Assays Using Seven Different Primer-Probe Sets and One Assay Kit. J. Clin. Microbiol. 2022, 58, e00557-20  DOI: 10.1128/JCM.00557-20
      There is no corresponding record for this reference.
    16. 16
      Avisdu 25 septembre 2020 de la Société Française de Microbiologie (SFM) relatifà l’interprétation de la valeur de Ct (estimation de la charge virale)obtenue en cas de RT-PCR SARS-CoV-2 positive sur les prélèvements cliniquesréalisés à des fins diagnostiques ou de dépistage. https://www.sfm-microbiologie.org/covid-19-fiches-et-documents-sfm/ (Version 3 du 7 octobre, 2020).
      There is no corresponding record for this reference.
    17. 17
      Institut Pasteur, Paris. Protocol: Real-time RT-PCR Assays for the Detection of SARS CoV-2. [Internet] [cited 2020 Aug 28]. https://www.who.int/docs/default-source/coronaviruse/real-time-rt-pcr-assays-for-the-detection-of-sars-cov-2-institut-pasteur-paris.pdf?sfvrsn=3662fcb6_2.
      There is no corresponding record for this reference.
    18. 18
      Arrêté du 18 décembre 2020 relatif à la classification du coronavirus SARS-CoV-2 dans la liste des agents biologiques pathogènes. https://www.legifrance.gouv.fr/jorf/article_jo/JORFARTI000042840497.
      There is no corresponding record for this reference.
    19. 19
      Szunerits, S.; Pagneux, Q.; Swaidan, A.; Mishyn, V.; Roussel, A.; Cambillau, C.; Devos, D.; Engelmann, I.; Alidjinou, E. K.; Happy, H. The role of the surface ligand on the performance of electrochemical SARS-CoV-2 antigen biosensors. Anal. Bioanal. Chem. 2021. DOI: 10.1007/s00216-020-03137-y .
      There is no corresponding record for this reference.
    20. 20
      Seo, G.; Lee, G.; Kim, M. J.; Baek, S.-H.; Choi, M.; Ku, K. B.; Lee, C.-S.; Jun, S.; Park, D.; Kim, H. G. Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor. ACS Nano 2020, 14, 5135– 5142,  DOI: 10.1021/acsnano.0c02823
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      Rapid Detection of COVID-19 Causative Virus (SARS-CoV-2) in Human Nasopharyngeal Swab Specimens Using Field-Effect Transistor-Based Biosensor
      Seo, Giwan; Lee, Geonhee; Kim, Mi Jeong; Baek, Seung-Hwa; Choi, Minsuk; Ku, Keun Bon; Lee, Chang-Seop; Jun, Sangmi; Park, Daeui; Kim, Hong Gi; Kim, Seong-Jun; Lee, Jeong-O.; Kim, Bum Tae; Park, Edmond Changkyun; Kim, Seung Il
      ACS Nano (2020), 14 (4), 5135-5142CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      Coronavirus disease 2019 (COVID-19) is a newly emerging human infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2, previously called 2019-nCoV). Based on the rapid increase in the rate of human infection, the World Health Organization (WHO) has classified the COVID-19 outbreak as a pandemic. Because no specific drugs or vaccines for COVID-19 are yet available, early diagnosis and management are crucial for contg. the outbreak. Here, we report a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clin. samples. The sensor was produced by coating graphene sheets of the FET with a specific antibody against SARS-CoV-2 spike protein. The performance of the sensor was detd. using antigen protein, cultured virus, and nasopharyngeal swab specimens from COVID-19 patients. Our FET device could detect the SARS-CoV-2 spike protein at concns. of 1 fg/mL in phosphate-buffered saline and 100 fg/mL clin. transport medium. In addn., the FET sensor successfully detected SARS-CoV-2 in culture medium (limit of detection [LOD]: 1.6 x 101 pfu/mL) and clin. samples (LOD: 2.42 x 102 copies/mL). Thus, we have successfully fabricated a promising FET biosensor for SARS-CoV-2; our device is a highly sensitive immunol. diagnostic method for COVID-19 that requires no sample pretreatment or labeling.
    21. 21
      Kilic, T.; Weissleder, R.; Lee, H. Molecular and Immunological Diagnostic Tests of COVID-19: Current Status and Challenges. iScience 2020, 23, 101406  DOI: 10.1016/j.isci.2020.101406
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      Molecular and Immunological Diagnostic Tests of COVID-19: Current Status and Challenges
      Kilic, Tugba; Weissleder, Ralph; Lee, Hakho
      iScience (2020), 23 (8), 101406CODEN: ISCICE; ISSN:2589-0042. (Elsevier B.V.)
      A review. Rapid spread of coronavirus disease 2019 (COVID-19) is ravaging the globe. Since its first report in Dec. 2019, COVID-19 cases have exploded to over 14 million as of July 2020, claiming more than 600,000 lives. Implementing fast and widespread diagnostic tests is paramount to contain COVID-19, given the current lack of an effective therapeutic or vaccine. This focuses on a broad description of currently available diagnostic tests to detect either the virus (SARS-CoV-2) or virus-induced immune responses. We specifically explain the working mechanisms of these tests and compare their anal. performance. These analyses will assist in selecting most effective tests for a given application, for example, epidemiol. or global pandemic research, population screening, hospital-based testing, home-based and point-of-care testing, and therapeutic trials. Finally, we lay out the shortcomings of certain tests and future needs.
    22. 22
      Jayamohan, H.; Lambert, C. J.; Sant, H. J.; Jafek, A.; Patel, D.; Feng, H.; Beeman, M.; Mahmood, T.; Nze, U.; Gale, B. K. SARS-CoV-2 pandemic: a review of molecular diagnostic tools including sample collection and commercial response with associated advantages and limitations. Anal. Bioanal. Chem. 2020, 1– 23,  DOI: 10.1007/s00216-020-02958-1
      There is no corresponding record for this reference.
    23. 23
      Scallan, M. F.; Dempsey, C.; MacSharry, J.; O’Callaghan, I.; O’Connor, P. M.; Horgan, C. P.; Durack, E.; Cotter, P. D.; Hudson, S.; Moynihan, H. A. Validation of a lysis buffer containing 4 M guanidinium thiocyanate (GITC)/Triton X-100 for extraction of SARS-CoV-2 RNA for COVID-19 testing: comparison of formulated lysis buffers containing 4 to 6 M GITC, Roche external lysis buffer and Qiagen RTL lysis buffer. bioRxiv 2020, 026435  DOI: 10.1101/2020.04.05.026435
      There is no corresponding record for this reference.
    24. 24
      Engelmann, I.; Benhalima, I.; Ouafi, M.; Pagneux, Q.; Boukherroub, R.; Hober, D.; Alidjinou, E. K.; Szunerits, S. Validation of a specimen transport buffer for stabilization of SARS-CoV-2 RNA at room temperature for molecular diagnostics,2020, unpublished results.
      There is no corresponding record for this reference.
    25. 25
      Auerswald, H.; Yann, S.; Dul, S.; In, S.; Dussart, P.; Martin, N. J.; Karlsson, E. A.; Garcia-Rivera, J. A. Assessment of Inactivation Procedures for SARS-CoV-2. BioRxiv 2020, 20, 533  DOI: 10.1101/2020.05.28.120444
      There is no corresponding record for this reference.
    26. 26
      van Bockel, D.; Munier, C. M. L.; Turville, S.; Badman, S. G.; Walker, G.; Stella, A. O.; Anupriya Aggarwal, A.; Yeang, M.; Condylios, A.; Kelleher, A. D. Evaluation of Commercially Available Viral TransportMedium (VTM) for SARS-CoV-2 Inactivation and Usein Point-of-Care (POC) Testing. Viruses 2020, 12, 1208  DOI: 10.3390/v12111208
      26
      Evaluation of commercially available viral transport medium (VTM) for SARS-CoV-2 inactivation and use in point-of-care (POC) testing
      van Bockel, David; Munier, C. Mee Ling; Turville, Stuart; Badman, Steven G.; Walker, Gregory; Stella, Alberto Ospina; Aggarwal, Anupriya; Yeang, Malinna; Condylios, Anna; Kelleher, Anthony D.; Applegate, Tanya L.; Vallely, Andrew; Whiley, David; Rawlinson, William; Cunningham, Phillip; Kaldor, John; Guy, Rebecca
      Viruses (2020), 12 (11), 1208CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      Crit. to facilitating SARS-CoV-2 point-of-care (POC) testing is assurance that viruses present in specimens are inactivated onsite prior to processing. Here, we conducted expts. to det. the virucidal activity of com. available Viral Transport Mediums (VTMs) to inactivate SARS-CoV-2. Independent testing methods for viral inactivation testing were applied, including a previously described World Health Organization (WHO) protocol, in addn. to a buffer exchange method where the virus is phys. sepd. from the VTM post exposure. The latter method enables sensitive detection of viral viability at higher viral titer when incubated with VTM. We demonstrate that VTM formulations, Primestore Mol. Transport Medium (MTM) and COPAN eNAT completely inactivate high-titer SARS-CoV-2 virus (>1 × 107 copies/mL) and are compatible with POC processing. Furthermore, full viral inactivation was rapidly achieved in as little as 2 min of VTM exposure. We conclude that adding certain VTM formulations as a first step post specimen collection will render SARS-CoV-2 non-infectious for transport, or for further in-field POC mol. testing using rapid turnaround GeneXpert platforms or equiv.
    27. 27
      Welch, S. R.; Davies, K. A.; Buczkowski, H.; Hettiarachchi, N.; Green, N.; Arnold, U.; Jones, M.; Hannah, M. J.; Evans, R.; Burton, C. Analysis of Inactivation of SARS-CoV-2 by Specimen Transport Media, Nucleic Acid Extraction Reagents, Detergents, and Fixatives. J. Clin. Microbiol. 20220, e01713– e01720,  DOI: 10.1128/JCM.01713-20
      There is no corresponding record for this reference.
    28. 28
      Patterson, E.; Prince, T.; Anderson, E. R.; Casas-Sanchez, A.; Smith, S. L.; Cansado-Utrilla, C.; Solomon, T.; Griffith, M.; Acosta-Serrano, A.; Turtle, L. Methods of Inactivation of SARS-CoV-2 for Downstream Biological Assays. J. Infect. Dis. 2020, 222, 1462– 1467,  DOI: 10.1093/infdis/jiaa507
      28
      Methods of inactivation of SARS-CoV-2 for downstream biological assays
      Patterson, Edward I.; Prince, Tessa; Anderson, Enyia R.; Casas-Sanchez, Aitor; Smith, Shirley L.; Cansado-Utrilla, Cintia; Solomon, Tom; Griffiths, Michael J.; Acosta-Serrano, Alvaro; Turtle, Lance; Hughes, Grant L.
      Journal of Infectious Diseases (2020), 222 (9), 1462-1467CODEN: JIDIAQ; ISSN:1537-6613. (Oxford University Press)
      The scientific community has responded to the coronavirus disease 2019 (COVID-19) pandemic by rapidly undertaking research to find effective strategies to reduce the burden of this disease. Encouragingly, researchers from a diverse array of fields are collectively working towards this goal. Research with infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is undertaken in high-containment labs.; however, it is often desirable to work with samples at lower-containment levels. To facilitate the transfer of infectious samples from high-containment labs., we have tested methods commonly used to inactivate virus and prep. the sample for addnl. expts. Incubation at 80°C, a range of detergents, Trizol reagents, and UV energies were successful at inactivating a high titer of SARS-CoV-2. Methanol and paraformaldehyde incubation of infected cells also inactivated the virus. These protocols can provide a framework for inhouse inactivation of SARS-CoV-2 in other labs., ensuring the safe use of samples in lower-containment levels.
    29. 29
      Darnell, M. E.; Subbarao, K.; Feinstone, S. M.; Taylor, D. R. Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV. J. Virol. Methods 2004, 121, 85– 91,  DOI: 10.1016/j.jviromet.2004.06.006
      29
      Inactivation of the coronavirus that induces severe acute respiratory syndrome, SARS-CoV
      Darnell, Miriam E. R.; Subbarao, Kanta; Feinstone, Stephen M.; Taylor, Deborah R.
      Journal of Virological Methods (2004), 121 (1), 85-91CODEN: JVMEDH; ISSN:0166-0934. (Elsevier B.V.)
      Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by a novel coronavirus termed SARS-CoV. Due to the severity of this disease, the World Health Organization (WHO) recommends that manipulation of active viral cultures of SARS-CoV be performed in containment labs. at biosafety level 3 (BSL3). The virus was inactivated by UV light (UV) at 254 nm, heat treatment of 65 or greater, alk. (pH > 12) or acidic (pH < 3) conditions, formalin and glutaraldehyde treatments. We describe the kinetics of these efficient viral inactivation methods, which will allow research with SARS-CoV contg. materials, that are rendered non-infectious, to be conducted at reduced safety levels.
    30. 30
      Rabenau, H. F.; Biesert, L.; Schmidt, T.; Bauer, G.; Cinatl, J.; Doerr, H. W. SARS-coronavirus (SARS-CoV) and the safety of a solvent/detergent (S/D) treated immunoglobulin preparation. Biologicals 2005, 33, 95– 99,  DOI: 10.1016/j.biologicals.2005.01.003
      30
      SARS-coronavirus (SARS-CoV) and the safety of a solvent/detergent (S/D) treated immunoglobulin preparation
      Rabenau, H. F.; Biesert, L.; Schmidt, T.; Bauer, G.; Cinatl, J.; Doerr, H. W.
      Biologicals (2005), 33 (2), 95-99CODEN: BILSEC; ISSN:1045-1056. (Elsevier B.V.)
      SARS-coronavirus (SARS-CoV) is a newly emerged, highly pathogenic agent that caused over 8000 human infections with nearly 800 deaths between Nov. 2002 and Sept. 2003. While direct person-to-person transmission via respiratory droplets accounted for most cases, other modes have not been ruled out. SARS-CoV viremia does not seem to reach high titers, however, it has to be excluded that virus transmission may occur via blood transfusion or application of therapeutic plasma products, e.g. fresh-frozen plasma or single components derived thereof. Manufg. processes of all plasma derivs. are required to comprise dedicated virus inactivation/removal steps. Treatment with a mixt. of solvent and detergent (SD) has successfully been applied to inactivate the most members of the transfusion-relevant viruses without affecting therapeutic properties of the products. The SD treatment irreversibly disrupts the lipid envelope of viruses such as HIV, HBV, HCV, HGV, and CMV. Here, the authors evaluated the manufg. process of an Ig prepn. (OCTAGAM, manufd. by Octapharma Pharmazeutika Produktionsges. m.b.H., Vienna, Austria) for its capacity to inactivate the SARS-CoV. The results demonstrate that SARS-CoV was completely inactivated below the limit of detection. This was found to occur within 1 min of SD treatment.
    31. 31
      Jureka, A.; Silvas, J. A.; Basler, C. F. Propagation, Inactivation, and Safety Testing of SARS-CoV-2. Viruses 2020, 12, 622  DOI: 10.3390/v12060622
      31
      Propagation, inactivation, and safety testing of SARS-CoV-2
      Jureka, Alexander S.; Silvas, Jesus A.; Basler, Christopher F.
      Viruses (2020), 12 (6), 622CODEN: VIRUBR; ISSN:1999-4915. (MDPI AG)
      In late 2019, a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, the capital of the Chinese province Hubei. Since then, SARS-CoV-2 has been responsible for a worldwide pandemic resulting in over 4 million infections and over 250,000 deaths. The pandemic has instigated widespread research related to SARS-CoV-2 and the disease that it causes, COVID-19. Research into this new virus will be facilitated by the availability of clearly described and effective procedures that enable the propagation and quantification of infectious virus. As work with the virus is recommended to be performed at biosafety level 3, validated methods to effectively inactivate the virus to enable the safe study of RNA, DNA, and protein from infected cells are also needed. Here, we report methods used to grow SARS-CoV-2 in multiple cell lines and to measure virus infectivity by plaque assay using either agarose or microcryst. cellulose as an overlay as well as a SARS-CoV-2 specific focus forming assay. We also demonstrate effective inactivation by TRIzol, 10% neutral buffered formalin, beta propiolactone, and heat.
    32. 32
      Engelmann, I.; Benhalima, I.; Ouafi, M.; Pagneux, Q.; Boukherroub, R.; Hober, D.; Alidjinou, E. K.; Szunerits, S., Validation of a specimen transport buffer for stabilization of SARS-CoV-2 RNA at room temperature for molecular diagnostics. Diagnostics 2021, submitted.
      There is no corresponding record for this reference.
    33. 33
      Wrapp, D.; De Vlieger, D. Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies. Cell 2020, 181, 1004– 1015,  DOI: 10.1016/j.cell.2020.04.031
      33
      Structural Basis for Potent Neutralization of Betacoronaviruses by Single-Domain Camelid Antibodies
      Wrapp, Daniel; De Vlieger, Dorien; Corbett, Kizzmekia S.; Torres, Gretel M.; Wang, Nianshuang; Van Breedam, Wander; Roose, Kenny; van Schie, Loes; Hoffmann, Markus; Pohlmann, Stefan; Graham, Barney S.; Callewaert, Nico; Schepens, Bert; Saelens, Xavier; McLellan, Jason S.
      Cell (Cambridge, MA, United States) (2020), 181 (5), 1004-1015.e15CODEN: CELLB5; ISSN:0092-8674. (Cell Press)
      Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, resp. Crystal structures of these VHHs bound to their resp. viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a mol. basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these mols. may serve as useful therapeutics during coronavirus outbreaks.
    34. 34
      Ke, Z.; Oton, J.; Qu, K.; Cortese, M.; Zila, V.; McKeane, L.; Nakane, T.; Zinanov, J.; Neufeldt, C. J.; Cerikan, B. Structures and distribution of SARS-CoV-2 spike proteins on inatact virions. Nature 2020, 588, 498– 502,  DOI: 10.1038/s41586-020-2665-2
      34
      Structures and distributions of SARS-CoV-2 spike proteins on intact virions
      Ke, Zunlong; Oton, Joaquin; Qu, Kun; Cortese, Mirko; Zila, Vojtech; McKeane, Lesley; Nakane, Takanori; Zivanov, Jasenko; Neufeldt, Christopher J.; Cerikan, Berati; Lu, John M.; Peukes, Julia; Xiong, Xiaoli; Krausslich, Hans-Georg; Scheres, Sjors H. W.; Bartenschlager, Ralf; Briggs, John A. G.
      Nature (London, United Kingdom) (2020), 588 (7838), 498-502CODEN: NATUAS; ISSN:0028-0836. (Nature Research)
      Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of sol., overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy, but the structure and distribution of S on the virion surface remain unknown. We applied cryo-electron microscopy and tomog. to image intact SARS-CoV-2 virions and det. the high-resoln. structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
    35. 35
      Liu, C.; Mendonca, L.; Yang, Y.; Gao, Y.; Shen, G.; Liu, J.; Ni, T.; Liu, C.; Tang, X.; Wei, J. The Architecture of Inactivated SARS-CoV-2 with Postfusion Spikes Revealed by Cryo-EM and Cryo-ET. Structure 2020, 28, 1218.e4– 1224.e4,  DOI: 10.1016/j.str.2020.10.001
      There is no corresponding record for this reference.
    36. 36
      Gao, Q.; Bao, L.; Mao, H.; Wang, L.; Xu, K.; Yang, M.; Li, Y.; Zhu, L.; Wang, N.; Lv, Z. Development of an inactivated vaccine candidate for SARS-CoV-2. Science 2020, 369, 77– 81,  DOI: 10.1126/science.abc1932
      36
      Development of an inactivated vaccine candidate for SARS-CoV-2
      Gao, Qiang; Bao, Linlin; Mao, Haiyan; Wang, Lin; Xu, Kangwei; Yang, Minnan; Li, Yajing; Zhu, Ling; Wang, Nan; Lv, Zhe; Gao, Hong; Ge, Xiaoqin; Kan, Biao; Hu, Yaling; Liu, Jiangning; Cai, Fang; Jiang, Deyu; Yin, Yanhui; Qin, Chengfeng; Li, Jing; Gong, Xuejie; Lou, Xiuyu; Shi, Wen; Wu, Dongdong; Zhang, Hengming; Zhu, Lang; Deng, Wei; Li, Yurong; Lu, Jinxing; Li, Changgui; Wang, Xiangxi; Yin, Weidong; Zhang, Yanjun; Qin, Chuan
      Science (Washington, DC, United States) (2020), 369 (6499), 77-81CODEN: SCIEAS; ISSN:1095-9203. (American Association for the Advancement of Science)
      The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented public health crisis. Because of the novelty of the virus, there are currently no SARS-CoV-2-specific treatments or vaccines available. Therefore, rapid development of effective vaccines against SARS-CoV-2 are urgently needed. Here, we developed a pilot-scale prodn. of PiCoVacc, a purified inactivated SARS-CoV-2 virus vaccine candidate, which induced SARS-CoV-2-specific neutralizing antibodies in mice, rats, and nonhuman primates. These antibodies neutralized 10 representative SARS-CoV-2 strains, suggesting a possible broader neutralizing ability against other strains. Three immunizations using two different doses, 3 or 6μg per dose, provided partial or complete protection in macaques against SARS-CoV-2 challenge, resp., without observable antibody-dependent enhancement of infection. These data support the clin. development and testing of PiCoVacc for use in humans.
    37. 37
      Gao, Q.; Bao, L.; Mao, H.; Wang, L.; Xu, K.; Yang, M.; Li, Y.; Zhu, L.; Wang, N.; Lv, Z. Development of an inactivated vaccine candidate for SARS-CoV-2. Science 2020, 369, eabc1932  DOI: 10.1126/science.abc1932
      There is no corresponding record for this reference.
    38. 38
      Dewar, R.; Baunoch, D.; Wojno, K.; Parkash, V.; Khosravi-Far, R. Viral transportation in covid-19 pandemic: Inactivated virus transportation should be implemented for safe transportation and handling at diagnostics laboratories. Arch. Pathol. Lab. Med. 2020, 16, 32298137  DOI: 10.5858/arpa.2020-0175-L
      There is no corresponding record for this reference.
    39. 39
      Understanding Cycle Threshold Ct in SARS-CoV-2 RT-PCR. https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/926410/Understanding_Cycle_Threshold__Ct__in_SARS-CoV-2_RT-PCR_.pdf (last downloaded Jan 4, 2021).
      There is no corresponding record for this reference.
    40. 40
      Byrne, A. W.; McEveoy, D.; Collins, A. B.; Hunt, K.; Casey, M.; Barber, A.; Butler, F.; Griggin, J.; Lane, E. A.; McAloon, C. Inferred duraiton of infectious periode of SARS-CoV-2: rapid scoping review and analysis of avalaible evidence for asymptpomatic and symptomatic CAOVID-19 cases. BMJ Open 2020, 10, e039856  DOI: 10.1136/bmjopen-2020-039856
      There is no corresponding record for this reference.
    41. 41
      Weiss, A.; Jellingsø, M.; Sommer, M. O. A. Spatial and temporal dynamics of SARS-CoV-2 in COVID-19 patients: A systematic review and meta-analysis. EBioMedicine 2020, 58, 102916  DOI: 10.1016/j.ebiom.2020.102916
      41
      Spatial and temporal dynamics of SARS-CoV-2 in COVID-19 patients: A systematic review and meta-analysis
      Weiss Anne; Jellingso Mads; Sommer Morten Otto Alexander
      EBioMedicine (2020), 58 (), 102916 ISSN:.
      BACKGROUND: The spatial and temporal dynamics of SARS-CoV-2 have been described in case series and retrospective studies. In this study, we provide a coherent overview of the duration of viral detection and viral RNA load in COVID-19 patients, stratified by specimen type, clinical severity, and age. METHOD: We systematically searched PubMed/MEDLINE and Cochrane review database for studies published between 1.11.2019 and 23.04.2020. We pooled the data of selected studies (22/7226 (650 patients) for meta-analysis) to estimate duration of viral detection and visualized viral load over time. FINDINGS: Our analysis showed consistent viral detection from specimen from the upper respiratory tract (URT), the lower respiratory tract (LRT), and faeces, irrespective of the clinical severity of COVID-19. Our analysis suggests that SARS-CoV-2 persists for a longer duration in the LRT compared to the URT in adult patients (5•7 days in mild; 5•9 days in moderate-severe patients). The differences in the duration of viral detection between mild and moderate-severe patients is limited in the LRT, but an indication of longer duration of viral detection for moderate-severe patients was observed in feces (15 days in mild vs. 21 days in moderate-severe patients) and the URT (12 days in mild vs. 16 days in moderate-severe patients). Further, viral load was demonstrated to peak in earlier stages of infection in the URT compared to LRT. INTERPRETATION: This review may aid mathematical modelling and help in defining appropriate endpoints for clinical trails with antivirals in COVID-19. FUNDING: The project has received funding support from Innovation Fund Denmark.
    42. 42
      Cevik, M.; Tate, M.; Lloyd, O.; Maraolo, A. E.; Schafers, J.; Ho, A. SARS-CoV-2, SARS-CoV, and MERS-CoV viral load dynamics, duration of viral shedding, and infectiousness: a systematic review and meta-analysis. Lancet Microbe 2020, 2, e13– e22,  DOI: 10.1016/S2666-5247(20)30172-5
      There is no corresponding record for this reference.
    43. 43
      He, X.; Lau, E. H. Y.; Wu, P.; Deng, X.; Wang, J.; Hao, X.; Lau, Y. C.; Wong, J. Y.; Guan, Y.; Tan, X. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat. Med. 2020, 26, 672– 675,  DOI: 10.1038/s41591-020-0869-5
      43
      Temporal dynamics in viral shedding and transmissibility of COVID-19
      He, Xi; Lau, Eric H. Y.; Wu, Peng; Deng, Xilong; Wang, Jian; Hao, Xinxin; Lau, Yiu Chung; Wong, Jessica Y.; Guan, Yujuan; Tan, Xinghua; Mo, Xiaoneng; Chen, Yanqing; Liao, Baolin; Chen, Weilie; Hu, Fengyu; Zhang, Qing; Zhong, Mingqiu; Wu, Yanrong; Zhao, Lingzhai; Zhang, Fuchun; Cowling, Benjamin J.; Li, Fang; Leung, Gabriel M.
      Nature Medicine (New York, NY, United States) (2020), 26 (5), 672-675CODEN: NAMEFI; ISSN:1078-8956. (Nature Research)
      Abstr.: We report temporal patterns of viral shedding in 94 patients with lab.-confirmed COVID-19 and modeled COVID-19 infectiousness profiles from a sep. sample of 77 infector-infectee transmission pairs. We obsd. the highest viral load in throat swabs at the time of symptom onset, and inferred that infectiousness peaked on or before symptom onset. We estd. that 44% (95% confidence interval, 25-69%) of secondary cases were infected during the index cases' presymptomatic stage, in settings with substantial household clustering, active case finding and quarantine outside the home. Disease control measures should be adjusted to account for probable substantial presymptomatic transmission.
    44. 44
      Kim, S. E.; Jeong, H.; Yu, Y.; Shin, S. U.; Kim, S. I.; Oh, T. H.; Kim, U. J.; Kang, S.-J.; Jang, H.-C.; Jung, S.-I. Viral kinetics of SARS-CoV-2 in asymptomatic carriers andpresymptomatic patients. Int. J. Infect. Dis. 2020, 95, 441– 443,  DOI: 10.1016/j.ijid.2020.04.083
      44
      Viral kinetics of SARS-CoV-2 in asymptomatic carriers and presymptomatic patients
      Kim, Seong Eun; Jeong, Hae Seong; Yu, Yohan; Shin, Sung Un; Kim, Soosung; Oh, Tae Hoon; Kim, Uh Jin; Kang, Seung-Ji; Jang, Hee-Chang; Jung, Sook-In; Park, Kyung-Hwa
      International Journal of Infectious Diseases (2020), 95 (), 441-443CODEN: IJIDF3; ISSN:1201-9712. (Elsevier Ltd.)
      From a total of 71 lab.-confirmed cases, three presymptomatic patients and 10 patients with entirely asymptomatic infections were identified. In two of the three incubation period patients, the viral titer in the presymptomatic period was very high (Ct value < 20). The median no. of days to first neg. RT-PCR in the asymptomatic carriers was 4.5 (range 2.5-9), and all asymptomatic carriers reached a first RT-PCR Ct > 35 within 14 days after diagnosis. Patients who have COVID-19 may already be infectious before there are symptoms, and 14 days of isolation after diagnosis may be sufficient in entirely asymptomatic cases.
    45. 45
      Walsh, K. A.; Jordan, K.; Clyne, B.; Rohde, D.; Drummond, L.; Bryne, P.; Ahern, S.; Carty, P. G.; O’Brien, K. K.; O’Murchu, E. SARS-CoV-2 detection, viral load and infectivity over the course of an infection. J. Infect. 2020, 81, 357– 371,  DOI: 10.1016/j.jinf.2020.06.067
      45
      SARS-CoV-2 detection, viral load and infectivity over the course of an infection
      Walsh, Kieran A.; Jordan, Karen; Clyne, Barbara; Rohde, Daniela; Drummond, Linda; Byrne, Paula; Ahern, Susan; Carty, Paul G.; O'Brien, Kirsty K.; O'Murchu, Eamon; O'Neill, Michelle; Smith, Susan M.; Ryan, Mairin; Harrington, Patricia
      Journal of Infection (2020), 81 (3), 357-371CODEN: JINFD2; ISSN:1532-2742. (Elsevier B.V.)
      A review. To summarise the evidence on the detection pattern and viral load of SARS-CoV-2 over the course of an infection (including any asymptomatic or pre-symptomatic phase), and the duration of infectivity. A systematic literature search was undertaken in PubMed, Europe PubMed Central and EMBASE from 30 Dec. 2019 to 12 May 2020. We identified 113 studies conducted in 17 countries. The evidence from upper respiratory tract samples suggests that the viral load of SARS-CoV-2 peaks around symptom onset or a few days thereafter, and becomes undetectable about two weeks after symptom onset; however, viral loads from sputum samples may be higher, peak later and persist for longer. There is evidence of prolonged virus detection in stool samples, with unclear clin. significance. No study was found that definitively measured the duration of infectivity; however, patients may not be infectious for the entire duration of virus detection, as the presence of viral RNA may not represent transmissible live virus. There is a relatively consistent trajectory of SARS-CoV-2 viral load over the course of COVID-19 from respiratory tract samples, however the duration of infectivity remains uncertain.
    46. 46
      Jefferson, T.; Spencer, E. A.; Brassey, J.; Heneghan, C. Viral cultures for COVID-19 infectivity assessment – a systematic review (Update 4. medRxiv 2020,  DOI: 10.1101/2020.08.04.20167932
      There is no corresponding record for this reference.
    47. 47
      Engelmann, I.; Alidjinou, E. K. Spatial and temporal virus load dynamics of SARS-CoV-2: A single center cohort study, 2020, submitted for publication.
      There is no corresponding record for this reference.
    48. 48
      Spearman, C. The Method of “Right and Wrong Cases” (Constant Stimuli) without Gauss’s Formula. Br. J. Psychol. 1908, 2, 227– 242,  DOI: 10.1111/j.2044-8295.1908.tb00176.x
      There is no corresponding record for this reference.
    49. 49
      Kärber, G. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch. Exp. Pathol. Pharmakol. 1931, 162, 480– 483,  DOI: 10.1007/BF01863914
      There is no corresponding record for this reference.
    50. 50
      https://www.lgcstandards-atcc.org/support/faqs/48802/Converting%20TCID50%20to%20plaque%20forming%20units%20PFU-124.aspx.
      There is no corresponding record for this reference.
    51. 51
      Singanayagam, A.; Patel, M.; Charlett, A.; Bernal, J. L.; Silba, V.; Ellis, J.; Ladhani, S.; Zambon, M.; Gopal, R. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Eurosurveillance 2020, 25, 2001483  DOI: 10.2807/1560-7917.ES.2020.25.32.2001483
      51
      Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020
      Singanayagam, Anika; Patel, Monika; Charlett, Andre; Bernal, Jamie Lopez; Saliba, Vanessa; Ellis, Joanna; Ladhani, Shamez; Zambon, Maria; Gopal, Robin
      Eurosurveillance (2020), 25 (32), 2001483CODEN: EUROGD; ISSN:1560-7917. (European Centre for Disease Prevention and Control)
      Severe acute respiratory syndrome coronavirus 2 viral load in the upper respiratory tract peaks around symptom onset and infectious virus persists for 10 days in mild-to-moderate coronavirus disease (n = 324 samples analyzed). RT-PCR cycle threshold (Ct) values correlate strongly with cultivable virus. Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset; it is similar in asymptomatic and symptomatic persons. Asymptomatic persons represent a source of transmissible virus.
    52. 52
      La Scola, B.; Le Bideau, M.; Andreani, J.; Hoang, V. T.; Grimaldier, C.; Colson, P.; Gautret, P.; Raolt, D. Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards. Eur. J. Clin. Microbiol. Infect. Dis. 2020, 39, 1059– 1061,  DOI: 10.1007/s10096-020-03913-9
      52
      Viral RNA load as determined by cell culture as a management tool for discharge of SARS-CoV-2 patients from infectious disease wards
      La Scola, Bernard; Le Bideau, Marion; Andreani, Julien; Hoang, Van Thuan; Grimaldier, Clio; Colson, Philippe; Gautret, Philippe; Raoult, Didier
      European Journal of Clinical Microbiology & Infectious Diseases (2020), 39 (6), 1059-1061CODEN: EJCDEU; ISSN:0934-9723. (Springer)
      Abstr.: In a preliminary clin. study, we obsd. that the combination of hydroxychloroquine and azithromycin was effective against SARS-CoV-2 by shortening the duration of viral load in Covid-19 patients. It is of paramount importance to define when a treated patient can be considered as no longer contagious. Correlation between successful isolation of virus in cell culture and Ct value of quant. RT-PCR targeting E gene suggests that patients with Ct above 33-34 using our RT-PCR system are not contagious and thus can be discharged from hospital care or strict confinement for non-hospitalized patients.
    53. 53
      Bullard, J.; Dust, K.; Funk, D.; Strong, J. E.; Alexander, D.; Garnett, L.; Boodman, C.; Bello, A.; Hedley, A.; Schiffman, Z. Predicting infectious SARS-CoV-2 from diagnostic samples. Clin. Infect. Dis. 2020, 71, 2663– 2666,  DOI: 10.1093/cid/ciaa638
      53
      Predicting infectious severe acute respiratory syndrome coronavirus 2 from diagnostic samples
      Bullard, Jared; Dust, Kerry; Funk, Duane; Strong, James E.; Alexander, David; Garnett, Lauren; Boodman, Carl; Bello, Alexander; Hedley, Adam; Schiffman, Zachary; Doan, Kaylie; Bastien, Nathalie; Li, Yan; Van Caeseele, Paul G.; Poliquin, Guillaume
      Clinical Infectious Diseases (2020), 71 (10), 2663-2666CODEN: CIDIEL; ISSN:1537-6591. (Oxford University Press)
      Background: Reverse-transcription polymerase chain reaction (RT-PCR) has become the primary method to diagnose viral diseases, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). RT-PCR detects RNA, not infectious virus; thus, its ability to det. duration of infectivity of patients is limited. Infectivity is a crit. determinant in informing public health guidelines/interventions. Our goal was to det. the relationship between E gene SARS-CoV-2 RT-PCR cycle threshold (Ct) values from respiratory samples, symptom onset to test (STT), and infectivity in cell culture. Methods: In this retrospective cross-sectional study, we took SARS-CoV-2 RT-PCR-confirmed pos. samples and detd. their ability to infect Vero cell lines. Results: Ninety RT-PCR SARS-CoV-2-pos. samples were incubated on Vero cells. Twenty-six samples (28.9%) demonstrated viral growth. Median tissue culture infectious dose/mL was 1780 (interquartile range, 282-8511). There was no growth in samples with a Ct > 24 or STT > 8 days. Multivariate logistic regression using pos. viral culture as a binary predictor variable, STT, and Ct demonstrated an odds ratio (OR) for pos. viral culture of 0.64 (95% confidence interval [CI],.49-.84; P <.001) for every 1-unit increase in Ct. Area under the receiver operating characteristic curve for Ct vs pos. culture was OR, 0.91 (95% CI,.85-.97; P <.001), with 97% specificity obtained at a Ct of > 24. Conclusions: SARS-CoV-2 Vero cell infectivity was only obsd. for RT-PCR Ct < 24 and STT < 8 days. Infectivity of patients with Ct > 24 and duration of symptoms > 8 days may be low. This information can inform public health policy and guide clin., infection control, and occupational health decisions. Further studies of larger size are needed.
    54. 54
      Singanayagam, A.; Patel, M.; Charlett, A.; Lopez Bernal, J.; Saliba, V.; Ellis, J.; Ladhani, S.; Zambon, M.; Gopal, R. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Eurosurveillance 2020, 25, 2001483  DOI: 10.2807/1560-7917.ES.2020.25.32.2001483
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      Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020
      Singanayagam, Anika; Patel, Monika; Charlett, Andre; Bernal, Jamie Lopez; Saliba, Vanessa; Ellis, Joanna; Ladhani, Shamez; Zambon, Maria; Gopal, Robin
      Eurosurveillance (2020), 25 (32), 2001483CODEN: EUROGD; ISSN:1560-7917. (European Centre for Disease Prevention and Control)
      Severe acute respiratory syndrome coronavirus 2 viral load in the upper respiratory tract peaks around symptom onset and infectious virus persists for 10 days in mild-to-moderate coronavirus disease (n = 324 samples analyzed). RT-PCR cycle threshold (Ct) values correlate strongly with cultivable virus. Probability of culturing virus declines to 8% in samples with Ct > 35 and to 6% 10 days after onset; it is similar in asymptomatic and symptomatic persons. Asymptomatic persons represent a source of transmissible virus.
    55. 55
      Wölfel, R.; Corman, V. M.; Guggemos, W.; Seilmaier, M.; Zange, S.; Müller, M. A.; Niemeyer, D.; Jones, T. C.; Vollmar, P.; Rothe, C. Virological assessment of hospitalized patients with COVID-2019. Nature 2020, 581, 465– 469,  DOI: 10.1038/s41586-020-2196-x
      55
      Virological assessment of hospitalized patients with COVID-2019
      Wolfel Roman; Zange Sabine; Vollmar Patrick; Ehmann Rosina; Zwirglmaier Katrin; Corman Victor M; Muller Marcel A; Niemeyer Daniela; Jones Terry C; Bleicker Tobias; Brunink Sebastian; Schneider Julia; Drosten Christian; Guggemos Wolfgang; Seilmaier Michael; Wendtner Clemens; Jones Terry C; Rothe Camilla; Hoelscher Michael
      Nature (2020), 581 (7809), 465-469 ISSN:.
      Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 2019(1,2). Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses(3). This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung(2,4); the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 2003(5). However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission(6-8). There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 10(8) RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
    56. 56
      Xiao, A. T.; Tong, Y. X.; Zhang, S. Profile of RT-PCR for SARS-CoV-2: A Preliminary Study From 56 COVID-19 Patients. Clin. Infect. Dis. 2020, 71, 1149– 2251,  DOI: 10.1093/cid/ciaa460
      There is no corresponding record for this reference.
    57. 57
      Omar, S.; Bartz, C.; Becker, S.; Basenach, S.; Pfeifer, S.; Trapp, C.; Hamm, H.; Schlichting, H. C.; Friederichs, M.; Koch, U. Duration of SARS-CoV-2 RNA detection in COVID-19 patients in home isolation, Rhineland-Palatinate, Germany, 2020 – an interval-censored survival analysis. Eurosurveillance 2020, 25, 2001292  DOI: 10.2807/1560-7917.ES.2020.25.30.2001292
      57
      Duration of SARS-CoV-2 RNA detection in COVID-19 patients in home isolation, Rhineland-Palatinate, Germany, 2020 - an interval-censored survival analysis
      Omar, Sarah; Bartz, Christoph; Becker, Sabine; Basenach, Silke; Pfeifer, Sandra; Trapp, Corinna; Hamm, Hildegard; Schlichting, Hans Christoph; Friederichs, Magdalena; Koch, Ulrich; Jestrabek, Christian; Hilger, Ernst; Vogt, Manfred; Jahn, Klaus; Chen, Simiao; Baernighausen, Till; Zanger, Philipp
      Eurosurveillance (2020), 25 (30), 2001292CODEN: EUROGD; ISSN:1560-7917. (European Centre for Disease Prevention and Control)
      The authors analyzed consecutive RT-qPCR results of 537 symptomatic coronavirus disease (COVID-19) patients in home quarantine. Resp. 2, 3, and 4 wk after symptom onset, 50%, 25% and 10% of patients had detectable RNA from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). In patients with mild COVID-19, RNA detection is likely to outlast currently known periods of infectiousness by far and fixed time periods seem more appropriate in detg. the length of home isolation than lab.-based approaches.
    58. 58
      World Health Organization. Laboratory Testing for 2019 Novel Coronavirus (2019-nCoV) in Suspected Human Cases. 2020, https://www.who.int/publications/i/item/10665-3315012020.
      There is no corresponding record for this reference.
    59. 59
      Hayden, R. T.; Caliendo, A. M. Persistent Challenges of Interassay Variability in Transplant Viral Load Testing. J. Clin. Microbiol. 2020, 58, 00782-20  DOI: 10.1128/JCM.00782-20
      There is no corresponding record for this reference.
    60. 60
      Engelmann, I.; Alidjinou, E. K.; Lazrek, M.; Ogiez, J.; Pouillaude, J. M.; Chazard, E.; Dewilde, A.; Hober, D. Comparison of two commercial quantitative PCR assays and correlation with the first WHO International Standard for human CMV. Diagn. Microbiol. Infect. Dis. 2018, 91, 27– 33,  DOI: 10.1016/j.diagmicrobio.2017.12.021
      60
      Comparison of two commercial quantitative PCR assays and correlation with the first WHO International Standard for human CMV
      Engelmann, Ilka; Alidjinou, Enagnon Kazali; Lazrek, Mouna; Ogiez, Judith; Pouillaude, Jean-Marie; Chazard, Emmanuel; Dewilde, Anny; Hober, Didier
      Diagnostic Microbiology and Infectious Disease (2018), 91 (1), 27-33CODEN: DMIDDZ; ISSN:0732-8893. (Elsevier)
      Comparability between CMV assays could be facilitated by the first WHO International Std. for human CMV (std.). Std. dilns. were submitted to nucleic acid extn. with Versant kPCR Mol. systems SP or MagNA Pure LC System followed by the kPCR PLX CMV DNA (kPCR) or the CMV R-gene assay (R-gene), resp.; 139 clin. specimens were tested. Both assays correlated well with the std. (R2 > 0.96) and a matrix effect was obsd. Quant. results correlated reasonably between both assays for whole blood (R2 = 0.79) and well for other specimen types (R2 = 0.93). Quantification differences were within one log10 of the averaged log10 results for 25/27 blood specimens and for 32/33 other specimens. Calibration to the std. did not increase this percentage. In conclusion, results of both assays showed reasonable correlation with each other and good correlation with the std. Calibration to the std. did not improve comparability of quant. results.
    61. 61
      Okoye, N. C.; Adam P Barker, A. P.; Curtis, K.; Orlandi, R. R.; Snavely, E. A.; Wright, C.; Hanson, K. E.; Pearson, L. N. Performance Characteristics of BinaxNOW COVID-19 Antigen Card for Screening an Asymptomatic Individuals in a University Setting. J. Clin. Microbiol. 2021,  DOI: 10.1128/JCM.03282-20
      There is no corresponding record for this reference.
    62. 62
      Gremmels, H.; Winkel, B. M. F.; Schuurman, R.; Rosingh, A.; Rigter, N. A. M.; Rodriguez, O.; Ubijaan, J.; Wensing, A. M. J.; Bonten, M. J. M.; Hofstra, L. M. Real-life validation of the Panbio COVID-19 antigen rapid test (Abbott) in community-dwelling subjects with symptoms of potential SARS-CoV-2 infection. EClinicalMedicine 2020,  DOI: 10.1016/j.eclinm.2020.100677
      There is no corresponding record for this reference.
    63. 63
      Kohmer, N.; Toptan, T.; Pallas, C.; Karaca, O.; Pfeiffer, A.; Westhaus, S.; Widera, M.; Berger, A.; Hoehl, S.; Kammel, M. The Comparative Clinical Performance of Four SARS-CoV-2 Rapid Antigen Tests and Their Correlation to Infectivity In Vitro. J. Clin. Med. 2021, 10, 328,  DOI: 10.3390/jcm10020328
      There is no corresponding record for this reference.
    64. 64
      Roussel, A.; Pagneux, Q.; Cambillau, C.; Engelmann, I.; Alidjinou, E. K.; Ogiez, J.; Boukherroub, R.; Devos, D.; Szunerits, S. A fast, cheap and reliable SARS-CoV-2 detection method based on a portable voltammetric device functionalized with nanobodies, 2021, submitted for publication.
      There is no corresponding record for this reference.
    65. 65
      Ryan, D. J.; Toomey, S.; Madden, S. F.; Casey, M.; Breathnach, O. S.; Morris, P. G.; Grogan, L.; Branagan, P.; Costello, R. W.; De Barra, E. Use of exhaled breath condensate (EBC) in the diagnosis of SARS-COV-2 (COVID-19). Thorax 2021, 76, 86– 88,  DOI: 10.1136/thoraxjnl-2020-215705
      65
      Use of exhaled breath condensate (EBC) in the diagnosis of SARS-COV-2 (COVID-19)
      Ryan Daniel J; Toomey Sinead; Hennessy Bryan T; Ryan Daniel J; Casey Michelle; Branagan Peter; Costello Richard W; Hurley Killian; Gunaratnam Cedric; McElvaney Noel G; OBrien Michael Emmet; Sulaiman Imran; Morgan Ross K; Madden Stephen F; Breathnach Oscar S; Morris Patrick G; Grogan Liam; Hennessy Bryan T; De Barra Eoghan
      Thorax (2021), 76 (1), 86-88 ISSN:.
      False negatives from nasopharyngeal swabs (NPS) using reverse transcriptase PCR (RT-PCR) in SARS-CoV-2 are high. Exhaled breath condensate (EBC) contains lower respiratory droplets that may improve detection. We performed EBC RT-PCR for SARS-CoV-2 genes (E, S, N, ORF1ab) on NPS-positive (n=16) and NPS-negative/clinically positive COVID-19 patients (n=15) using two commercial assays. EBC detected SARS-CoV-2 in 93.5% (29/31) using the four genes. Pre-SARS-CoV-2 era controls (n=14) were negative. EBC was positive in NPS negative/clinically positive patients in 66.6% (10/15) using the identical E and S (E/S) gene assay used for NPS, 73.3% (11/15) using the N/ORF1ab assay and 14/15 (93.3%) combined.