This page will be updated on 28 February. In the meantime, check new papers at https://covidreference.com/top10.
Guglielmi G. Rapid coronavirus tests: a guide for the perplexed. Nature 2021, published 9 February. Full-text: https://www.nature.com/articles/d41586-021-00332-4
Rapid tests, which typically mix nasal or throat swabs with liquid on a paper strip and return results within half an hour, are thought of as tests of infectiousness, not of infection. They can detect only high viral loads, so they will miss many people with lower levels of the SARS-CoV-2 virus. But the hope is that they will help to curb the pandemic by quickly identifying the most contagious people, who might otherwise unknowingly pass on the virus.
Teo AKJ, Choudhury Y, Tan IB, et al. Saliva is more sensitive than nasopharyngeal or nasal swabs for diagnosis of asymptomatic and mild COVID-19 infection. Sci Rep 11, 3134 (2021). Full-text: https://doi.org/10.1038/s41598-021-82787-z
Li Yang Hsu, Alvin Kuo Jing Teo and colleagues recruited 200 subjects, of which 91 and 46 were tested twice and thrice respectively. In total, 62,0%, 44,5%, and 37,7% of saliva, nasopharyngeal (NP) swab and self-administered nasal (SN) swab samples were positive. The percentage of test-positive saliva was higher than NP and SN swabs. The authors conclude that saliva is an alternative sample for COVID-19 screening and diagnosis.
Du Z, Pandey A, Bai Y, et al. Comparative cost-effectiveness of SARS-CoV-2 testing strategies in the USA: a modelling study. Lancet Public Health 2021, published 4 February. Full-text: https://www.thelancet.com/journals/lanpub/article/PIIS2468-2667(21)00002-5/fulltext
In communities where the virus is spreading rapidly, weekly testing coupled with a 2-week isolation period after a positive test is advisable. Where non-pharmacological measures are substantially curtailing the spread of the virus, monthly testing with a 1-week isolation period after a positive test is expected to be the most optimal strategy, according to this paper.
Vogels CBF, Breban M, Alpert T, et al. PCR assay to enhance global surveillance for SARS-CoV-2 variants of concern. medRxiv 2021, posted 1 February. Full-text: https://doi.org/10.1101/2021.01.28.21250486
Nathan Grubaugh, Chantal Vogels and colleagues identified a deletion in the ORF1a gene (ORF1a Δ3675-3677) in the new “trio infernale” variants B117, B1351, and P1, which has not yet been widely detected in other SARS-CoV-2 lineages. Using ORF1a Δ3675-3677 as the primary target and spike Δ69-70 to differentiate, they designed and validated an open source PCR assay to detect the new variants. The authors assure that their assay can be rapidly deployed in laboratories around the world to enhance surveillance for any local emergence of B117, B1351, P1.
Scholkmann F, Restin T, Ferrari M, Quaresima V. The Role of Methemoglobin and Carboxyhemoglobin in COVID-19: A Review. J Clin Med. 2020 Dec 25;10(1):50. PubMed: https://pubmed.gov/33375707. Full-text: https://doi.org/10.3390/jcm10010050
MetHb and COHb can be elevated in COVID-19 patients and should be checked routinely in order to provide adequate medical treatment, as well as to avoid misinterpretation of fingertip pulse oximetry readings, which can be inaccurate and unreliable in case of elevated MetHb and COHb levels in the blood.
Bal A, Destras G, Gaymard A. Two-step strategy for the identification of SARS-CoV-2 variant of concern 202012/01 and other variants with spike deletion H69–V70, France, August to December 2020. Eurosurveillance Volume 26, Issue 3, 21/Jan/2021. https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2021.26.3.2100008
A technical paper, reporting a two-step strategy that enables the timely detection of VOC 202012/01, as well as other variants carrying ΔH69/ΔV70. This strategy allowed the first detection of the VOC 202012/01 in France.
Li Y, Lai D, Lei Q, et al. Systematic evaluation of IgG responses to SARS-CoV-2 spike protein-derived peptides for monitoring COVID-19 patients. Cell Mol Immunol (2021). Full-text: https://doi.org/10.1038/s41423-020-00612-5
Yang Li and colleagues from Shanghai identified and verified eight peptides derived from the S protein that may be of high diagnostic value. These peptides might be used in different circumstances alone or in combination as candidates to generate immunoassays for monitoring COVID-19. In comparison to the current protein-based immunoassays, peptide-based assays could be highly affordable and accessible.
Goh YS, Chavatte JM, Jieling AL, et al. Sensitive detection of total anti-Spike antibodies and isotype switching in asymptomatic and symptomatic COVID-19 patients. Cell Reports January 16, 2021. Full-text: https://doi.org/10.1016/j.xcrm.2021.100193
This flow cytometry assay based on the full-length SARS-CoV-2 S protein (SFB assay) allows the detection of a wider repertoire of antibodies such as antibodies binding to various domains and conformational epitopes of the S protein. Of note, the SFB assay was able to detect 97% of the pre-/asymptomatic infections.
Prince-Guerra JL, Almendares O, Nolen LD, et al. Evaluation of Abbott BinaxNOW Rapid Antigen Test for SARS-CoV-2 Infection at Two Community-Based Testing Sites — Pima County, Arizona, November 3–17, 2020. MMWR Morb Mortal Wkly Rep. ePub: 19 January 2021. Full-text: http://dx.doi.org/10.15585/mmwr.mm7003e3
Sensitivity of the BinaxNOW antigen test, compared with polymerase chain reaction testing, was lower when used to test specimens from asymptomatic (35,8%) than from symptomatic (64,2%) persons, but specificity was high. Sensitivity was higher for culture-positive specimens (92,6% and 78,6% for those from symptomatic and asymptomatic persons, respectively); however, some antigen test-negative specimens had culturable virus.
Zhang Z, Bi Q, Fang S. Insight into the practical performance of RT-PCR testing for SARS-CoV-2 using serological data: a cohort study. Lancet Microbe January 19, 2021.ft https://doi.org/10.1016/S2666-5247(20)30200-7
Even rigorous RT-PCR testing protocols (as used in Shenzhen, China) might miss some SARS-CoV-2 infections, perhaps in part due to difficulties in determining the timing of testing in asymptomatic individuals for optimal sensitivity. In this study, 40 (4,5%) of 880 RT-PCR-negative close contacts were later found to be positive on total antibody ELISA.
Vogl T, Leviatan S, Segal E. SARS-CoV-2 antibody testing for estimating COVID-19 prevalence in the population. Cell Reports Med 2021, published 14 January. Full-text: https://www.cell.com/cell-reports-medicine/fulltext/S2666-3791(21)00002-1
ELISAs/CLIAs appear currently as the method of choice for monitoring the population-wide spread of SARS-CoV-2 in a post-lockdown world. This is the conclusion of Eran Segal, Thomas Vogl and Sigal Leviatan who review different types of antibody tests and their application for population-scale testing.
Butler-Laporte G, Lawandi A, Schiller I, et al. Comparison of Saliva and Nasopharyngeal Swab Nucleic Acid Amplification Testing for Detection of SARS-CoV-2: A Systematic Review and Meta-analysis. JAMA Intern Med. 2021 Jan 15. PubMed: https://pubmed.gov/33449069. Full-text: https://doi.org/10.1001/jamainternmed.2020.8876
Saliva instead of nasopharyngeal swab – the future of SARS-CoV-2 testing? In this review and meta-analysis, Guillaume Butler-Laporte suggest that saliva diagnostic accuracy is similar to that of nasopharyngeal swab, especially in the ambulatory setting.
Bastos ML, Perlman-Arrow S, Menzies D, Campbell JR. The Sensitivity and Costs of Testing for SARS-CoV-2 Infection With Saliva Versus Nasopharyngeal Swabs : A Systematic Review and Meta-analysis. Ann Intern Med. 2021 Jan 12. PubMed: https://pubmed.gov/33428446. Full-text: https://doi.org/10.7326/M20-6569
Same topic. Saliva sampling seemed to be a similarly sensitive and less costly alternative that might soon replace nasopharyngeal swabs for collection of clinical samples for SARS-CoV-2 testing.
Everything you always wanted to know about pre-test probability, test sensitivity and test specificity* (*But Were Afraid to Ask). Jessica Watson and Alex Richter have prepared an interactive page where you can define any of these values. Get a feel for it.
Barclay RA, Akhrymuk I, Patnaik A, et al. Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types. Sci Rep. 2020 Dec 30;10(1):22425. PubMed: https://pubmed.gov/33380736. Full-text: https://doi.org/10.1038/s41598-020-78771-8
To improve detection of SARS-CoV-2 when used with CDC-recommended assays, researchers from Ceres Nanosciences developed affinity-capture hydrogel particles (Nanotrap particles) that capture and concentrate the virus prior to RNA purification. A 5-min Nanotrap particle capture step substantially increases the sensitivity of SARS-CoV-2 RT-PCR assays when used in conjunction with either commercial RNA extraction kits or a simple heat and detergent extraction method in both saliva and transport medium samples. Furthermore, the authors identified viral RNA in several diagnostic remnant samples that previously had tested negative for SARS-CoV-2.
Pilarowski G, Marquez C, Rubio L, et al. Field performance and public health response using the BinaxNOW TM Rapid SARS-CoV-2 antigen detection assay during community-based testing. Clin Infect Dis 2020, published 27 December. Full-text: https://doi.org/10.1093/cid/ciaa1890
SARS-CoV-2 pandemic control calls for fast, low-barrier, high-performing field assays accessible to people who will not otherwise be tested or who will receive results too late for results to make a difference. Here, Diane Havlir, Genay Pilarowski and colleagues evaluated the Abbott BinaxNOWTM COVID-19 antigen card rapid assay performance for detection of persons with high levels of virus and measured the time to isolation in a community walk-up “test and respond” program. Among 3302 persons tested by BinaxNOWTM and RT-PCR in a community setting, rapid assay sensitivity was 100%/98.5%/89% using RT-PCR Ct thresholds of 30, 35 and 0. The specificity was 99.9%. Performance was high across ages and those both with and without symptoms. Rapid results may permit immediate public health action.
Rathe JA, Hemann EA, Eggenberger J, et al. SARS-CoV-2 Serologic Assays in Control and Unknown Populations Demonstrate the Necessity of Virus Neutralization Testing. J Infect Dis 2020, published 25 December. Full-text: https://doi.org/10.1093/infdis/jiaa797
How does serologic antibody testing outcome link with virus neutralization of SARS-CoV-2? To answer this question, Jennifer Rathe et al. compared serum Ig levels across platforms of viral antigens and antibodies with 15 positive and 30 negative SARS-CoV-2 controls followed by viral neutralization assessment. After applying these platforms to a clinically relevant cohort of 114 individuals with unknown histories of SARS-CoV-2 infection, they confirmed that no single serologic assay provides perfect prediction for viral neutralizing ability. Spike IgG3 provided the highest accuracy for predicting serologically positive individuals with virus neutralization activity.
Rosado J, Pelleau S, Cockram C, et al. Multiplex assays for the identification of serological signatures of SARS-CoV-2 infection: an antibody-based diagnostic and machine learning study. Lancet Microbe 2020, published 21 December. Full-text: https://doi.org/10.1016/S2666-5247(20)30197-X
Would you like to classify individuals who were infected more than 6 months ago and measure perform serological surveys in very low transmission settings? Then measure IgG and IgM antibody responses to 1) seven SARS-CoV-2 spike or nucleoprotein antigens, 2) two antigens for the nucleoproteins of the 229E and NL63 seasonal coronaviruses, and 3) three non-coronavirus antigens up to 39 days after symptom onset from 215 adults – and start your machine learning algorithms. A paper by Michael White, Jason Rosado and colleagues from the Pasteur Institute in Paris.
Lean FZX, Lamers MM, Smith SP, et al. Development of immunohistochemistry and in situ hybridisation for the detection of SARS-CoV and SARS-CoV-2 in formalin-fixed paraffin-embedded specimens. Sci Rep 10, 21894 (2020). Full-text: https://doi.org/10.1038/s41598-020-78949-0
Have you ever needed to detect SARS-CoV and SARS-CoV-2 viral products in formalin-fixed paraffin-embedded (FFPE) specimens? Fabian Lean and colleagues show you how. The authors conclude that the diverse techniques for virus detection and control material generation could be applied to investigations of coronavirus pathogenesis and therapeutic research in animal models.
Topol EJ. Is my cough COVID-19? Lancet 2020, published 12 December. Full-text: https://doi.org/10.1016/S0140-6736(20)32589-7
Imagine that even if you have no symptoms of COVID-19, the sound of your forced cough transmitted to your smartphone or smart speaker then processed by an algorithm, could provide a 98·5% accurate diagnosis. Here, Eric Topol describes this science fiction scenario while commenting on a study by Laguarta et al. [Laguarta J, Hueto F, Subirana B. Covid-19 artifical intelligence diagnosis using only cough recordings. IEEE Open J Eng Med Biol 2020, published 29 September. Full-text: https://doi.org/10.1109/OJEMB.2020.3026928], who trained their MIT Open Voice model and built a data collection pipeline of COVID-19 cough recordings through their website (opensigma.mit.edu) between April and May 2020 and created the largest audio COVID-19 cough dataset reported to date with 5320 subjects. Result: COVID-19 sensitivity of 98,5% with a specificity of 94,2% (AUC: 0.97). For asymptomatic subjects it achieves sensitivity of 100% with a specificity of 83,2%.
Yang S, Stanzione N, Uslan DZ, Garner OB, de St Maurice A. Clinical and Epidemiologic Evaluation of Inconclusive COVID-19 PCR Results Using a Quantitative Algorithm. Am J Clin Pathol. 2020 Dec 4:aqaa251. PubMed: https://pubmed.gov/33274731. Full-text: https://doi.org/10.1093/ajcp/aqaa251
Most PCR assays are designed to detect two or more specific target gene regions. An inconclusive result can rarely occur when only one of the targets is detected (< 1%). Shangxin Yang developed a quantitative algorithm to assess and interpret inconclusive PCR results, by combining laboratory, clinical, and epidemiologic data. They determined 5 (28%) of 18 (CDC assay) and 20 (39%) of 51 (TaqPath assay) cases to be false positive. Lowering the cycle threshold cut-off from 40 to 37 in the TaqPath assay resulted in a dramatic reduction of the false-positive rate to 14%. Testing of asymptomatic individuals was associated with a significantly higher probability of having a false-positive result.
Hodges G, Pallisgaard J, Schjerning Olsen AM, et al. Association between biomarkers and COVID-19 severity and mortality: a nationwide Danish cohort study. BMJ Open. 2020 Dec 2;10(12):e041295. PubMed: https://pubmed.gov/33268425. Full-text: https://doi.org/10.1136/bmjopen-2020-041295
In a large study from Denmark, analyzing all patients aged ≥ 18 years admitted to hospital with COVID-19 from 27th of February to 1st of May 2020, with available biochemistry data, of the 1310 patients admitted to hospital (54,6% men; median age 73,6 years), 352 (26,9%) experienced the composite endpoint and 263 (20,1%) died. Elevated levels of CRP, leucocytes, procalcitonin, urea, troponins and D-dimer, and low levels of eGFR were associated with higher standardized absolute risk of death/ICU admission within 30 days.
Wang D, He S, Wang X, et al. Rapid lateral flow immunoassay for the fluorescence detection of SARS-CoV-2 RNA. Nat Biomed Eng 2020, published 3 December. Full-text: https://doi.org/10.1038/s41551-020-00655-z
The authors describe an inexpensive amplification-free nucleic acid immunoassay for the fluorescence detection of SARS-CoV-2 RNA in less than an hour. In a multi-hospital randomized double-blind trial involving 734 samples (593 throat swabs and 141 sputum), the assay achieved sensitivities of 100% and specificities of 99% for both types of sample.
Larremore DB, Toomre D, Parker R. Modeling the effectiveness of olfactory testing to limit SARS-2-CoV transmission. medRxiv 2020, posted 2 December. Full-text: https://doi.org/10.1101/2020.11.30.20241174
Olfactory dysfunction – hyposmia or anosmia – occurs in up to 80% of SARS-CoV-2 infected individuals if standardized olfactory dysfunction test is performed, even in those who remain otherwise asymptomatic. Here, the authors from the University of Colorado Boulder suggest that mass-produced, low-cost and self-administered olfactory tests might reduce the spread of SARS-CoV-2. Interesting approach but attention: this is just a model.
Dugdale CM, Anahtar MN, Chiosi JJ, et al. Clinical, laboratory, and radiologic characteristics of patients with initial false-negative SARS-CoV-2 nucleic acid amplification test results. Open Forum Infect Dis 2020, published 24 November. Full-text: https://doi.org/10.1093/ofid/ofaa559
People with COVID-19 symptoms who have a negative PCR test should be retested within two weeks, especially in areas with sustained community transmission of SARS-CoV-2. This is the message Caitlin Dugdale and colleagues from the Massachusetts General Hospital, Boston, after finding 60 positives of 2699 subjects (2,2%) on a second test. Most of these subjects had symptoms (52/60; 87%) and chest radiography (19/32; 59%) consistent with COVID-19. A total of 60% had their initial test either ≤ 1 day (n = 18) or > 7 days (n = 18) after symptom onset.
Klumpp-Thomas C, Kalish H, Hicks J, et al. D614G Spike Variant Does Not Alter IgG, IgM, or IgA Spike Seroassay Performance. J Infect Dis 2020, published 1 December. Full-text: https://doi.org/10.1093/infdis/jiaa743
Does an individual exposed to one variant of a virus have cross-reactive memory to the second? Probably yes. Carleen Klumpp-Thomas and colleagues from NIH analyzed the serologic ELISA reactivity of both variants and found that antibodies from 88 donors from a high-incidence population reacted toward both the original spike and the D614 spike variant. This suggests that use of the full spike protein construct should not impact seroassay performance or “miss” seropositive samples. However, the fact that D614 and G614 both elicited seropositivity is perhaps expected, given that the human immune response is polyclonal.
Myhre PL, Prebensen C, Strand H, et al. Growth Differentiation Factor 15 Provides Prognostic Information Superior to Established Cardiovascular and Inflammatory Biomarkers in Unselected Patients Hospitalized With COVID-19. Circulation 2020 Dec;142(22):2128-2137. PubMed: https://pubmed.gov/33058695. Full-text: https://doi.org/10.1161/CIRCULATIONAHA.120.050360
Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor β superfamily. Expression in pathological states is highly regulated through several pathways including inflammation, oxidative stress, and hypoxia and elevated concentrations of circulating GDF-15 have been identified in multiple disease entities. Among 123 hospitalized COVID-19 patients from Oslo, Norway, GDF-15 was elevated in the majority of patients. Higher concentrations were associated with SARS-CoV-2 viremia, hypoxemia, and worse outcome. The prognostic value of GDF-15 was additional and superior to established cardiovascular and inflammatory biomarkers.
Rieser M, Wirth L, Pollmeier L, et al. Serum protein profiling reveals a specific upregulation of the immunomodulatory protein progranulin in COVID-19. J Inf Dis November 29, 2020. Full-text: https://doi.org/10.1093/infdis/jiaa741
Another biomarker. The immunomodulatory protein progranulin (GRN) is a pleiotrophic growth factor and immunoregulatory molecule expressed in a broad range of tissues and cell types such as epithelia, bone marrow and various immune cells including T cells, DCs, monocytes and macrophages. In a prospective single-center registry, Marina Rieser and colleagues from Freiburg, Germany included 24 SARS-CoV-2 positive patients and 61 patients with “similar symptoms and severity of disease but negative for SARS-CoV-2” (cancer, cardiac disease, and others) admitted to the emergency department and compared their serum protein expression profiles. Of note, no differences in IL-6 expression between SARS-CoV-2-positive and -negative patients were observed. In contrast, a specific upregulation of GRN was found which was associated with COVID-19 severity. Among the proteins showing the most significant positive correlation with GRN were GDF-15, urokinase-type plasminogen activator (uPA) and urokinase-type plasminogen activator receptor (uPAR).
Alexandersen S, Chamings A, Bhatta TR. SARS-CoV-2 genomic and subgenomic RNAs in diagnostic samples are not an indicator of active replication. Nat Commun 11, 6059 (2020). Full-text: https://doi.org/10.1038/s41467-020-19883-7
In patients with mild or moderate SARS-CoV-2 infection, a positive RT-PCR test 10 days or more after the onset of symptoms is generally not considered proof of infectiousness. But why do tests continue to be positive? Now, Soren Alexandersen, Anthony Chamings and Tarka Raj Bhatta provide a preliminary answer. Subgenomic RNAs, like virion RNA, are rather stable and are likely protected from nucleases by cellular membranes. This information may pave the way for development of better strategies to define active SARS-CoV-2 infection as opposed to extended presence of what most likely represent highly stable virus genomic and subgenomic RNAs present in, and at least in part protected by, cellular membranes. The message for national Health Services: Don’t lock your citizens down for weeks and weeks.
Oved K, Olmer L, Shemer-Avni Y, et al. Multi-center nationwide comparison of seven serology assays reveals a SARS-CoV-2 non-responding seronegative subpopulation. EClinical Med November 19, 2020. Full-text: https://doi.org/10.1016/j.eclinm.2020.100651
Serology assays from Roche, Abbott, Diasorin, BioMerieux, Beckman-Coulter, Siemens, and Mt. Sinai ELISA were used to analyze negative samples from 2391 individuals representative of the Israeli population, and 698 SARS-CoV-2 PCR positive patients. Immunoassay sensitivities were between 81,5% – 89,4% while specificities were between 97,7% – 100%, resulting in a profound impact on the expected Positive Predictive Value in low (< 15%) prevalence scenarios. No meaningful increase was detected in the false positive rate in children compared to adults. A positive correlation between disease severity and antibody titers, and no decrease in antibody titers in the first 8 weeks after PCR positivity was observed. The authors also identified a subgroup of symptomatic SARS-CoV-2 positive patients (~5%), who remained seronegative across a wide range of antigens, isotypes, and technologies.
Qian J, Boswell SA, Chidley C, et al. An enhanced isothermal amplification assay for viral detection. Nat Commun 11, 5920 (2020). Full-text: https://doi.org/10.1038/s41467-020-19258-y
Michael Springer, Jason Qian and colleagues from Harvard Medical School report the development of a molecular diagnostic test for SARS-CoV-2 based on an enhanced recombinase polymerase amplification (eRPA) reaction. eRPA has a detection limit on patient samples down to 5 viral copies, requires minimal instrumentation, and is highly scalable and inexpensive. The protocol reported here was developed and optimized in less than 3 weeks, with an additional 4 weeks for sample preparation optimization, and patient sample acquisition. Good news for future pandemics.
Arizti-Sanz J, Freije CA, Stanton AC, et al. Streamlined inactivation, amplification, and Cas13-based detection of SARS-CoV-2. Nat Commun 11, 5921 (2020). Full-text: https://doi.org/10.1038/s41467-020-19097-x
Cameron Myhrvold, Jon Arizti-Sanz and colleagues from Princeton University describe SHINE (Streamlined Highlighting of Infections to Navigate Epidemics), an extraction-free, rapid, and sensitive detection tool for SARS-CoV-2 RNA. The results can be visualized with an in-tube fluorescent readout — reducing contamination risk as amplification reaction tubes remain sealed — and interpreted by a companion smartphone application. Validation on 50 nasopharyngeal patient samples showed 90% sensitivity and 100% specificity with a sample-to-answer time of 50 min.
Karp DG, Danh K, Espinoza NF, et al. A serological assay to detect SARS-CoV-2 antibodies in at-home collected finger-prick dried blood spots. Sci Rep 10, 20188 (2020). Full-text: https://doi.org/10.1038/s41598-020-76913-6
The authors report a self-collection kit for at-home finger-prick dried blood spot collection. The results suggest 100% sensitivity and specificity. If proven successful at a large scale, such methods could facilitate the conduct of unbiased serosurveys in hard-to-reach populations.
Gniffke EP, Harrington WE, Dambrauskas N, et al. Plasma From Recovered COVID-19 Patients Inhibits Spike Protein Binding to ACE2 in a Microsphere-Based Inhibition Assay. J Infect Dis. 2020 Nov 13;222(12):1965-1973. PubMed: https://pubmed.gov/32798222 . Full-text: https://doi.org/10.1093/infdis/jiaa508
A new microsphere-based flow cytometry assay that quantifies the ability of plasma to inhibit the binding of spike protein to ACE2. Plasma from 22 patients who had recovered from mild COVID-19 and expressed anti–spike protein trimer immunoglobulin G (IgG) inhibited ACE2–spike protein binding to a greater degree than controls. The degree of inhibition was correlated with anti–spike protein IgG levels, neutralizing titers in a pseudotyped lentiviral assay, and the presence of fever during illness. This inhibition assay may be broadly useful to quantify the functional antibody response of patients recovered from COVID-19 or vaccine recipients in a cell-free assay system.
Escribano P, Álvarez-Uría A, Alonso R, et al. Detection of SARS-CoV-2 antibodies is insufficient for the diagnosis of active or cured COVID-19. Sci Rep 10, 19893 2020. Full-text: https://doi.org/10.1038/s41598-020-76914-5
Using Abbott´s SARS-CoV-2 IgG assay and the PanbioTM COVID-19 IgG/IgM device, the authors found that serum IgG detection alone is insufficient for the diagnosis of active or cured COVID-19, with sensitivity values that range between 60 and 75%, respectively. Detection of IgM adds limited value to the performance of serological strategies.
Wang H, Hogan CA, Miller JA, et al. Performance of nucleic acid amplification tests for detection of severe acute respiratory syndrome coronavirus 2 in prospectively pooled specimens. Emerg Infect Dis. 2012 Jan. Full-text: https://doi.org/10.3201/eid2701.203379
Pooled nucleic acid amplification tests (NATs) could increase availability of testing at a decreased cost. However, it is not that trivial, and the effect of dilution on analytical sensitivity through sample pooling has not been well characterized. Hannah Wang and colleagues from Stanford tested 1648 prospectively pooled specimens by using 3 different NATs. Positive percent agreement (PPA) of pooled versus individual testing ranged from 71,7% to 82,6% for pools of 8 and from 82,9% to 100,0% for pools of 4. PPA was dependent on the proportion of tests with positive results, cycle threshold distribution, and assay limit of detection. False negative results occurred exclusively in pools containing samples with low estimated viral load (Ct > 34).
Manabe YC, Sharfstein JS, Armstrong K. The Need for More and Better Testing for COVID-19. JAMA November 13, 2020. Full-text: https://doi.org/10.1001/jama.2020.21694
In their viewpoint, Yukari C. Manabe, Joshua S. Sharfstein and Katrina Armstrong argue that it is more accurate to consider testing less of a prevention strategy than a mitigation strategy. Testing in the absence of other proven prevention strategies is unable to prevent outbreaks. Even as tests become faster with higher sensitivity and specificity, social distancing, mask wearing, and avoidance of large indoor and outdoor gatherings must remain central to any public health strategy. Even the perfect test cannot go it alone.
Liotti FM, Menchinelli G, Marchetti S, et al. Assessment of SARS-CoV-2 RNA Test Results Among Patients Who Recovered From COVID-19 With Prior Negative Results. JAMA Intern Med, November 12, 2020. Full-text: https://doi.org/10.1001/jamainternmed.2020.7570
Many patients who recovered from COVID-19 may still be positive (albeit at lower levels) for SARS-CoV-2 RNA, but only a minority of the patients may carry replicating SARS-CoV-2 in the respiratory tract. Flora Marzia Liotti and colleagues from Rome, Italy analyzed nasal/oropharyngeal swab samples of 176 recovered patients with no fever and with 2 negative RT-PCR results for SARS-CoV-2 RNA 24 hours apart. In total, 32 of 176 samples (18%) tested positive for total SARS-CoV-2 RNA. All had low viral loads and only one of the 32 samples (3.1%) had replicative SARS-CoV-2 RNA.
Zilla M, Wheeler BJ, Keetch C, et al. Variable Performance in 6 Commercial SARS-CoV-2 Antibody Assays May Affect Convalescent Plasma and Seroprevalence Screening. American Journal of Clinical Pathology, aqaa228. Full-text: https://doi.org/10.1093/ajcp/aqaa228
Megan Zilla and colleagues from Pittsburgh have compared six SARS-CoV-2 antibody assays, namely Beckman Coulter, Euroimmun (IgG, IgA), Roche, and Siemens (Centaur, Vista). Assays were assessed for specificity (n=184), sensitivity (n=154), and seroconversion in a defined cohort with clinical correlates and molecular SARS-CoV-2 results. Assay specificity was 99% or greater for all assays except the Euroimmun IgA (95%). Sensitivity at more than 21 days from symptom onset were 84%, 95%, 72%, 98%, 67%, and 96% for Beckman Coulter, Centaur, Vista, Roche, Euroimmun IgA, and Euroimmun IgG, respectively. These finding raises concerns that seroprevalence studies may vary significantly based on the serologic assay utilized, even when the assays are from reliable manufacturers with proven methodologies and have similar targets and initial specificity and sensitivity measures.
Chan CW, Shahul S, Coleman C, et al. Evaluation of the Truvian Easy Check COVID-19 IgM/IgG Lateral Flow Device for Rapid Anti-SARS-CoV-2 Antibody Detection. American Journal of Clinical Pathology, 02 November 2020, aqaa22. Full-text: https://doi.org/10.1093/ajcp/aqaa221
Clarence W. Chan and colleagues from Chicago evaluated the analytical and clinical performance of the Truvian Easy Check COVID-19 IgM/IgG antibody test. This test was designed to detect the nucleocapsid and S1 spike protein RBD epitopes of SARS-CoV-2. Results are available at 10 minutes after test initiation for serum and possibly fingerstick blood samples. The Easy Check device showed excellent clinical and analytical performance; the test compares well with the Roche Elecsys anti-SARS-CoV-2 antibody assay. Of 99 patient samples that were positively confirmed by PCR for SARS-CoV-2, antibodies against the virus were detected by both tests in 88 of the samples, whereas 9 of the 99 samples eluded detection by both testing modalities. All samples from 41 prepandemic volunteers remained negative.
Ward H, Cooke G, Atchison C, et al. Declining prevalence of antibody positivity to SARS-CoV-2: a community study of 365 000 adults. [Preprint] 2020. Full-text: https://www.imperial.ac.uk/media/imperial-college/institute-of-global-health-innovation/MEDRXIV-2020-219725v1-Elliott.pdfdoi:10.1101/2020.10.26.20219725.
Not yet peer reviewed, but important data. Results from the large, nation-wide REACT (real time assessment of community transmission) antibody study which was led by Imperial College London, show that the national antibody prevalence in the UK was 6% around 12 weeks after the epidemic’s April peak. Since then, the rates had fallen to 4.4% 24 weeks after the peak. These data suggest that antibodies induced by natural infection may be short lived, as is the case for other seasonal coronaviruses.
Henss L, Scholz T, von Rhein C, et al. Analysis of humoral immune responses in SARS-CoV-2 infected patients. J Infect Dis. 2020 Oct 31:jiaa680. PubMed: https://pubmed.gov/33128369 . Full-text: https://doi.org/10.1093/infdis/jiaa680
Do previous coronavirus infections protect from severe courses? Lisa Henss and colleagues from Frankfurt University analyzed the humoral immune response of a cohort of 143 COVID-19 patients, using ELISA and neutralization assays. Disease severity correlated with the amount of SARS-CoV-2 specific IgG and IgA and the neutralization activity of the antibodies. Neutralizing titers of patients with mild disease were very low and higher titers were only detected in severe cases. Of note, compared to patients with mild-moderate disease, patients with severe disease had only weakly neutralizing antibodies against coronavirus-NL63. Although the numbers of severe cases were low, it remains tempting to speculate that preexisting immunity to NL63 or other common cold coronaviruses might reduce the risk of severe disease.
Fontana IC, Bongarzone S, Gee A. PET Imaging as a Tool for Assessing COVID-19 Brain Changes. Trends Neurosc October 29, 2020. Full-text: https://doi.org/10.1016/j.tins.2020.10.010
A brief overview of positron emission tomography (PET) applications that could advance current understanding of CNS pathophysiological alterations associated with SARS-CoV-2 infection. Apart from testing for brain changes during the acute phase of the disease, longitudinal assessment of COVID-19 patients using PET may be conducted to examine whether brain changes are transient or long-lasting.
Quer G, Radin JM, Gadaleta M, et al. Wearable sensor data and self-reported symptoms for COVID-19 detection. Nat Med 2020, published 29 October. Full-text: https://doi.org/10.1038/s41591-020-1123-x
Installing a smartphone app that collects smartwatch and activity tracker data and seeing one day a menu pop up: “You might be positive for COVID-19”? What would have seemed unachievable science-fiction a decade ago might be around the corner. Giorgio Quer and colleagues from Scripps Research Translational Institute would just need to confirm in a larger population that a combination of symptom and sensor can discriminate between symptomatic individuals who were positive or negative for COVID-19.
Rubin R. The Challenges of Expanding Rapid Tests to Curb COVID-19. JAMA October 21, 2020. Full-text: https://doi.org/10.1001/jama.2020.21106
BinaxNOW is one of 6 point-of-care rapid antigen tests that had received an Emergency Use Authorization from the FDA as of October 10. The Trump administration awarded a contract for $760 million to Abbott for delivery of 150 million tests (Abbott said it would ship 50 million tests per month beginning in October). However, rapid point-of-care tests alone can’t halt the spread of SARS-CoV-2: this interesting article describes how the rapid test BinaxNOW fueled the White House COVID-19 cluster.
Pettengill MA, McAdam AJ. Can We Test Our Way Out of the COVID-19 Pandemic? J Clin Microbiol 2020, published 21 October. Full-text: https://doi.org/10.1128/JCM.02225-20
Frequent, low-cost, universal testing for SARS-CoV-2 infection and quarantining those with a positive result has been suggested as a strategy to address the COVID-19 pandemic. Could such low-sensitivity daily tests (LSDT) help us ‘test our way out’ of the pandemic? The authors don’t think so and don’t mince their words: “Using testing to prevent transmission of SARS-CoV-2 on a large scale is like using the weather report to prevent global warming.” Find out why.
Azzi L. Saliva is the Key Element for SARS-CoV-2 Mass Screening. Clin Infect Dis 2020, published 21 October. Full-text: https://doi.org/10.1093/cid/ciaa1440
Saliva is the future of mass screening. In his comment on the paper by Isao Yokota et al. we presented on September 29, Lorenzo Azzi highlights the potential merits of saliva: it can be easily and non-invasively self-collected by the subject, thus avoiding the employment of skilled staff and the risk of viral transmission during the procedure, is more comfortable for the patient if compared with the nasopharyngeal swab, and for this reason is more frequently repeatable, with good compliance.
Yokota I, Shane PY, Okada K, et al. Mass screening of asymptomatic persons for SARS-CoV-2 using saliva. Clin Infect Dis. 2020 Sep 25:ciaa1388. PubMed: https://pubmed.gov/32976596. Full-text: https://doi.org/10.1093/cid/ciaa1388
We should not forget, though, that mass screening might not be easy to implement. Look at this: Pettengill MA, McAdam AJ. Can We Test Our Way Out of the COVID-19 Pandemic? J Clin Microbiol 2020, published 21 October. Full-text: https://doi.org/10.1128/JCM.02225-20
Lai CKC, Chen Z, Lui G, et al. Prospective Study Comparing Deep Throat Saliva With Other Respiratory Tract Specimens in the Diagnosis of Novel Coronavirus Disease 2019. J Infect Dis. 2020 Oct 13;222(10):1612-1619. PubMed: https://pubmed.gov/32738137. Full-text: https://doi.org/10.1093/infdis/jiaa487
Use sputum, not deep throat saliva. This study prospectively examined 563 serial samples collected during the virus shedding periods of 50 patients: 150 deep throat saliva (DTS, patients first cleared their throat by gargling with their own saliva, and then they spit out the DTS into a sterile bottle), 309 pooled-nasopharyngeal (NP) and throat swabs, and 104 sputum (self-collected, patients were asked to cough out sputum and spit into a sterile plastic bottle). Deep throat saliva had the lowest overall RT-PCR-positive rate (69% vs 89% for sputum and 81% for pooled NP and throat swabs) and the lowest viral RNA concentration (mean log copy/mL 3.54 vs 5.03 and 4.63, respectively).
Brandsma E, Verhagen HJMP, van de Laar TJW, et al. Rapid, sensitive and specific SARS coronavirus-2 detection: a multi-center comparison between standard qRT-PCR and CRISPR based DETECTR. J Infect Dis 2020. Full-text: https://doi.org/10.1093/infdis/jiaa641
We will need faster and cheaper alternatives to qRT-PCR. Here Emile van den Akker, Eelke Brandsma and colleagues compare DETECTR (a combination of isothermal reverse transcriptase loop mediated amplification [RT-LAMP] and subsequent Cas12 bystander nuclease activation by amplicon targeting ribonucleoprotein complexes) with qRT-PCR to diagnose COVID-19 on 378 patient samples. The authors report a 95% reproducibility between the two tests. DETECTR was 100% specific for SARS-CoV-2 relative to other human coronaviruses.
Procop GW, Brock JE, Reineks EZ, et al. A Comparison of Five SARS-CoV-2 Molecular Assays With Clinical Correlations. Am J Clin Pathol. 2020 Oct 5:aqaa181. PubMed: https://pubmed.gov/33015712 . Full-text: https://doi.org/10.1093/ajcp/aqaa181
A total of 239 specimens (168 contained SARS-CoV-2) were tested by five nucleic acid amplification test methods. The assays that lacked a nucleic acid extraction step produced more false-negative reactions than assays that included this step. The false-negative rates were 0% for the 2019-nCoV Real-Time RT-PCR Diagnostic Panel (CDC), 3.5% for TIB MOLBIOL Assay (Roche), 2.4% for Xpert Xpress SARS-CoV-2 (Cepheid), 11.9% for Simplexa COVID-19 Direct Kit (DiaSorin), and 16.7% for the ID Now COVID-19 (Abbott). Most false negatives were seen in patients with low viral loads.
Yilmaz A, Marklund E, Andersson M, et al. Upper respiratory tract levels of SARS-CoV-2 RNA and duration of viral RNA shedding do not differ between patients with mild and severe/critical COVID-19. J Infect Dis. 2020 Oct 6:jiaa632. PubMed: https://pubmed.gov/33020822 . Full-text: https://doi.org/10.1093/infdis/jiaa632
Aylin Yilmaz and colleagues from Gothenburg, Sweden have analyzed viral RNA loads over time from nasopharynx/throat in 56 patients with mild and severe/critical COVID-19. Neither the viral RNA loads in the upper respiratory tract, nor the time to viral RNA clearance differed between patients with mild or severe/critical disease.
Perreault J, FournierMJ, Beaudoin-Bussières G, et al. Waning of SARS-CoV-2 RBD antibodies in longitudinal convalescent plasma samples within four months after symptom onset. Blood October 1, 2020. Full-text: https://doi.org/10.1182/blood.2020008367
Josée Perreault and colleagues from Québec, Canada have performed a longitudinal analysis of the anti-RBD antibody response in 15 CCP donors (11 males, 4 females, median age of 56 years, no donor was hospitalized) who donated at least four times, during a time interval after symptom onset ranging from 33-77 days for the first donation to 66-114 days for the last donation. A decrease in anti-RBD antibody level between first and last donation was observed for all donors.
Ziegler K, Steininger P, Ziegler R, et al. SARS-CoV-2 samples may escape detection because of a single point mutation in the N gene. Euro Surveill. 2020;25(39). Full-text: https://doi.org/10.2807/1560-7917.ES.2020.25.39.2001650
Katharina Ziegler and colleagues found that a single nucleotide polymorphism (SNP) in the nucleoprotein gene of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) from a patient interfered with detection in a widely used commercial assay. Some 0.2% of the isolates in the EpiCoV database contain this SNP. Although SARS-CoV-2 was still detected by the other probe in the assay, this underlines the necessity of targeting two independent essential regions of a pathogen for reliable detection.
Sullivan CB, Schwalje AT, Jensen M, et al. Cerebrospinal Fluid Leak After Nasal Swab Testing for Coronavirus Disease 2019. JAMA Otolaryngol Head Neck Surg. October 1, 2020. Full-text: https://doi.org/10.1001/jamaoto.2020.3579
Christopher Blake Sullivan describes a case of a cerebrospinal fluid (CSF) leak after nasal testing for COVID-19. After nasal COVID-19 testing for an elective hernia repair, a woman in her 40s presented shortly after with unilateral rhinorrhea, headache, and vomiting. Of note, the patient had an undiagnosed skull base defect at the fovea ethmoidalis that was present on imaging dating back to 2017. The authors therefore speculate that the swab itself did not result in a violation of the bony skull base, but rather the invasive test caused trauma to the patient’s pre-existing encephalocele. However, adverse events may still occur owing to complex and delicate anatomy.
Mina MJ, Parker R, Larremore DB. Rethinking Covid-19 Test Sensitivity — A Strategy for Containment. NEJM September 30, 2020. Full-text: https://doi.org/10.1056/NEJMp2025631
Strong statement (overdue, IMHO): the frequent use of cheap, simple, rapid tests are essential, even if their analytic sensitivities are vastly inferior to those of benchmark tests. The key question is not how well molecules can be detected in a single sample – but how effectively infections can be detected in a population by the repeated use of a given test as part of an overall testing strategy – the sensitivity of the testing regimen.
Yokota I, Shane PY, Okada K, et al. Mass screening of asymptomatic persons for SARS-CoV-2 using saliva. Clin Infect Dis. 2020 Sep 25:ciaa1388. PubMed: https://pubmed.gov/32976596. Full-text: https://doi.org/10.1093/cid/ciaa1388
Self-collected saliva is a valuable specimen to detect SARS-CoV-2 in mass screening of asymptomatic persons. In this study including 1,924 individuals from Japan, the sensitivity of nucleic acid amplification testing with nasopharyngeal and saliva specimens were 86% (90% CI:77-93%) and 92% (90% CI:83-97%), respectively, with specificities greater than 99.9%. In positive individuals, viral load was highly correlated between NPS and saliva.
Clark AE, Lee FM. Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Screening With Specimen Pools: Time to Swim, or Too Deep for Comfort? Clinical Infectious Diseases28 September 2020. Full-text: https://doi.org/10.1093/cid/ciaa1145
Sample pooling is an option to reduce costs and speed results. In this approach, small volumes of samples from multiple patients are combined into a single test, resulting in substantial reagent savings. If the pooled sample returns a negative result, all patients with specimens comprising that pool are considered not to be infected. Andrew Clark and Francesca Lee discuss several caveats and argue that careful and rigorous investigation is necessary to assure that the pooling of specimens does not impact the analytical sensitivity of the assay.
Salvatore PP, Dawson P, Wadhwa A, et al. Epidemiological Correlates of PCR Cycle Threshold Values in the Detection of SARS-CoV-2. Clinical Infectious Diseases 28 September 2020. Full-text: https://doi.org/10.1093/cid/ciaa1469
Phillip P Salvatore and colleagues from CDC examined the relationship between Ct (Cycle Threshold) values and demographic, clinical, and epidemiological characteristics collected through participant interviews and daily symptom diaries. Among 93 household members (including index cases) who tested positive for SARS-CoV-2 by NP swab, Ct values were lowest (corresponding to higher viral RNA concentration) soon after symptom onset and are significantly correlated with time elapsed since onset (p < 0.001); within 7 days after symptom onset, the median Ct value was 26.5 compared with a median Ct value of 35.0 occurring 21 days after onset. Ct values were significantly lower among participants under 18 years of age (p = 0.01) and those reporting upper respiratory symptoms at the time of sample collection (p = 0.001). Ct rates were higher among participants reporting no symptoms (probably due to high Ct values among post-symptomatic participants).
Smyrlaki I, Ekman M, Lentini A, et al. Massive and rapid COVID-19 testing is feasible by extraction-free SARS-CoV-2 RT-PCR. Nat Commun 11, 4812 (2020). Full-text: https://doi.org/10.1038/s41467-020-18611-5
Scalable, rapid, and affordable COVID-19 diagnostics could help to limit the spread of SARS-CoV-2, consequently saving lives. Here, Björn Reinius, Ioanna Smyrlaki and colleagues explored procedures to circumvent RNA extraction by performing RT-PCR directly on heat-inactivated subject samples and sample lysates. Significant savings in time and cost are achieved through RNA-extraction-free protocols that are directly compatible with established PCR-based testing pipelines. The authors suggest that the direct method might be attractive in settings where repeated, cheaper, and quicker testing is desirable, for example in frequent testing of healthcare personnel.
Zamecnik CR, Rajan JV, Yamauchi KA, et al. ReScan, a Multiplex Diagnostic Pipeline, Pans Human Sera for SARS-CoV-2 Antigens. Cell Reports 2020, published 24 September. Full-text: https://doi.org/10.1016/j.xcrm.2020.100123
Identifying highly specific sets of antigens that are less cross-reactive with antibodies elicited by other common human CoV infections could improve future diagnostic SARS-CoV-2 tests. Here Michael Wilson, Colin Zamecnik and colleagues perform proteome-wide profiling of coronavirus antigens enriched by 98 COVID-19 patient sera and identify 9 antigens derived from 3 SARS-CoV-2 proteins as candidates for more specific, multiplexed SARS-CoV-2 serologic assays. The authors conclude that their proof-of-concept study might have broad applicability for other emerging infectious diseases or autoimmune diseases that lack valid biomarkers. Music from the future?
The National SARS-CoV-2 Serology Assay Evaluation Group. Performance characteristics of five immunoassays for SARS-CoV-2: a head-to-head benchmark comparison. Lancet September 23, 2020. Full-text: https://doi.org/10.1016/S1473-3099(20)30634-4
A benchmark study in immunoassay assessment. This UK group did a head-to-head assessment of five SARS-CoV-2 IgG assays, including four commercial assays (Abbott, LIAISON/DiaSorin, Elecsys/Roche, and Siemens), plus a novel in-house 384-well/Oxford ELISA in 976 (!) pre-pandemic blood samples and 536 (!) blood samples with confirmed SARS-CoV-2 infection. All assays had a high sensitivity (92.7-99.1%) and specificity (98.7-99.9%). The most sensitive test assessed was the in-house ELISA. The Siemens assay and Oxford immunoassay achieved 98% sensitivity/specificity without further optimization. However, a limitation of this work was the small number of pauci-symptomatic and asymptomatic cases analyzed.
Yang OO, Ibarrondo FJ. Loss of Anti-SARS-CoV-2 Antibodies in Mild Covid-19. Reply. N Engl J Med. 2020 Sep 23;383(16):10.1056/NEJMc2027051#sa4. PubMed: https://pubmed.gov/32966713. Full-text: https://doi.org/10.1056/NEJMc2027051
The controversy about antibody kinetics in patients with mild COVID-19 is ongoing. Some groups have reported a rapid decline, some have observed stability. According to the authors, several factors probably explain these apparent contradictions, including varying methods used and the heterogeneity among the study participants.
Patel MM, Thornburg NH, Stubblefield WB, et al. Change in Antibodies to SARS-CoV-2 Over 60 Days Among Health Care Personnel in Nashville, Tennessee. JAMA September 17, 2020. Full-text: https://doi.org/10.1001/jama.2020.18796
Your COVID pass expires after a few weeks: among 19 health care workers who had anti–SARS-CoV-2 antibodies detected at baseline, 8 (42%) had antibodies that persisted above the seropositivity threshold at 60 days, whereas 11 (58%) became seronegative. The consistency in decline in the signal-to-threshold ratio regardless of the baseline ratio and a higher proportion of asymptomatic participants becoming seronegative support the interpretation of a true decline over a 2-month period rather than an artifact of assay performance.
Norman M, Gilboa T, Ogata AF, et al. Ultrasensitive high-resolution profiling of early seroconversion in patients with COVID-19. Nat Biomed Eng (2020). https://doi.org/10.1038/s41551-020-00611-x
The authors describe an assay which uses dye-encoded antigen-coated beads to quantify the levels of immunoglobulin G (IgG), IgM and IgA antibodies against four SARS-CoV-2 antigens – as early as the day of the first positive nasopharyngeal PCR test after symptom onset. The ultra-sensitivity enables plasma to be diluted 4,000×, greatly reducing the degree of non-specific circulating immunoglobulin binding.
Ding X, Yin K, Li Z, et al. Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay. Nat Commun 11, 4711 (2020). https://doi.org/10.1038/s41467-020-18575-6
The authors describe an all-in-one dual CRISPR-Cas12a (AIOD-CRISPR) assay which allows all components to be incubated in one pot for CRISPR-based nucleic acid detection, enabling simple, all-in-one molecular diagnostics without the need for separate and complex manual operations. Furthermore, a low-cost hand warmer (~$0.3) was used as an incubator of the AIOD-CRISPR assay to detect clinical samples within 20 min, enabling an instrument-free, visual SARS-CoV-2 detection at the point of care.
Gibani MM, Toumazou C, Sohbati M, et al. Assessing a novel, lab-free, point-of-care test for SARS-CoV-2 (CovidNudge): a diagnostic accuracy study. Lancet Microbe 2020, published 17 September. Full-text: https://doi.org/10.1016/S2666-5247(20)30121-X
Access to rapid diagnosis is key to the control of the SARS-CoV-2 pandemic. In the future, point-of-care testing could relieve pressure on centralized laboratories and increase overall testing capacity. Here, Graham Cooke, Malick Gibani and colleagues describe a sensitive, specific, and rapid point of care test for the presence of SARS-CoV-2 without laboratory handling or sample pre-processing. Their CovidNudge test 2 had 94% sensitivity and 100% specificity when compared with standard laboratory-based RT-PCR.
Joung J, Ladha A, Saito M, et al. Detection of SARS-CoV-2 with SHERLOCK One-Pot Testing. N Engl J Med 2020, published 16 September. Fulltext: https://doi.org/10.1056/NEJMc2026172
CRISPR (clustered regularly interspaced short palindromic repeats)–based diagnostic tests are en vogue. Now Feng Zhang, Julia Joung and colleagues describe a simple SARS-CoV-2 test that combines simplified extraction of viral RNA with isothermal amplification and CRISPR-mediated detection. You’ll get the results in an hour with minimal equipment. First results (n = 202+/200-): sensitivity 93.1%, specificity 98.5%.
Guglielmi G. Fast coronavirus tests: what they can and can’t do. Nature 2020, published 16 September. Full-text: https://www.nature.com/articles/d41586-020-02661-2
Rapid antigen tests are designed to tell in a few minutes whether someone is infectious. Will they be game changers?
Guglielmi G. ‘We didn’t model that people would go to a party if they tested positive’. Nature 2020, published 11 September. Full-text: https://www.nature.com/articles/d41586-020-02611-y
Develop an RNA-based saliva test, deploy scalable testing as part of a campus’s effort to reopen as safely as possible, and build a model to figure out how the epidemic might evolve on the campus. If you forget to model that some people might choose to go to a party although they know that they are positive, your model won’t work. Listen to chemist Martin Burke.
Poon KS, Tee NW. Caveats of Reporting Cycles Threshold from SARS-CoV-2 Qualitative PCR Assays: A Molecular Diagnostic Laboratory Perspective. Clinical Infectious Diseases, 15 September 2020, ciaa1399. Full-text: https://doi.org/10.1093/cid/ciaa1399
A note of caution regarding Ct values as a surrogate indicator of ‘quantity’ in a qualitative PCR assay (“viral load”). Lok-Siong Poon and Nancy Wen-Sim Tee argue that results are not portable across different assays, different gene targets and different specimen types.
Procop GW, Shrestha NK, Vogel S, et al. A Direct Comparison of Enhanced Saliva to Nasopharyngeal Swab for the Detection of SARS-CoV-2 in Symptomatic Patients. J Clin Microbiol Sep 2020, JCM.01946-20. Full-text: https://doi.org/10.1128/JCM.01946-20
The next study showing that saliva works as well as NPS (although the overall viral load in saliva is somewhat lower). An “enhanced” saliva specimen (strong sniff, elicited cough, and collection of saliva/secretions) was collected without transport media prior to nasopharyngeal swab from 216 patients with symptoms deemed consistent with COVID-19. There was a 100% positive agreement (38/38 positive specimens) and 99.4% negative agreement (177/178 negative specimens). The one discrepant specimen had the presence of SARS-CoV-2 confirmed in the saliva specimen using an alternate FDA EUA assay. The overall mean difference in crossing threshold (Ct) values for the positive NPS and saliva specimens was -3.61 (remember: low Ct = higher viral load, 95% CI -5.78 to -1.44).
Van Praet JT, Coene A, Van De Moortele K et al. Comparison of four commercial SARS-CoV-2 IgG immuno-assays in RT-PCR negative patients with suspect CT findings. Infection (2020). Full-text: https://doi.org/10.1007/s15010-020-01523-3
If PCR is negative, serology testing may be helpful. Jens T. Van Praet and colleagues analyzed 17 patients with suspicious CT findings but (repeatedly) negative nasopharyngeal PCR screening. After disease duration of at least 14 days, 12/17 were found to be positive by different anti-SARS-CoV-2 IgG immunoassays. Clinical specificity was suboptimal in some N-based ELISA kits.
Fauter M, Viel S, Zaepfel S, et al. Low glycosylated ferritin is a sensitive biomarker of severe COVID-19. Cell Mol Immunol (2020). Full-text: https://doi.org/10.1038/s41423-020-00544-0
Tromberg BJ, Schwetz, TA, Pérez-Stable EJ, et al. Rapid Scaling Up of Covid-19 Diagnostic Testing in the United States — The NIH RADx Initiative. N Engl J Med 2020, published 10 September. Full-text: https://doi.org/10.1056/NEJMsr2022263
Expanding the capacity, throughput, and speed of returning results will contribute to curb the COVID-19 pandemic. Francis Collins, Bruce Tromberg and colleagues describe the US Rapid Acceleration of Diagnostics (RADx) program, its goals, and its focus on underserved populations.
Watson J, Richter A, Deeks J. Testing for SARS-CoV-2 antibodies. BMJ September 8, 2020. Full-text: https://doi.org/10.1136/bmj.m3325
This brilliant article offers an approach to antibody testing in individuals with and without symptoms suggestive of current or past SARS-CoV-2 infection. The sensitivity and specificity of antibody tests vary over time and results should be interpreted in the context of clinical history. The authors give a practical overview of the pitfalls of antibody testing and how to communicate risk and uncertainty.
Behrens GM, Cossmann a, Stakov MV, et al. Strategic Anti-SARS-CoV-2 Serology Testing in a Low Prevalence Setting: The COVID-19 Contact (CoCo) Study in Healthcare Professionals. Infect Dis Ther 2020 Sep 4. Full-text: https://doi.org/10.1007/s40121-020-00334-1
At Hannover Medical School, Georg Behrens and colleagues have screened 1080 samples from 217 HCP for anti-SARS CoV-2 (S1) IgG. Only one out of eight initial positive results were confirmed by alternative serology tests or showed in vitro neutralisation against live SARS-CoV-2. When assessing anti-SARS-CoV-2 immune status in individuals with low pre-test probability, it may be better to confirm positive results from single measurements by alternative serology tests or functional assays.
Jacobs JL, Mellors JW. Detection of SARS-CoV-2 RNA in Blood of Patients with COVID-19: What Does It Mean? Clinical Infectious Diseases, 08 September 2020- Full-text: https://doi.org/10.1093/cid/ciaa1316
Jana Jacobs and John Mellors (among the first researchers in the early 90’s who established HIV viral load as a strong predictor of progression) say: we still don’t know enough. According to this brief review, the clinical significance of SARS-CoV-2 “RNAaemia” needs to be defined.
Ogata AF, Maley AM, Wu C, et al. Ultra-sensitive Serial Profiling of SARS-CoV-2 Antigens and Antibodies in Plasma to Understand Disease Progression in COVID-19 Patients with Severe Disease. Clin Chem 2020, published 8 September. Full-text: https://doi.org/10.1093/clinchem/hvaa213
We all know about SARS-CoV-2 PCR and serology tests. What about SARS-CoV-2 antigen assays? David Walt, Alana Ogata and colleagues saw the potential to identify active infection and monitor disease progression. Now they describe Single Molecule Array (Simoa) assays to quantitatively detect SARS-CoV-2 spike, S1 subunit, and nucleocapsid antigens in the plasma of 64 COVID-19 positive patients, 17 COVID-19 negative patients, and 34 pre-pandemic patients. They detected SARS-CoV-2 S1 and N antigens in 41 out of 64 COVID-19 positive patients. In these patients, full antigen clearance in plasma was observed a mean of 5 ± 1 days after seroconversion. Importantly, the correlation between patients with high concentrations of S1 antigen and ICU admission (77%) and time to intubation (within one day) was statistically significant. SARS-CoV-2 viral antigens associated with disease progression, such as respiratory failure, in patients with COVID-19? Congratulations!
Crone MA, Priestman M, Ciechonska M, et al. A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics. Nat Commun 11, 4464 (2020). Full-text: https://doi.org/10.1038/s41467-020-18130-3
Diagnostic testing is essential not only for the identification of infection in patients but also for tracking and containment of viral spread within communities, and daily screening of medical frontline workers. Here, Paul Freemont, Michael Crone and colleagues present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly scaled and that is operational in two London hospitals with a testing capacity of 2000 samples per day. The authors report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP.
Tang MS, Case JB, Franks CE, et al. Association between SARS-CoV-2 neutralizing antibodies and commercial serological assays. Clin Chem 2020, published 7 September. Full-text: https://doi.org/10.1093/clinchem/hvaa211
Are the results of commercially available SARS-CoV-2 serological assays correlated to the presence of neutralizing antibodies? (Remember: methods for the detection and quantification of neutralizing antibodies are relatively low-throughput and limited to Biosafety Level 3-equipped research laboratories!) Christopher Farnsworth, Mei San Tang and colleagues looked for neutralizing antibodies to SARS-CoV-2 in specimens from 48 patients with PCR-confirmed COVID-19 and a positive result by the Roche Elecsys anti-SARS-CoV-2 (nucleocapsid), Abbott SARS-CoV-2 IgG (nucleocapsid), or EUROIMMUN (EI) SARS-CoV-2 IgG (S1) assays. They found modest correlation, but poor concordance and NPA between the Roche, Abbott and EI SARS-CoV-2 assays for the detection of SARS-CoV-2 neutralizing antibodies.
[Sentence of the day: “The emergence of commercially available serological assays for the detection of antibodies to SARS-CoV-2 has outpaced scientific understanding of their immunological meaning and their value in clinical decision making.”]
Weiss S, Klingler J, Hioe C, et al. A High Through-Put Assay For Circulating Antibodies Directed Against The S Protein Of Severe Acute Respiratory Syndrome Coronavirus 2 (Sars-Cov-2). J Infect Dis. 2020 Aug 29:jiaa531. PubMed: https://pubmed.gov/32860510. Full-text: https://doi.org/10.1093/infdis/jiaa531
Svenja Weiss and colleagues developed a Luminex binding assay to assess simultaneously the presence of COVID-19-specific antibodies in human serum and plasma. Clear differentiation was achieved between specimens from infected and uninfected subjects, and a wide range of serum/plasma antibody levels were delineated in infected subjects. Since the Luminex Ab assay can simultaneously test qualitatively and quantitatively for RBD and S protein Abs and can be performed in < 2.5 hours with 5 – 10 ng of antigen per test, it provides a platform that will result in cost savings and the processing of large numbers of samples per day.
Wyllie AL, Fournier J, Casanovas-Massana A, et al. Saliva or Nasopharyngeal Swab Specimens for Detection of SARS-CoV-2. N Engl J Med 2020, published 28 August. Full-text: https://doi.org/10.1056/NEJMc2016359
Detecting more SARS-CoV-2 RNA copies in saliva specimens than in nasopharyngeal swab specimens? Finding that a higher percentage of saliva samples than nasopharyngeal swab samples are positive up to 10 days after the COVID-19 diagnosis? Screening 495 asymptomatic health care workers who provided both saliva and nasopharyngeal samples and discovering, to your surprise, more positive results in the saliva samples? That’s what Nathan Grubaugh, Anne Wyllie and colleagues reported in a letter to the NEJM. Read more about fewer direct interactions between health care workers and patients, a possibly lower risk of nosocomial infection and lower demands for personal protective equipment.
Vogels CBF, Brackney D, Wang J, et al. SalivaDirect: Simple and sensitive molecular diagnostic test for SARS-CoV-2 surveillance. medRxiv 2020, posted 4 August. Full-text: https://doi.org/10.1101/2020.08.03.20167791
More information about the previous article (same research group). Here Nathan Grubaugh, Anne Wyllie, Chantal Vogels and colleagues explain how SalivaDirect might improve accessibility, scalability and cost ($1.29-$4.37/sample) of SARS-CoV-2 testing. The test has been granted an emergency use authorization by the U.S. Food and Drug Administration (FDA) on August 15. This paper has not yet been peer-reviewd.
Lepak AJ, Chen DJ, Buys A, et al. Utility of Repeat Nasopharyngeal SARS-CoV-2 RT-PCR Testing and Refinement of Diagnostic Stewardship Strategies at a Tertiary Care Academic Center in a low Prevalence Area of the United States. Open Forum Infectious Diseases, August 27, 2020. Full-text: https://doi.org/10.1093/ofid/ofaa388
The clinical sensitivity of PCR testing may be higher than previously believed. Among a total of 660 patients who had more than one SARS-CoV-2 PCR test performed, the initial test was negative in 638. There were only 6 negative-to-positive conversions (0.9%). All 6 were outpatients undergoing a “person under investigation” work-up 5-17 days after an initial negative result. In > 260 inpatients with repeat testing, the authors found no instances of negative-to-positive conversion including those undergoing PUI or asymptomatic evaluation.
Perera RAPM, Tso E, Tsang OTY, et al. SARS-CoV-2 Virus Culture and Subgenomic RNA for Respiratory Specimens from Patients with Mild Coronavirus Disease. Emerg Infect Dis. 2020 Aug 4;26(11). PubMed: https://pubmed.gov/32749957. Full-text: https://doi.org/10.3201/eid2611.203219
Viral RNA detection by RT-PCR does not prove the presence of infectious virus; culture isolation of virus is a better indication of contagiousness. Now Malik Peiris, Ranawaka Perera and colleagues attempt viral isolation in 68 specimens from 35 patients at different times after symptom onset to define the kinetics of viral isolation in upper respiratory specimens. Their findings suggest that patients with mild or moderate illness might be less contagious 8 days after symptom onset. Mildly ill patients who have clinically recovered and are not immunocompromised might therefore be discharged from containment > 9 days after symptom onset, as long as they are not being discharged into settings that contain other highly vulnerable persons.
Ren L, Fan G, Wu W, et al. Antibody Responses and Clinical Outcomes in Adults Hospitalized with Severe COVID-19: A Post hoc Analysis of LOTUS China Trial. Clin Infect Dis. 2020 Aug 25:ciaa1247. PubMed: https://pubmed.gov/32840287. Full-text: https://doi.org/10.1093/cid/ciaa1247
A retrospective analysis of patients from the LOTUS trial which showed no effect of lopinavir/r in patients with severe COVID-19. From 191 patients included in the trial, a total of 576 blood and 576 throat swabs samples were taken at days 1, 5, 10, 14, 21 and 28 after recruitment, until death or discharge, whichever came first. IgM, IgG against N, S and RBD and NAbs developed in most patients but did not correlate clearly with clinical outcomes. The levels of IgG antibodies against N, S and RBD were related to viral clearance.
Del Valle DM, Kim-Schulze S, Huang HH, et al. An inflammatory cytokine signature predicts COVID-19 severity and survival. Nat Med. 2020 Aug 24. PubMed: https://pubmed.gov/32839624. Full-text: https://doi.org/10.1038/s41591-020-1051-9
Can inflammatory cytokine levels can help predict disease course? Probably yes. Upon admission to the Mount Sinai Health System in New York, cytokines were measured in 1484 patients. When adjusting for disease severity, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF-α serum levels remained independent and significant predictors of disease severity and death. These findings were validated in a second cohort of 231 patients. Diane Marie del Valle and colleagues propose that serum IL-6 and TNF-α levels should be considered in the management and treatment of patients with COVID-19 to stratify prospective clinical trials, guide resource allocation and inform therapeutic options.
Stites EC, Wilen CB. The Interpretation of SARS-CoV-2 Diagnostic Tests. Med 2020, published 21 August. Full-text: https://doi.org/10.1016/j.medj.2020.08.001
There is evidence that physicians struggle with proper probabilistic test interpretation. Here, Edward Stites and Craig Wilen provide answers to questions such as, Should asymptomatic individuals be tested? What does it mean to test positive (or negative)? How shall we interpret tests for “immunity passports”? They review the general principles of SARS-CoV-2 test interpretation and warn that improper utilization can potentially have unintended negative consequences.
Kiran U, Gokulan CG, Kuncha SK, et al. Easing diagnosis and pushing the detection limits of SARS-CoV-2. Biology Methods and Protocols, August 20, 2020. Full-text: https://doi.org/10.1093/biomethods/bpaa017
A more effective and reliable method of SARS-CoV-2 detection. Udan Kiray and colleagues show that the currently used and most reliable RT-PCR based SARS-CoV-2 procedure can be further simplified to make it faster, safer, and economical by eliminating the RNA isolation step.
Hachim A, Kavian N, Cohen CA et al. ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection. Nat Immunol 2020, published 17 August. Full-text: https://doi.org/10.1038/s41590-020-0773-7
A broader landscape of antibody responses to a range of viral proteins may help in detecting the immunogenicity of SARS-CoV-2 infection and understanding pathogenesis and immunity. Sophie Valkenburg, Niloufar Kavian, Asmaa Hachim and colleagues used the luciferase immunoprecipitation system (LIPS) assay to assess the antibody responses to a panel of 15 SARS-CoV-2 antigens; four structural proteins (S, N, M and E), three S subunits (S1, S2 and S2′), the seven available ORFs (ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8 and ORF10) and one relevant NSP within ORF1ab (NSP1). Their data suggest that the combinational use of ORF3b, ORF8 and N may be a high-performing marker of infection at early and late time points.
González-González E, Trujillo-de Santiago G, Lara-Mayorga IM. Portable and accurate diagnostics for COVID-19: Combined use of the miniPCR thermocycler and a well-plate reader for SARS-CoV-2 virus detection. PLOS August 13, 2020. Full-text: https://doi.org/10.1371/journal.pone.0237418
These Mexican researchers demonstrate the use of the mini-PCR, a commercial compact and portable PCR device recently available on the market, in combination with a commercial well-plate reader as a diagnostic system. The accuracy and simplicity of this diagnostics strategy may provide a cost-efficient and reliable alternative for COVID-19 pandemic testing, particularly in underdeveloped regions but also for deployment in point-of-care SARS-CoV-2 detection.
Singanayagam A, Patel M, Charlett A, et al. Duration of infectiousness and correlation with RT-PCR cycle threshold values in cases of COVID-19, England, January to May 2020. Eurosurveillance August 13, 2020, 25, Issue 32. Full-text: https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.32.2001483
Using the Ct values from semi-quantitative RT-PCR can be a valuable proxy for infectious virus detection and may help to inform decision-making on infection control. Culturing virus from 324 samples (253 cases) that tested positive by RT-PCR, this large study found a strong relationship between Ct value and ability to recover infectious virus. The study also adds to the evidence on duration of infectiousness following mild-to-moderate COVID-19. At 10 days after symptom onset, probability of culturing virus declined to 6%. Of note, Ct values and the presence of infectious virus were similar in samples from asymptomatic and pre-symptomatic persons.
Kilic T, Weissleder R, Lee H. Molecular and Immunological Diagnostic Tests of COVID-19: Current Status and Challenges. iScience. 2020 Jul 25;23(8):101406. PubMed: https://pubmed.gov/32771976. Full-text: https://doi.org/10.1016/j.isci.2020.101406
We are presenting this article more than two weeks after publication – an almost inexcusable delay. Allocate at least half an hour to read the review by Tugba Kilic, Ralph Weissleder and Hakho Lee – it’s 16 pages. The authors describe currently available tests to detect either the virus (SARS-CoV-2) or virus-induced immune responses, explaining how the tests work and comparing their performance (see the graphical abstract). Discover also the shortcomings of certain tests and future needs.
Sapkota D, Søland TM, Galtung HK, et al. COVID-19 salivary signature: diagnostic and research opportunities. J Clin Pathol 2020 Aug 7. Full-text: https://doi.org/10.1136/jclinpath-2020-206834
As a non-invasive approach with possibility for self-collection, saliva collection circumvents to a great extent the limitations associated with the use of nasopharyngeal/oropharyngeal swabs. This review summarizes the clinical and scientific basis for the potential use of saliva for COVID-19 diagnosis and disease monitoring. Additionally, Dipak Sapkota and colleagues discuss saliva-based biomarkers and their potential clinical and research applications related to COVID-19.
Lai CKC, Chen Z, Lui G, et al. Prospective study comparing deep-throat saliva with other respiratory tract specimens in the diagnosis of novel coronavirus disease (COVID-19). J Infect Dis 2020, published 1 August. Full-text: https://doi.org/10.1093/infdis/jiaa487
Self-collected specimens for SARS-CoV-2 diagnosis could one day avoid infectious exposure to healthcare workers. Now Paul Chan and colleagues perform a prospective study in two regional hospitals in Hong Kong, examining 563 serial samples collected during the viral shedding period of 50 patients: 150 deep throat saliva (DTS), 309 pooled-nasopharyngeal (NP) and throat swabs, and 104 sputum. (Instructions for deap throat saliva: first clear your throat by gargling with your own saliva, and then spit out the DTS into a sterile bottle.) Deep throat saliva produced the lowest viral RNA concentration and RT-PCR positive rate compared to conventional respiratory specimens.
Tu YP, Jennings R, Hart B, et al. Swabs Collected by Patients or Health Care Workers for SARS-CoV-2 Testing. N Engl J Med. 2020 Jul 30;383(5):494-496. PubMed: https://pubmed.gov/32492294. Full-text: https://doi.org/10.1056/NEJMc2016321
Ethan Berke et al. show the clinical usefulness of tongue, nasal, or mid-turbinate samples collected by patients as compared with nasopharyngeal samples collected by health care workers for the diagnosis of COVID-19. When a nasopharyngeal sample collected by a health care worker was used as the comparator, the estimated sensitivities of the tongue, nasal, and mid-turbinate samples collected by the patients were 89.8% (one-sided 97.5% confidence interval [CI], 78.2 to 100.0), 94.0% (97.5% CI, 83.8 to 100.0), and 96.2% (97.5% CI, 87.0 to 100.0), respectively. Adoption of patient sampling techniques may reduce use of personal protective equipment and provide a more comfortable patient experience.
Tan CW, Chia WN, Qin X, et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction. Nat Biotechnol (2020). Full-text: https://doi.org/10.1038/s41587-020-0631-z
“A robust serological test to detect neutralizing antibodies to SARS-CoV-2 is urgently needed to determine not only the infection rate, herd immunity and predicted humoral protection, but also vaccine efficacy during clinical trials and after large-scale vaccination.” To avoid neutralization tests that require live pathogen and a biosafety level 3 laboratory (BSL3), the authors propose a test based on antibody-mediated blockage of the interaction between the angiotensin-converting enzyme 2 (ACE2) receptor protein and the receptor-binding domain. The test achieved 99.93% specificity and 95–100% sensitivity. Time to completion: 1–2 h. Lab requirement: BSL2.
Mallapaty S. The mathematical strategy that could transform coronavirus testing. Nature. 2020 Jul;583(7817):504-505. PubMed: https://pubmed.gov/32651561. Full-text: https://doi.org/10.1038/d41586-020-02053-6
Widespread testing is needed to get SARS-CoV-2 outbreaks under control but in many regions, there’s a shortage of the chemicals needed to run diagnostics. The solution: pooling samples from many people to save time and resources.
McCulloch DJ, Kim AE, Wilcox NC. Collected Nasopharyngeal Swabs for Detection of SARS-CoV-2 Infection. JAMA 2020;3(7):e2016382. Full-text: https://doi.org/10.1001/jamanetworkopen.2020.16382
Home self-collected swabs may increase testing access while minimizing exposure risk to health care workers and depletion of personal protective equipment, allowing for early community detection of COVID-19. The authors provided participants with test kits for unsupervised home self-collection of a mid-nasal swab. Home swab performance was compared with clinician-collected nasopharyngeal swabs collected by medical assistants and nurses. Compared with clinician swabs, sensitivity and specificity of home swabs was 80.0% and 97.9%, respectively. Unsupervised home mid-nasal swab collection was comparable to clinician-collected nasopharyngeal swab collection for detection of SARS-CoV-2 in symptomatic patients, particularly those with higher viral loads.
Tromberg, BJ, Schwetz TA, Pérez-Stable EJ, et al. Rapid Scaling Up of Covid-19 Diagnostic Testing in the United States — The NIH RADx Initiative. N Engl J Med 2020, published July 22. Full-text: https://doi.org/10.1056/NEJMsr2022263
Earlier in 2020, the COVID-19 epidemic in the US was fueled by a severe (scandalous?) lack of testing capacity. The authors describe the role of the NIH in the effort to increase the range and availability of diagnostic SARS-CoV-2 tests.
Collier Dam Assennato SM, Warne B, et al. Point of care nucleic acid testing for SARS-CoV-2 in hospitalised patients: a clinical validation trial and implementation study. Cell Rep Med 2020, July 15, 2020. Full-text: https://doi.org/10.1016/j.xcrm.2020.100062
This will be the future (at least in hospitals). A point of care (POC) nucleic acid amplification testing (NAAT) was evaluated in 149 participants with parallel combined nasal/throat swabbing for POC versus standard time to result was 2.6 versus 26.4 hours. In an implementation study, POC testing increases isolation room availability, avoids bed closures, allows discharge to care homes and expedites access to hospital procedures.
Mallapaty S. The mathematical strategy that could transform coronavirus testing. Nature 10 July 2020. Full-text: https://www.nature.com/articles/d41586-020-02053-6
If you are interested in math, then this article is for you (everyone else should avoid it). Beautiful mental exercise about how to best pool samples from as many people as possible, in order to save time and/or resources. It’s not that trivial. Some sophisticated strategies are discussed.
Fung B, Gopez A, Servellita V, et al. Direct Comparison of SARS-CoV-2 Analytical Limits of Detection across Seven Molecular Assays. J Clin Microbiol. 2020 Jul 10:JCM.01535-20. PubMed: https://pubmed.gov/32651238. Full-text: https://jcm.asm.org/content/early/2020/07/09/JCM.01535-20
The authors have determined analytical limits of detection for seven SARS-CoV-2 assays using serial dilutions of pooled patient material quantified with droplet digital PCR. Limits of detection ranged from ≤ 10-74 copies/mL for commercial high-throughput laboratory analyzers (Roche Cobas, Abbott m2000, Hologic Panther Fusion) and 167-511 copies/mL for sample to answer (Diasorin Simplexa, Genmark ePlex) and point-of-care instruments (Abbott ID NOW). The CDC assay yielded limits of detection ranging from 85-499 copies/mL, depending on the extraction method and thermocycler used.
Mathers AJ. The practical challenges of making clinical use of the quantitative value for SARS-CoV-2 viral load across several dynamics. Clin Infect Dis. 2020 Jul 10:ciaa958. Full-text: https://doi.org/10.1093/cid/ciaa958
Review of several hurdles and nuances which need to be addressed to deploy Ct value as a meaningful clinical metric. Facing the variability of specimen collection and different diagnostic platforms with varying sensitivity, laboratory professionals will need to develop a standard equivalency across their own diagnostic platforms and specimen types.
Dharavath B, Yadav N, Desai N, et al. A one-step, one-tube real-time RT-PCR based assay with an automated analysis for detection of SARS-CoV-2. Heliyon. July 07, 2020. Full-text: https://doi.org/10.1016/j.heliyon.2020.e04405
The authors present a rapid, easy-to-implement real-time PCR based assay with automated analysis using a novel COVID qPCR Analyzer tool with graphical user interface to analyze the raw qRT-PCR data in an unbiased manner at a cost of less than $3 per reaction and turn-around time of less than 2h, to enable in-house SARS-CoV-2 testing across laboratories.
Wang Y, Zhang L, Sang L, et al. Kinetics of viral load and antibody response in relation to COVID-19 severity. J Clin Invest. 2020 Jul 7:138759. PubMed: https://pubmed.gov/32634129. Full-text: https://doi.org/10.1172/JCI138759
In 8/12 patients with severe COVID-19, viral shedding was shown in a variety of tissues for 20~40 days post-onset of disease; in contrast 9/11 patients with mild disease had viral shedding restricted to the respiratory tract and had no detectable virus RNA after 10 days post-onset.
Mei Q, Li J, Du R, et al. Assessment of patients who tested positive for COVID-19 after recovery. Lancet Inf Dis 2020, July 06, 2020. Full-text: https://doi.org/10.1016/S1473-3099(20)30433-3
In this study, 23 (3%) of 651 patients tested positive on a retest for SARS-CoV-2 by RT-qPCR assay in a routine health check. Of note, 52% had IgG anti-viral antibodies and 30% had IgM antibodies, indicating partial immune system recognition of SARS-CoV-2. Of note, 35% of patients had one or more COVID-19-related symptoms, questioning the usefulness of viral antibodies in COVID-19 clearance.
Schmidt M, Hoehl S, Berger A, et al. Novel multiple swab method enables high efficiency in SARS-CoV-2 screenings without loss of sensitivity for screening of a complete population. Transfusion. 2020 Jul 6. PubMed: https://pubmed.gov/32627200. Full-text: https://doi.org/10.1111/trf.15973
The authors present a novel alternate multiple swab protocol that is based on incubation of a respiratory swab first in a single-sample tube, and then again in a multiple-sample tube. No significant difference in the amount of virus was detected by NAT in the single-sample or multiple-swab tube. The novel protocol was able to reduce the total number of required NAT tests by up to 80%, without loss of diagnostic sensitivity.
Bastos ML, Tavaziva G, Abidi SK, et al. Diagnostic accuracy of serological tests for covid-19: systematic review and meta-analysis. BMJ July 1, 2020; 370. Full-text: https://doi.org/10.1136/bmj.m2516 l (Important)
Systematic review of 40 studies on sensitivity and specificity, stratified by method of serological testing (enzyme linked immunosorbent assays, ELISAs), lateral flow immunoassays (LFIAs), or chemiluminescent immunoassays, CLIAs). The pooled sensitivity of ELISAs measuring IgG or IgM was 84.3% (95% confidence interval 75.6% to 90.9%), of LFIAs was 66.0% (49.3% to 79.3%), and of CLIAs was 97.8% (46.2% to 100%). According to the authors, higher quality clinical studies assessing the diagnostic accuracy of serological tests for COVID-19 are urgently needed. Currently, available evidence does not support the continued use of existing point-of-care serological tests.
Shi J, Han D, Zhang R, et al. Molecular and Serological Assays for SARS-CoV-2: Insights from Genome and Clinical Characteristics. Clinical Chemistry Jul 5, 2020. Full-text: https://doi.org/10.1093/clinchem/hvaa122
This comprehensive review summarizes the principles and related details of PCR and serological assays for SARS-CoV-2 as well as the quality assurance measures for these assays.
Guo X, Jie Y, Chen P, et al. Upper Respiratory Tract Viral RNA Load at Hospital Admission is Associated with COVID-19 Disease Severity. Open Forum Infectious Diseases Jul 5, 2020. Full-text: https://doi.org/10.1093/ofid/ofaa282
The next study reporting that initial viral load is positive correlated to illness severity. Among 195 patients, the two conversely correlated indexes for initial viral load, log10 (copies/mL) and Ct value, were found to be respective significantly positive and negative correlated to severity.
Dong Y, Chi X, Hai H, et al. Antibodies in the breast milk of a maternal woman with COVID-19. Emerg Microbes Infect. 2020 Dec;9(1):1467-1469. PubMed: https://pubmed.gov/32552365. Full-text: https://doi.org/10.1080/22221751.2020.1780952
Case report of an infected mother, in which IgG and IgA antibodies were detected in breast milk, indicating the potential immune protection for the neonates. The infant negative for SARS-CoV-2 at birth had elevated IgG in serum but it quickly decayed.
Tollånes MC, Bakken Kran AM, Abildsnes E, Jenum PA, Breivik AC, Sandberg S. Evaluation of eleven rapid tests for detection of antibodies against SARS-CoV-2. Clin Chem Lab Med. 2020 Jun 29. PubMed: https://pubmed.gov/32598303. l (Important)
Sensitivity of rapid tests is at best moderate: the authors evaluated diagnostic performance of eleven rapid tests for detection of antibodies to SARS-CoV-2 in 20 hospitalized patients with PCR-confirmed COVID-19, 23 recovered outpatients with former PCR-confirmed COVID-19, and 49 participants with suspected COVID-19 presenting at a primary care emergency room. All eleven tests detected antibodies in hospitalized COVID-19 patients, though with varying sensitivities. In former outpatients recovered from COVID-19, there were differences between tests in the immunoglobulin type G (IgG) sensitivity, with five tests having a sensitivity below 65%. In participants with suspected COVID-19 infection, the rapid tests had very low sensitivities.
Magleby R, Westblade LF, Trzebucki A, et al. Impact of SARS-CoV-2 Viral Load on Risk of Intubation and Mortality Among Hospitalized Patients with Coronavirus Disease 2019. Clin Infect Dis. 2020 Jun 30:ciaa851. PubMed: https://pubmed.gov/32603425. Full-text: https://doi.org/10.1093/cid/ciaa851
Viral load matters: admission SARS-CoV-2 viral load among hospitalized patients with COVID-19 independently correlated with the risk of intubation and in-hospital mortality. In 678 patients with COVID-19, higher viral load was associated with increased age, comorbidities, smoking status, and recent chemotherapy. In-hospital mortality was 35.0% with a high viral load (Ct < 25; n = 220), 17.6% with a medium viral load (Ct 25-30; n=216), and 6.2% with a low viral load (Ct > 30; n = 242; P < 0.001). The risk of intubation was also higher in patients with a high viral load (29.1%), compared to those with a medium (20.8%) or low viral load (14.9%; P < 0.001). High viral load was independently associated with mortality (adjusted odds ratio 6.05; 95% CI: 2.92-12.52) and intubation (aOR 2.73; 95% CI: 1.68-4.44) in multivariate models.
Paden CR, Tao Y, Queen K, et al. Rapid, sensitive, full-genome sequencing of severe acute respiratory syndrome coronavirus 2. Emerg Infect Dis. 2020, Jul 1, 2020. https://doi.org/10.3201/eid2610.201800
Validated protocols are described for generating high-quality, full-length genomes from primary samples. One protocol uses multiplex reverse transcription PCR, followed by MinION or MiSeq sequencing; the other uses singleplex, nested reverse transcription PCR and Sanger sequencing. These protocols enable sensitive virus sequencing in different laboratory environments.
Choe PG, Kang CK, Suh HJ, Jung J, Kang EK, Lee SY, et al. Antibody responses to SARS-CoV-2 at 8 weeks postinfection in asymptomatic patients. Emerg Infect Dis. 2020 Sep [date cited]. Full-text: https://doi.org/10.3201/eid2610.202211
The authors compared levels of SARS-CoV-2 neutralizing antibodies in recovery plasma from 7 completely asymptomatic patients with those in symptomatic patients in South Korea. Serologic diagnostic testing was positive for 71% (5/7) of completely asymptomatic patients, but neutralizing antibody response occurred in all 7 patients.
Pinninti S, Trieu C, Pati SK; et al. Comparing Nasopharyngeal and Mid-Turbinate Nasal Swab Testing for the Identification of SARS-CoV-2. Clin Inf Dis 29 June 2020. Full-text: https://doi.org/10.1093/cid/ciaa882
Mid-turbinate nasal swab is not sufficient. Testing of paired MT nasal and nasopharyngeal (NP) swabs, collected by trained personnel from 40 patients with COVID-19 showed more NP (76/95, 80%) than MT swabs tested positive (61/95, 64%; p=0.02). Among samples collected a week after study enrollment, fewer MT than NP samples were positive (45% vs 76%; p=0.001). Patients whose NP swabs are PCR-positive but have a lower viral load as suggested by high CT values (> 30), may often test negative by MT swab.
Nicol T, Lefeuvre C, Serri O, et al. Assessment of SARS-CoV-2 serological tests for the diagnosis of COVID-19 through the evaluation of three immunoassays: Two automated immunoassays (Euroimmun and Abbott) and one rapid lateral flow immunoassay (NG Biotech). J Clin Virol. 2020 Jun 15;129:104511. PubMed: https://pubmed.gov/32593133. Full-text: https://doi.org/10.1016/j.jcv.2020.104511
Lateral flow immunoassay (LFIA) can be used easily as point of care tests or in the laboratory, with a result in less than 15 min. In this study, a LFIA (NG-Test®) was reliable and accurate. The authors compared LFIA and two immunoassays (Abbott CLIA and Euroimmun ELISA) in 293 specimens. Overall sensitivity for IgG was equivalent (around 80%) among all tests and reached 100% > 14 days after onset of symptoms. Overall specificity for IgG was greater for CLIA and LFIA (more than 98%) compared to ELISA (95.8%). Specificity was significantly different between IgA ELISA (78.9%) and IgM LFIA (95.8%) (p < 0.05).
Mak GC, Cheng PK, Lau SS, et al. Evaluation of rapid antigen test for detection of SARS-CoV-2 virus. J Clin Virol. 2020 Jun 8;129:104500. PubMed: https://pubmed.gov/32585619. Full-text: https://doi.org/10.1016/j.jcv.2020.104500
Bad performance of the commercially available rapid BIOCREDIT COVID-19 antigen test. This test was 10,000 fold less sensitive than RT-PCR and detected between 11.1 % and 45.7 % of RT-PCR-positive samples from COVID-19 patients. It serves only as adjunct to RT-PCR test because of the potential for false-negative results.
Ben-Ami R, Klochendler A, Seidel M, et al. Large-scale implementation of pooled RNA extraction and RT-PCR for SARS-CoV-2 detection. Clin Microbiol Infect. 2020 Jun 22:S1198-743X(20)30349-9. PubMed: https://pubmed.gov/32585353. Full-text: https://doi.org/10.1016/j.cmi.2020.06.009
Due to the overwhelming use of SARS-CoV-2 RT-PCR tests worldwide, availability of test kits has become a major bottleneck. The authors show how to overcome these challenges by pooling samples, performing RNA extraction and RT-PCR in pools. A comparison of 184 samples tested individually and in pools of 8 samples, showed that test results were not significantly affected.
Amanat F, White KM, Miorin L, et al. An In Vitro Microneutralization Assay for SARS-CoV-2 Serology and Drug Screening. Curr Protoc Microbiol. 2020 Sep;58(1):e108. PubMed: https://pubmed.gov/32585083. Full-text: https://doi.org/10.1002/cpmc.108
A new microneutralization assay is described in detail. This assay can be used to assess in a quantitative manner if antibodies or drugs can block entry and/or replication of SARS-CoV-2 in vitro. Compared to the most common neutralization assay, the plaque reduction neutralization test (PRNT), more samples can be analyzed. Compared to RBD-ACE2 inhibition assays, the test will also detect neutralizing antibodies binding to epitopes outside of the RBD. Different virus isolates can be used, and the assay can likely be adapted for staining antibodies other than mAbs (e.g., polyclonal sera, antibodies targeting S or M, etc.).
Deeks JJ, Dinnes J, Takwoingi Y, et al. Antibody tests for identification of current and past infection with SARS-CoV-2. Cochrane Database Syst Rev. 2020 Jun 25;6:CD013652. PubMed: https://pubmed.gov/32584464. Full-text: https://doi.org/10.1002/14651858.CD013652
This Cochrane analysis on 57 publications with 15,976 samples says that the sensitivity of antibody tests is too low in the first week from symptom onset to have a primary role in the diagnosis of COVID-19. However, these tests may still have a role complementing other testing in individuals presenting later, when RT-PCR tests are negative, or are not done. Antibody tests are likely to have a useful role for detecting previous SARS-CoV-2 infection if used 15 or more days after the onset of symptoms. Data beyond 35 days post-symptom onset is scarce. According to the authors, studies of the accuracy of COVID-19 tests require considerable improvement. Studies must report data on sensitivity disaggregated by time from onset of symptoms. Updates of this living systematic review are planned.
Lerner AMذ, Eisinger RW, Lowy DR et al. The COVID-19 Serology Studies Workshop: Recommendations and Challenges. Immunity June 23, 2020. Full-text: https://doi.org/10.1016/j.immuni.2020.06.012
Summary of a virtual workshop convened on May 7, 2020 by leading US experts (from NIAID and CDC) in the field. Recommendations for advancing serology assays and conducting crucial serology field studies to advance our understanding of immunity to SARS-CoV-2 will lead to protection and duration of protection, including the correlation between serological test results and risk of reinfection.
Hu X, Zhang R, An T, et al. Impact of Heat-Inactivation on the detection of SARS-CoV-2 IgM and IgG Antibody by ELISA. Clin Chim Acta. 2020 Jun 19:S0009-8981(20)30294-1. PubMed: https://pubmed.gov/32569631. Full-text: https://doi.org/10.1016/j.cca.2020.06.032
Sera inactivated by heating may minimize the risk of virus contamination of laboratory staff. In this study in 62 patients, heat-activation at 56℃ for 30 minutes did not impair the diagnostic efficacy of SARS-CoV-2 IgM or IgG antibodies (ELISA-immunoassay).
Jääskeläinen AJ, Kuivanen S, Kekäläinen E, et al. Performance of six SARS-CoV-2 immunoassays in comparison with microneutralisation. J Clin Virol. 2020 Jun 15;129:104512. PubMed: https://pubmed.gov/32563180. Full-text: https://doi.org/10.1016/j.jcv.2020.104512
“Variable” performance means that some performed badly: Among 62 COVID-19 patients with neutralising antibodies, the specificity and sensitivity values of the commercial antibody tests were as follows: 95%/81% (Abbott Architect), 95%/44% (Diasorin Liaison), 68%/88% (Euroimmun IgA), 87%/71% (Euroimmun IgG), 74%/56% (Acro IgG), 70%/46% (Acro 2019 IgM), 98%/72% (Xiamen IgG), and 89%/81% (Xiamen IgM). The variable performance highlights the need for laboratories to carefully consider their testing process in order to optimize the overall performance of SARS-CoV-2 serodiagnostics.
Atum M, Boz AAE, Çakır B, et al. Evaluation of Conjunctival Swab PCR Results in Patients with SARS-CoV-2 Infection. Ocul Immunol Inflamm. 2020 Jun 22:1-4. PubMed: https://pubmed.gov/32569495. Full-text: https://doi.org/10.1080/09273948.2020.1775261
Don’t use conjunctival swabs, even in patients with conjunctivitis. Among 40 patients (10 with conjunctivitis) who tested positive by RT-PCR of nasopharyngeal and oropharyngeal swabs, conjunctival swab rRT‐PCR was positive for 3 patients (one with conjunctivitis).
Münchhoff M, Mairhofer H, Nitschko H, et al. Multicentre comparison of quantitative PCR-based assays to detect SARS-CoV-2, Germany, March 2020. Eurosurveillance 2020, June 18. 25(24). Full-text: https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.24.2001057
The authors compared 11 different RT-PCR test systems used in seven diagnostic laboratories in Germany in March 2020. The majority of RT-PCR assays detected ca 5 RNA copies per reaction, reflecting a high sensitivity and their suitability for screening purposes worldwide. A reduced sensitivity was noted for the original Charité RdRp gene confirmatory protocol, which may have impacted the confirmation of some cases in the early weeks of the pandemic. The CDC N1 primer/probe set was sensitive and robust for detection of SARS-CoV-2 in nucleic acid extracts from respiratory material, stool and serum from COVID-19 patients.
Chi Y, Ge Y, Wu B, et al. Serum Cytokine and Chemokine profile in Relation to the Severity of Coronavirus disease 2019 (COVID-19) in China. J Infect Dis. 2020 Jun 21:jiaa363. PubMed: https://pubmed.gov/32563194. Full-text: https://doi.org/10.1093/infdis/jiaa363
In this study, the authors detected the serum levels of 48 cytokines and chemokines (!) in a cohort of 74 patients including asymptomatic, mild, moderate and severe cases with laboratory confirmed COVID-19 in Jiangsu, China. IL-6, IL-7, IL-10, IL-18, G-CSF, M-CSF, MCP-1, MCP-3, IP-10, MIG, and MIP-1α were found to be associated with the severity of COVID-19. Some cytokines were significantly higher in men and many were elevated in asymptomatic patients.
Long Q, Tang X, Shi Q et al. Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat Med 2020. Full-text: https://doi.org/10.1038/s41591-020-0965-6 l (Important)
“COVID-19 passes” will last a few weeks, at least in patients with mild symptoms: Compared to symptomatic patients, 37 asymptomatic patients had a significantly longer duration of viral shedding. The virus-specific IgG levels were significantly lower in the acute phase. IgG levels and neutralizing antibodies started to decrease within 2–3 months after infection. Of note, 40% became seronegative (13% of the symptomatic group) for IgG in the early convalescent phase.
Patel MR, Carroll D, Ussery E, et al. Performance of oropharyngeal swab testing compared to nasopharyngeal swab testing for diagnosis of COVID-19 -United States, January-February 2020. Clin Infect Dis. 2020 Jun 16:ciaa759. PubMed: https://pubmed.gov/32548635. Full-text: https://doi.org/10.1093/cid/ciaa759
Among persons with specimens collected early in the course of illness, SARS-CoV-2 RNA diagnostic results were highly concordant between OP and NP swabs (95.2%). However, NP swab Ct values were lower (indicating more virus) in 66.7% of concordant-positive pairs, suggesting NP swabs may more accurately detect SARS-CoV-2.
Kam KG, Yung CF, Maiwald M, et al. Clinical Utility of Buccal Swabs for Severe Acute Respiratory Syndrome Coronavirus 2 Detection in Coronavirus Disease 2019–Infected Children. J Ped Inf Dis 2020, Jun 13. Full-text: https://doi.org/10.1093/jpids/piaa068
Best conclusion of the day: “Buccal swabs are not good” as COVID-19 screening specimens in children. In 11 children positive via nasopharyngeal swabs, 2 remained negative using buccal swabs. There was a general trend for buccal specimens to contain lower SARS-CoV-2 viral loads (higher Ct values) compared with nasopharyngeal specimens. The sensitivity of buccal swabs compared with nasopharyngeal swabs ranged from 25% to 71.4% on different days of collection during the first week of illness/diagnosis. Buccal SARS-CoV-2 was undetectable by day 8 of admission/diagnosis, although the nasopharyngeal SARS-CoV-2 was still detectable.
Lamb LE, Bartolone SN, Ward E, Chancellor MB. Rapid detection of novel coronavirus/Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by reverse transcription-loop-mediated isothermal amplification. PLoS One. 2020 Jun 12;15(6):e0234682. PubMed: https://pubmed.gov/32530929. Full-text: https://doi.org/10.1371/journal.pone.0234682. eCollection 2020
The authors describe a “fast and robust assay for detection of SARS-CoV-2 in 30–45 minutes”. This simple assay (Reverse Transcription Loop-Mediated Isothermal Amplification, RT-LAMP) could be used outside of a central laboratory on various types of biological samples. This assay can be completed by individuals without specialty training or equipment and may provide a new diagnostic strategy for combatting the spread of SARS-CoV-2 at the point-of-risk. However, numbers of tested samples were low. Sensitivity and specificity have to be tested in larger populations.
Chia WN, Tan CW, Foo R, et al. Serological differentiation between COVID-19 and SARS infections. Emerg Microbes Infect. 2020 Jun 12:1-23. PubMed: https://pubmed.gov/32529906. Full-text: https://doi.org/10.1080/22221751.2020.1780951
The authors examined the performance of N, S1 and RBD proteins from SARS-CoV-2 and SARS-CoV in four different test platforms. Results show that the RBD protein provides the best specificity, whereas the N protein of either virus is not suitable to detect virus-specific antibodies due to a very high level of cross-reactivity.
Caini S, Bellerba F, Corso F, et al. Meta-analysis of diagnostic performance of serological tests for SARS-CoV-2 antibodies up to 25 April 2020 and public health implications. Eurosurveillance 2020, June 11. Volume 25, Issue 23. Full-text: https://www.eurosurveillance.org/content/10.2807/1560-7917.ES.2020.25.23.2000980 l (Important)
This review of the diagnostic accuracy of SARS-CoV-2 serological tests includes 9 studies, using different test kits. Random-effects models yielded a summary sensitivity of 82% for IgM, and 85% for IgG and total antibodies. For specificity, the pooled estimates were 98% for IgM and 99% for IgG and total antibodies. In populations with ≤ 5% of seroconverted individuals, the positive predictive value would be ≤ 88%. According to the authors, serological tests should be used for prevalence surveys only in hard-hit areas.
Hung IF, Cheng VC, Li X. SARS-CoV-2 shedding and seroconversion among passengers quarantined after disembarking a cruise ship: a case series. Lancet Inf Dis, June 12, 2020. Full-text: https://doi.org/10.1016/S1473-3099(20)30364-9
Among 215 adult (≥ 18 years) passengers from Hong Kong who had been on board the Diamond Princess cruise ship and who had been found to be PCR-negative before disembarking, 9 became positive during quarantine. Those with evidence of pneumonia on imaging tended to have an increased antibody response. However, positive IgG or IgM confirmed infection of COVID-19 in both symptomatic and asymptomatic patients.
Liu ZL, Liu Y, Wan LG, et al. Antibody profiles in mild and severe cases of COVID-19. Clinical Chemistry 10 June 2020. Full-text: https://doi.org/10.1093/clinchem/hvaa137
This study analysed antibody response in 192 RT-PCR confirmed COVID-19 patients, using two commercial microparticle chemiluminescence immunoassays (Wantai). Patients were stratified by disease severity. Severe cases had significantly higher IgM titers than mild cases after day 6 post-onset. Strikingly, 34% and 14% of mild patients were consistently serologically negative for IgM and total antibody, respectively.
Chew KL, Tan SS, Saw S, et al. Clinical evaluation of serological IgG antibody response on the Abbott Architect for established SARS-CoV-2 infection. Clin Microbiol June 09, 2020. Full-text: https://doi.org/10.1016/j.cmi.2020.05.036
Residual sera from 177 symptomatic COVID-19 patients, and 163 non-COVID-19 patients were tested for antibody with the Abbott SARS-CoV-2 IgG assay. Specificity of the assay was 100%. The clinical sensitivity varied depending on time from onset of symptoms, increasing with longer periods since onset of clinical illness. The clinical sensitivity at ≤ 6 days was 8.6%, 7-13 days: 43.6%, 14-20 days: 84.0%, and ≥ 21 days: 84.4%.
Wong MC, Huang J, Lai C, et al. Detection of SARS-CoV-2 RNA in fecal specimens of patients with confirmed COVID-19: a meta-analysis. J Infection, June 11, 2020. Full-text: https://doi.org/10.1016/j.jinf.2020.06.012
In this meta-analysis of 17 studies, the pooled detection rate of fecal SARS-CoV-2 RNA was 43.7% and 33.7% by patient and number of specimens, respectively. Female individuals (59.6% vs. 53.5%), those who presented with gastrointestinal symptoms (77.1% vs. 57.7%), and patients with more severe disease (68.3% vs. 34.6%) tended to have a higher detection rate.
Grifoni E, Valoriani A, Cei F. Interleukin-6 as prognosticator in patients with COVID-19. J Infection 2020, June 8. Full-text: https://doi.org/10.1016/j.jinf.2020.06.008
According to this study analysing 77 patients, IL-6 levels at hospital admission seem to be a good ”prognosticator” for the combined endpoint progression to severe disease and/or in-hospital mortality, and it seems to be the best prognosticator for negative outcome.
Theel ES, Harring J, Hilgart H, Granger D. Performance Characteristics of Four High-Throughput Immunoassays for Detection of IgG Antibodies against SARS-CoV-2. J Clin Microbiol. 2020 Jun 8:JCM.01243-20. PubMed: https://pubmed.gov/32513859. Full-text: https://doi.org/10.1128/JCM.01243-20 l (Important)
Head-to-head comparison of four high-throughput, commercially available anti-SARS-CoV-2 IgG serologic tests from Abbott Laboratories, Epitope Diagnostics Inc, Euroimmun, and Ortho-Clinical Diagnostics, using serially collected acute and convalescent sera from both hospitalized patients and out-patients with RT-PCR confirmed COVID-19. All four immunoassays performed similarly with respect to sensitivity in COVID-19 hospitalized patients, and except for the Epitope assay, also in individuals with milder forms of the infection. The Abbott and Ortho-Clinical immunoassays provided the highest overall specificity, of over 99%.
Cheng MP, Yansouni CP, Basta NE, et al. Serodiagnostics for Severe Acute Respiratory Syndrome–Related Coronavirus-2. Annals Int Med 2020, Jun 4. Full-text: https://doi.org/10.7326/M20-2854
For SARS-CoV-2, the accuracy of antibody test results and the appropriate test interpretation both depend on clinical context. This article discusses key use cases for SARS-CoV-2 antibody detection tests and their application to serologic studies, reviews currently available assays, highlights key areas of ongoing research, and proposes potential strategies for test implementation. This review also includes a decision tree for interpreting antibody test results.
Long DR, Gombar S, Hogan CA. Occurrence and Timing of Subsequent SARS-CoV-2 RT-PCR Positivity Among Initially Negative Patients. Clinical Infectious Diseases 2020, June 7. Full-text: https://doi.org/10.1093/cid/ciaa722
If the first PCR is negative, a second PCR only yields a small number of positive results. Using data for 20,912 patients, authors analyzed the frequency of SARS-CoV-2 RT-PCR test discordance among individuals initially testing negative by nasopharyngeal swab who were retested on clinical grounds within 7 days. The frequency of subsequent positivity within this window was only 3.5% and similar across institutions.
Hung DL, Li X, Chiu KH, et al. Early Morning Versus Spot Posterior Oropharyngeal Saliva for Diagnosis of SARS-CoV-2 Infection: Implication of Timing of Specimen Collection for Community-wide Screening. Open Forum Infectious Diseases, June 7. Full-text: https://doi.org/10.1093/ofid/ofaa210
The cycle threshold (Ct) PCR values (low = more virus) were compared in posterior oropharyngeal saliva, collected at five different time points within the same day from 18 COVID-19 patients. There was an overall trend of lower Ct values from specimens collected in the early morning, with a gradual decrease of viral load towards night time. Eight out of 13 subjects had highest viral load in the early morning than the rest of the four time points. The results suggest a diurnal variation of viral shedding from the upper respiratory tract.
Tu YP, Jennings R, Hart B, et al. Swabs Collected by Patients or Health Care Workers for SARS-CoV-2 Testing. N Engl J Med. 2020 Jun 3. PubMed: https://pubmed.gov/32492294. Full-text: https://doi.org/10.1056/NEJMc2016321 l (Important)
Let the patients do their own swabs! A total of 530 patients with upper respiratory infection were provided with instructions and asked to collect tongue, nasal, and mid-turbinate samples. A nasopharyngeal (NP) sample was then collected from the patient by a HCW. When this NP sample was used as the comparator, the estimated sensitivities of the tongue, nasal, and mid-turbinate samples collected by the patients were 89.8%, 94.0% and 96.2%, respectively. This study shows the clinical usefulness of these samples. This may reduce PPE use and provide a more comfortable patient experience.
Woloshin S, Patel N, Kesselheim AS. False Negative Tests for SARS-CoV-2 Infection – Challenges and Implications. N Engl J Med. 2020 Jun 5. PubMed: https://pubmed.gov/32502334. Full-text: https://doi.org/10.1056/NEJMp2015897
Important article on false negative tests (which are frequent), with several conclusions. According to the authors, FDA should ensure that manufacturers provide details of tests’ clinical sensitivity and specificity at the time of market authorization. It will also be important to develop prediction rules for estimating the pre-test probability of infection (for asymptomatic and symptomatic people) to allow calculation of post-test probabilities after positive or negative results.
Tang MS, Hocl KG, Logsdon NM, et al. Clinical Performance of the Roche SARS-CoV-2 Serologic Assay. Clinical Chemistry, June 2, 2020. hvaa132, https://doi.org/10.1093/clinchem/hvaa132.
The authors compared the clinical performance of several serologic assays (Abbott, EUROIMMUN and the Roche Elecsys assay). The Abbott assay demonstrated the fewest false negative results > 14d post-symptom onset and the fewest false positive results. While the Roche assay detected more positive results earlier after onset of symptoms, none of the assays demonstrated high enough clinical sensitivity before day 14 from symptom onset to diagnose acute infection. This is bad news because we still have to rely on PCR during the first two weeks.
Bullard J, Dust K, Funk D, et al. Predicting infectious SARS-CoV-2 from diagnostic samples. Clinical Infectious Diseases 2020, May 22 2020. Full-text: https://doi.org/10.1093/cid/ciaa638
This retrospective cross-sectional study determined PCR positive samples for their ability to infect cell lines. Of 90 samples, only 29% demonstrated viral growth. There was no growth in samples with a Ct > 24 (the lower the cycle threshold, the higher the viral load) or duration of symptoms > 8 days. This is very good news, because positive PCR does not mean infectivity. And infectivity duration is short.
Hao S, Lian J, Lu Y, et al. Decreased B cells on admission was associated with prolonged viral RNA shedding from respiratory tract in Coronavirus Disease 2019: a case control study. J Infect Dis. 2020 May 31. PubMed: https://pubmed.gov/32474608. Full-text: https://doi.org/10.1093/infdis/jiaa311
In 104 patients, a decrease in B cells was independently associated with prolonged viral RNA shedding. The viral RNA shedding from respiratory tract in patients with normal B cell count was significantly shorter than patients with decreased B cell on admission (median 11 vs 16 days). This is good news, because these observations may help to individualize monitoring of COVID-19 patients.
Ojha V, Mani A, Pandey NN, Sharma S, Kumar S. CT in coronavirus disease 2019 (COVID-19): a systematic review of chest CT findings in 4410 adult patients. Eur Radiol. 2020 May 30. PubMed: https://pubmed.gov/32474632. Full-text: https://doi.org/10.1007/s00330-020-06975-7
A total of 45 studies comprising 4,410 (!) patients were included in this review. Ground glass opacities (GGOs), whether isolated (50%) or coexisting with consolidations (44%) in bilateral and subpleural distribution, were the most prevalent chest CT findings in adult COVID-19 patients. Follow-up CT shows a progression of GGOs into a mixed pattern, reaching a peak at 10-11 days, before gradually resolving or persisting as patchy fibrosis. Younger people tend to have more GGOs. Older or sicker people tend to have more extensive involvement with consolidations. This is good news, because it’s good to see that there are nerds out there (like us) who have nothing better to do than look through 4,410 CT scans.
Basu A, Zinger T, Inglima K, et al. Performance of Abbott ID NOW COVID-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a New York City academic institution. J Clin Microbiol. 2020 May 29. PubMed: https://pubmed.gov/32471894. Full-text: https://doi.org/10.1128/JCM.01136-20
The authors evaluated the recently released Abbott ID NOW COVID-19 assay (uses isothermal nucleic acid amplification of the RdRp viral target) which is capable of producing positive results in as little as 5 minutes. Results were compared with RT-PCR Cepheid Xpert Xpress SARS-CoV-2 using nasopharyngeal swabs. Regardless of method of collection and sample type, the rapid test had negative results in a third of the samples that tested positive by PCR when using nasopharyngeal swabs in viral transport media and 45% when using dry nasal swabs. This is good news, well, because this is the-good-news-day. However, these rapid tests (if further improved) may find their role in clinical settings such as emergency departments where rapid diagnosis is crucial.
Traugott M, Aberle SW, Aberle JH, et al. Performance of SARS-CoV-2 antibody assays in different stages of the infection: Comparison of commercial ELISA and rapid tests. J Infect Dis. 2020 May. PubMed: https://pubmed.gov/32473021. Full-text: https://doi.org/10.1093/infdis/jiaa305
Four commercial ELISAs and two rapid tests performed well in 77 patients with PCR-confirmed SARS-CoV-2 infection, grouped by intervals from symptom onset. While test sensitivities were low (<40%) within the first 5 days post disease onset, IgM-, IgA- and total antibody-ELISAs increased in sensitivity to >80% between the 6th and 10th day post-symptom onset. The evaluated tests (including IgG and rapid tests) provided positive results in all patients at or after the 11th day post-onset of disease.
Wu J, Liu X, Liu J, et al. Coronavirus Disease 2019 Test Results After Clinical Recovery and Hospital Discharge Among Patients in China. JAMA Netw Open. 2020;3(5):e209759. Full-text: https://doi.org/10.1001/jamanetworkopen.2020.9759
In this cross-sectional study, 10 of 60 patients previously diagnosed with and treated for COVID-19 had RT-PCR test results positive for SARS-CoV-2 from 4 to 24 days after index hospital discharge. In total, 6/10 patients had positive anal swab results. However, the infectivity remains unclear, as infectious viruses have not been isolated from stool samples. Positive results were presumed to be persistent viral shedding rather than reinfection.
Han MS, Byun JH, Cho Y, Rim JH. RT-PCR for SARS-CoV-2: quantitative versus qualitative. Lancet Infect Dis. 2020 May 20:S1473-3099(20)30424-2. PubMed: https://pubmed.gov/32445709. Full-text: https://doi.org/10.1016/S1473-3099(20)30424-2
Should we measure the “viral load”? Unfortunately, there is wide heterogeneity and inconsistency of the standard curves calculated from studies that provided Ct values from serial dilution samples and the estimated viral loads. According to the authors, precautions are needed when interpreting the Ct values of SARS-CoV-2 RT-PCR results shown in COVID-19 publications to avoid misunderstanding of viral load kinetics for comparison across different studies.
Luo X, Zhou W, Yan X, et al. Prognostic value of C-reactive protein in patients with COVID-19. Clin Infect Dis. 2020 May 23:ciaa641. PubMed: https://pubmed.gov/32445579. Full-text: https://doi.org/10.1093/cid/ciaa641
In 359 patients, CRP performed better than other parameters (age, neutrophil count, platelet count) in predicting adverse outcome. Besides, admission serum CRP level was identified as a moderate discriminator of disease severity.
Tom MR, Mina MJ. To Interpret the SARS-CoV-2 Test, Consider the Cycle Threshold Value. Clin Infect Dis. 2020 May 21:ciaa619. PubMed: https://pubmed.gov/32435816. Full-text: https://doi.org/10.1093/cid/ciaa619 l (Important)
A positive RT-qPCR result may not necessarily mean the person is still infectious or that they still have any meaningful disease. The RNA could be from nonviable virus and/or the amount of live virus may be too low for transmission. RT-qPCR provides quantification by first reverse transcribing RNA into DNA, and then performing qPCR where a fluorescence signal increases proportionally to the amount of amplified nucleic acid. The test is positive if the fluorescence reaches a specified threshold within a certain number of PCR cycles (Ct value, inversely related to the viral load). Many qPCR assays use a Ct cut-off of 40, allowing detection of very few starting RNA molecules. The authors suggest to use this Ct value or to calculate viral load which can help to refine decision-making (re: shorter isolation etc.).
Li Y, Zhao K, Wei H, et al. Dynamic Relationship Between D-dimer and COVID-19 Severity. Br J Haematol 2020 May 18. Full-text: https://doi.org/10.1111/bjh.16811
Testing coagulation profile for ten consecutive days since admission in 279 COVID-19 patients, this study gives some insights into the dynamic changes of D-dimer level that are of prognostic value.
Krammer F, Simon V. Serology assays to manage COVID-19. Science 15 May 2020. Full-text: https://doi.org/10.1126/science.abc1227
Nice overview on different platforms, including binding assays such as enzyme-linked immunosorbent assays (ELISAs), lateral flow assays, or Western blot–based assays. In addition, functional assays that test for virus neutralization, enzyme inhibition, or bactericidal assays can also inform on antibody-mediated immune responses. Many caveats and open questions with regard to antibody testing are also discussed.
Teng J, Dai J, Su Y, et al. Detection of IgM and IgG antibodies against SARS-CoV-2 in patients with autoimmune diseases. Lancet Rheumatology 2020, May 18. Full-text: https://www.thelancet.com/journals/lanrhe/article/PIIS2665-9913(20)30128-4/fulltext.
No cross-reactivity between autoantibodies and SARS-CoV-2 antibodies: in 290 older serum samples from patients with rheumatoid arthritis, systemic lupus erythematosus, and Sjogren’s syndrome, no IgG and IgM antibodies against SARS-CoV-2 were detected.
Lv H, Wu, NC, Tsang OT, et al. Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections. Open Access Published: May 17, 2020. Full-text: https://doi.org/10.1016/j.celrep.2020.107725
While cross-reactivity in antibody binding to the spike protein is common, cross-neutralization of the two live SARS viruses may be rare, indicating the presence of a non-neutralizing antibody response to conserved epitopes in the spike.
Gupta S, Parker J, Smits S, Underwood J, Dolwani S. Persistent viral shedding of SARS-CoV-2 in faeces – a rapid review. Colorectal Dis. 2020 May 17. PubMed: https://pubmed.gov/32418307. Full-text: https://doi.org/10.1111/codi.15138
Combining study results of 26 studies, 54% of those patients tested for fecal RNA were positive. Duration of fecal viral shedding ranged from 1 to 33 days after a negative nasopharyngeal swab. Evidence is still insufficient to suggest that COVID-19 is transmitted via fecally shed virus.
Joung J, Ladha A, Saito M, et al. Point-of-care testing for COVID-19 using SHERLOCK diagnostics. Full-text: https://doi.org/10.1101/2020.05.04.20091231.
Point-of-care testing is based on easy-to-use devices to facilitate testing outside laboratory settings. They are eagerly awaited. On May 6, the FDA granted an emergency use authorization for a clustered regularly interspaced short palindromic repeats (CRISPR)-based SARS-CoV-2 fluorescent assay marketed by Sherlock Biosciences. This method gives results in an hour and has successfully diagnosed 12 positive and 5 negative COVID-19 patients, with at least 2 of 3 replicates scoring positive in infected persons. However, use still remains limited to laboratories certified to perform high-complexity tests. On May 6, FDA also authorized Quidel’s Sofia 2 SARS Antigen Fluorescent Immunoassay. The test must be read on a dedicated analyser and detects SARS-CoV-2 nucleocapsid protein from nasopharyngeal swabs in 15 min. According to the manufacturer, the assay demonstrated acceptable clinical sensitivity and detected 47/59 infections (80%). Unfortunately, no peer-reviewed papers have been published to date.
Kucirka LM, Lauer SA, Laeyendecker O, et al. Variation in False-Negative Rate of Reverse Transcriptase Polymerase Chain Reaction–Based SARS-CoV-2 Tests by Time Since Exposure. Annals Int Med 2020, May 13. https://doi.org/10.7326/M20-1495. Full-text: https://www.acpjournals.org/doi/10.7326/M20-1495 l (Important)
The authors estimated the false-negative rate by day since infection, reviewing 7 studies with a total of 1,330 respiratory samples analyzed by RT-PCR. Over the 4 days before symptom onset, the rate decreased from 100% to 67%. On the day of symptom onset (day 5), the rate was 38%, decreased to 20% (day 8) and then began to increase again, from 21% (day 9) to 66% (day 21). If clinical suspicion is high, infection should not be ruled out on the basis of RT-PCR alone. The false-negative rate is lowest 3 days after onset of symptoms, or approximately 8 days after exposure.
Mathur F, Mathur S. Antibody Testing For Covid-19: Can It Be Used As A Screening Tool In Areas With Low Prevalence? American Journal of Clinical Pathology 2020, May 15. Full-text: https://academic.oup.com/ajcp/advance-article/doi/10.1093/ajcp/aqaa082/5837473
Answer is: probably no, because specificity is not 100%. Average sensitivity and specificity of FDA-approved antibody tests is 84.9% and 98.6%, respectively. Given the variable prevalence of COVID-19 (1%-15%) in different places, the positive predictive value can be statistically as low as 30% to 50% in areas with low prevalence.
Amanat F, Stadlbauer D, Strohmeier S, et al. A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat Med. 2020 May 12. PubMed: https://pubmed.gov/32398876. Full-text: https://doi.org/10.1038/s41591-020-0913-5
A simple solution is the use of a binding assay, e.g. an enzyme-linked immunosorbent assay (ELISA), with recombinant antigen as substrate, especially if ELISA results correlate with neutralization assay results. The authors report the development of such an assay and provide a protocol for both recombinant antigen production as well as the ELISA methodology. The method is based on reactivity to the immunogenic S protein of the virus, is relatively simple and quick in its execution and can be performed at biosafety level 2 as it does not involve live virus.
Kirkcaldy RD, King BA, Brooks JT. COVID-19 and Postinfection Immunity: Limited Evidence, Many Remaining Questions. JAMA. 2020 May 11. PubMed: https://pubmed.gov/32391855. Full-text: https://doi.org/10.1001/jama.2020.7869
After reading this viewpoint on the knowledge gaps on post-infection immunity, you will realize that any “COVID pass” would be about as accurate as issuing a certificate that she or he is “a kind person”. J
However, Chile is poised to become the first country to provide COVID passes to people who have recovered from the infection. We’ll see how this works.
Kandemirli SG, Dogan L, Sarikaya ZT, et al. Brain MRI Findings in Patients in the Intensive Care Unit with COVID-19 Infection. Radiology. 2020 May 8:201697. PubMed: https://pubmed.gov/32384020. Full-text: https://doi.org/10.1148/radiol.2020201697
A brain MRI was performed in 27/50 patients with neurologic symptoms. The most common imaging finding was cortical signal abnormalities on FLAIR images (10/27, 37%), accompanied by cortical diffusion restriction or leptomeningeal enhancement. However, the complex clinical course including comorbidities, long ICU stay with multidrug regimens, and respiratory distress with hypoxia episodes can all act as confounding factors – a clear cause-effect relationship between COVID-19 infection and MRI findings will be hard to establish.
Sama IE, Ravera A, Santema BT, et al. Circulating plasma concentrations of angiotensin-converting enzyme 2 in men and women with heart failure and effects of renin–angiotensin–aldosterone inhibitors. European Heart Journal 2020, May 10. Full-text: https://doi.org/10.1093/eurheartj/ehaa373
The first substantial study to examine the association between plasma ACE2 concentrations and the use of RAAS blockers in patients with cardiovascular disease. Authors measured ACE2 concentrations in 1485 men and 537 women with heart failure (index cohort, 11 European countries). Results were validated in 1123 men and 575 women (validation cohort from Scotland). In both cohorts, plasma concentrations of ACE2 were markedly higher in men than in women, but not the use of either an ACE inhibitor or an ARB. Data might explain the higher fatality rate of COVID-19 in men, but do not support the hypothesis that RAAS blockers increase the vulnerability for COVID-19.
Cai XF, Chen J, Hu JL, et al. A Peptide-based Magnetic Chemiluminescence Enzyme Immunoassay for Serological Diagnosis of Coronavirus Disease 2019 (COVID-19). J Infect Dis. 2020 May 8. PubMed: https://pubmed.gov/32382737. Full-text: https://doi.org/10.1093/infdis/jiaa243
A new antibody assay, based on a peptide from the S protein, which was screened out from 20 candidate peptides deduced from the genomic sequence. Using a synthetic peptide may enhance the stability and repeatability of the assay, and theoretically would be more specific. A high specificity was shown. Sensitivity was lower: in 276 infection-confirmed patients, IgG was detected in 71.4% and was higher than the detection rate of IgM (57.2%).
Sethuraman N, Jeremiah SS, Akihide Ryo A, et al. Interpreting Diagnostic Tests for SARS-CoV-2. JAMA May 6, 2020. Full-text: https://jamanetwork.com/journals/jama/fullarticle/2765837
Using available evidence, a clinically useful timeline of diagnostic markers for detection of COVID-19 is devised.
Zhang K, Liu X, Shen J, et al. Clinically Applicable AI System for Accurate Diagnosis, Quantitative Measurements and Prognosis of COVID-19 Pneumonia Using Computed Tomography. Cell April 29. Full-text: https://www.cell.com/cell/fulltext/S0092-8674(20)30551-1
A CT-based artificial intelligence (AI) system was shown to have the potential to assist in the early diagnosis and monitoring of pneumonia. For the classification model, 361,221 CT images from 2,246 patients including 752 NCP, 797 common pneumonia patients and 697 normal control patients were used for training. In brief, the AI system performance was overall superior to that of junior radiologists and comparable to mid-senior radiologists.
Yin L, Moi H, Shao J. Correlation between Heart fatty acid binding protein and severe COVID-19: A case-control study. PLOS One, 29 Apr 2020. https://doi.org/10.1371/journal.pone.0231687
Heart fatty acid-binding protein (HFABP), a serum biomarker for myocardial injury, is highly cardiac-specific. Elevated serum HFABP may be used as an indicator of severe COVID-19. This small retrospective analysis included 45 patients, in which HFABP was measured on the day of hospital admission. In the HFABP positive group (n = 15), severe illness was more common during hospitalization (87.5% vs 40%, p = 0.002).
Long QX, Liu BZ, Deng HJ, et al. Antibody responses to SARS-CoV-2 in patients with COVID-19. Nat Med. 2020 Jun;26(6):845-848. PubMed: https://pubmed.gov/32350462. Full-text: https://doi.org/10.1038/s41591-020-0897-1 ll (Outstanding)
One of the largest studies to date, reporting on acute antibody responses (using magnetic chemiluminescence enzyme immunoassay) in 285 patients (mostly non-severe COVID-19, 39 treated at ICU). Within 19 days after symptom onset, 100% of patients tested positive for antiviral IgG. Seroconversion for IgG and IgM occurred simultaneously or sequentially. Both IgG and IgM titers plateaued within 6 days after seroconversion. The median day of seroconversion for both IgG and IgM was 13 days post-symptom onset. No association between plateau IgG levels and clinical characteristics of the patients was found.
Altman DM, Douek DC, Boyton RJ. What policy makers need to know about COVID-19 protective immunity. Lancet April 27, 2020. Full-text: https://www.thelancet.com/journals/lancet/article/PIIS0140-6736(20)30985-5/fulltext
The bottom line of this comment: we don’t know enough. There is no certainty as to the immunological correlates of antiviral protection or the proportion of the population who must attain them, making it impossible to identify a point when this level of immunity has been reached.
Lipsitch M, Kahn R, Mina MJ. Antibody testing will enhance the power and accuracy of COVID-19-prevention trials. Nature Medicine 27 April 2020. Full-text: https://doi.org/10.1038/s41591-020-0887-3.
Many groups have initiated trials of prophylactic drugs and have envisioned efficacy trials of vaccine candidates. The authors argue for serological testing of trial participants at the start and end of these trials (and at intermediate points), in order to enhance the value and interpretability of these studies.
Qu J, Wu C, Li X. Profile of IgG and IgM antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Clinical Infectious Diseases. 2020. Full-text: https://doi.org/10.1093/cid/ciaa489
Seroconversion patterns of IgM and IgG antibodies using 347 serum samples from 41 patients. Using combined N and S proteins as capture antigen to increase sensitivity of their in-house assay, IgG and IgM antibodies were found in the majority of the patients during the first three weeks of the disease. Of note, the seroconversion time of IgG antibody was earlier than IgM. IgG antibody reached the highest concentration on day 30, while IgM antibody peaked on day 18, and then began to decline.
Torres R, Rinder HM. Double-Edged Spike: Are SARS-CoV-2 Serologic Tests Safe Right Now? Am J Clin Pathol. 2020 Apr 23. PubMed: https://pubmed.gov/32322898. Full-text: https://doi.org/10.1093/ajcp/aqaa071
Brief but excellent review on the pitfalls and problems of antibody tests. At present, a positive antibody result does not guarantee non-infectious status nor immunity. What about asymptomatic or minimally symptomatic persons? The molecular heterogeneity of SARS-CoV-2 subtypes, imperfect performance of available tests and cross-reactivity with seasonal CoVs are discussed.
Xiao AT, Tong YX, Zhang S. Profile of RT-PCR for SARS-CoV-2: a preliminary study from 56 COVID-19 patients. Clin Infect Dis. 2020 Apr 19. PubMed: https://pubmed.gov/32306036. Full-text: https://doi.org/10.1093/cid/ciaa460
The dynamics profile of SARS-CoV-2 shedding from 56 recovered patients. The negative results of RT-PCR test for SARS-CoV-2 (throat or deep nasal cavity swab samples) began to be dominant from week 4 after onset of symptoms and by the end of follow-up (6 weeks), all results of RT-PCR test were negative.
Yongchen Z, Shen H, Wang X, et al. Different longitudinal patterns of nucleic acid and serology testing results based on disease severity of COVID-19 patients. Emerg Microbes Infect. 2020 Apr 20:1-14. PubMed: https://pubmed.gov/32306864. Full-text: https://doi.org/10.1080/22221751.2020.1756699
Do asymptomatic individuals develop antibodies? Serial investigation on 21 individuals from Jiangsu Province, including 17 COVID-19 patients and 5 asymptomatic carriers, using gold immunochromatography assay supplied by Innovita (China). All of 17 symptomatic patients were seropositive by week 6. Only 1/5 asymptomatic cases generated SARS-CoV-2 specific antibody responses within the first 4 weeks.
Zheng S, Fan J, Yu F, et al. Viral load dynamics and disease severity in patients infected with SARS-CoV-2 in Zhejiang province, China, January-March 2020: retrospective cohort study. BMJ. 2020 Apr 21;369:m1443. PubMed: https://pubmed.gov/32317267. Full-text: https://doi.org/10.1136/bmj.m1443
Among 96 consecutively admitted patients (22 mild, 74 severe COVID-19), RNA viral load was measured in 3,497 respiratory, stool, serum, and urine samples. Infection was confirmed in all patients by testing sputum and saliva samples, in the stool of 59% and in the serum of 41%. The median duration of virus in stool (22 days) was significantly longer than in respiratory (18 days, severe cases: 21 days) and serum samples (16 days). However, the main limitation is that RNA PCR cannot distinguish between viable and non-viable virus.
Wilson NM, Norton A, Young FP, Collins DW. Airborne transmission of severe acute respiratory syndrome coronavirus-2 to healthcare workers: a narrative review. Anaesthesia. 2020 Apr 20. PubMed: https://pubmed.gov/32311771. Full-text: https://doi.org/10.1111/anae.15093
Evidence suggestive of airborne spread is growing. Authors discuss several ‘aerosol-generating procedures’ and current evidence (limited). A precautionary approach should be considered to assure healthcare worker safety.
Marty M, Chen K, Verrill KA. How to Obtain a Nasopharyngeal Swab Specimen. NEJM 2020. April 17, 2020. Full-text: https://www.nejm.org/doi/full/10.1056/NEJMvcm2010260?query=featured_home
It’s not that trivial to obtain a NP-swab. Watch this video on protection, preparation, equipment, handling, removing personal protective equipment, etc.
Xiang F, Wang X, He X, et al. Antibody Detection and Dynamic Characteristics in Patients with COVID-19. Clin Infect Dis. 2020 Apr 19. PubMed: https://pubmed.gov/32306047. Full-text: https://doi.org/10.1093/cid/ciaa461 l (Important)
More on antibodies, as a complementary approach for PCR. The seroconversion of specific IgM and IgG antibodies were observed as early as the 4th day after symptom onset. In the confirmed patients with COVID-19, sensitivity, specificity, positive predictive value of IgM were 77.3% (51/66), 100% and 100%, and those of IgG were 83.3% (55/66), 95.0% and 94.8%. Both antibodies performed well in serodiagnosis for COVID-19 and rely on great specificity. The antibodies against SARS-CoV-2 can be detected in the middle and later stage of the illness.
Brief but important comment on several papers reporting on prolonged viral shedding. PCR does not distinguish between infectious virus and non-infectious nucleic acid. This is well known from many viral infections such as Ebola or measles.
Abdalhamid B, Bilder CR, McCutchen EL, Hinrichs SH, Koepsell SA, Iwen PC. Assessment of Specimen Pooling to Conserve SARS CoV-2 Testing Resources. Am J Clin Pathol. 2020 Apr 18. PubMed: https://pubmed.gov/32304208. Full-text: https://doi.org/10.1093/ajcp/aqaa064
Experimental pools were created, mixing positive and negative nasopharyngeal specimens. Results: if the incidence rate of SARS-CoV-2 infection is 10% or less, group testing will result in the saving of reagents and personnel time with an overall increase in testing capability of at least 69%.
Comprehensive review on recent (last two weeks) developments in antibody testing, a very rapidly evolving field of research. Brief overview on promises and perils of different testing systems, including ELISA and lateral flow assays.
Older age is a risk factor for severe disease. This also applies to HCW. In China 11/23 deceased HCWs had been reactivated from retirement. In Italy, most of the 74 doctors who died were in their 60s, and only four were women. This brief letter to BMJ addresses this issue. Author declared the following competing interests: “I am an older, male GP”.
Iwen PC, Stiles KL, Pentella MA. Safety Considerations in the Laboratory Testing of Specimens Suspected or Known to Contain the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Am J Clin Pathol. 2020 Apr 15;153(5):567-570. PubMed: https://pubmed.gov/32190890. Full-text: https://doi.org/10.1093/ajcp/aqaa047
Brief review on laboratory biosafety practices necessary to safely process clinical specimens.
Huang Y, Chen S, Yang Z, et al. SARS-CoV-2 Viral Load in Clinical Samples of Critically Ill Patients. Am J Respir Crit Care Med. 2020 Apr 15. PubMed: https://pubmed.gov/32293905. Full-text: https://doi.org/10.1164/rccm.202003-0572LE
Small study on 16 critically ill patients, demonstrating higher viral load and prolonged shedding in lower respiratory tract specimens, as compared with upper respiratory tract specimens.
Kim H, Hong H, Yoon SH. Diagnostic Performance of CT and Reverse Transcriptase-Polymerase Chain Reaction for Coronavirus Disease 2019: A Meta-Analysis. Radiology. 2020 Apr 17:201343. PubMed: https://pubmed.gov/32301646. Full-text: https://doi.org/10.1148/radiol.2020201343
There is a big debate whether chest CT contributes to COVID-19 diagnosis. Chinese researchers say yes, everyone else says no. This meta-analysis found a high sensitivity but low specificity. In areas with low prevalence, chest CT has a low positive predictive value (1.5-30.7%).
He X, Lau EHY, Wu P, et al. Temporal dynamics in viral shedding and transmissibility of COVID-19. Nat Med. 2020 Apr 15. PubMed: https://pubmed.gov/32296168. Full-text: https://doi.org/10.1038/s41591-020-0869-5 ll (Outstanding)
Important work on viral shedding: this may begin 2 to 3 days before the appearance of the first symptoms and infectiousness profile may more closely resemble that of influenza than that of SARS. Analyzing a total of 414 throat swabs in 94 patients, the highest viral load was found at the time of symptom onset. Infectiousness started from 2.3 days (95% CI, 0.8–3.0 days) before symptom onset and peaked at 0.7 days (95% CI, −0.2–2.0 days) before symptom onset. The authors estimated that 44% (95%CI 25-69%) of secondary cases were infected during the index cases’ presymptomatic stage. Infectiousness was estimated to decline quickly within 7 days.
Raptis CA, Hammer MM, Short RG, et al. Chest CT and Coronavirus Disease (COVID-19): A Critical Review of the Literature to Date. AJR Am J Roentgenol. 2020 Apr 16:1-4. PubMed: https://pubmed.gov/32298149. Full-text: https://doi.org/10.2214/AJR.20.23202
A critical review concluding that current evidence does not substantiate the use of CT as a diagnostic test for COVID-19. At present, CT should be reserved for evaluation of complications of COVID-19 pneumonia or for assessment if alternative diagnoses are suspected.
Song C, Wang Y, Li W, et al. Absence of 2019 Novel Coronavirus in Semen and Testes of COVID-19 Patients. Biol Reprod. 2020 Apr 16. PubMed: https://pubmed.gov/32297920. Full-text: https://doi.org/10.1093/biolre/ioaa050
The virus was not found in the semen of 12 patients recovering from COVID-19 and in a testis sample of one deceased patient.
Scorzolini L, Corpolongo A, Castilletti C, Lalle E, Mariano A, Nicastri E. Comment of the potential risks of sexual and vertical transmission of Covid-19 infection. Clin Infect Dis. 2020 Apr 16. PubMed: https://pubmed.gov/32297915. Full-text: https://doi.org/10.1093/cid/ciaa445
In six women, SARS-CoV-2 was not detected in amniotic fluid, cord blood, neonatal throat swab, or breastmilk samples.
Cheng MP, Papenburg J, Desjardins M, et al. Diagnostic Testing for Severe Acute Respiratory Syndrome-Related Coronavirus-2: A Narrative Review. Ann Intern Med. 2020 Apr 13. PubMed: https://pubmed.gov/32282894. Full-text: https://doi.org/10.7326/M20-1301
Comprehensive review of the current array of tests for SARS-CoV-2, highlighting gaps in current diagnostic capacity, and proposing potential solutions.
Wang X, Yao H, Xu X, et al. Limits of Detection of Six Approved RT-PCR Kits for the Novel SARS-coronavirus-2 (SARS-CoV-2). Clin Chem. 2020 Apr 13. PubMed: https://pubmed.gov/32282874. Full-text: https://doi.org/10.1093/clinchem/hvaa099
Limits of detection of six commercial kits differed substantially (up to 16-fold difference), with the poorest limits likely leading to false-negative results when RT–PCR were used to detect SARS-CoV-2 infection. According to the authors, manufacturers should analyze the existing problems according to the clinical application and further improve their products.
Stam HJ, Stucki G, Bickenbach J. Covid-19 and Post Intensive Care Syndrome: A Call for Action. J Rehabil Med. 2020 Apr 14. PubMed: https://pubmed.gov/32286675. Full-text: https://doi.org/10.2340/16501977-2677
One aftershock of the pandemic will be the huge number of post-intensive care survivors who have been mechanically ventilated and will likely experience short- and medium-term consequences. These patients will require not only adequate screening but early rehabilitation and other interventions.
Guo WL, Jiang Q, Ye F, et al. Effect of throat washings on detection of 2019 novel coronavirus. Clin Infect Dis. 2020 Apr 9. PubMed: https://pubmed.gov/32271374. Full-text: https://doi.org/10.1093/cid/ciaa416
Throat washing may be used for monitoring due to its non-invasiveness and reliability. Throat washing was harvested by asking patients to oscillate over the posterior pharyngeal wall with 20 ml sterile normal saline. After 5-10 seconds, they spit out normal saline from their throat to a sterile container. In 24 paired throat washings and nasopharyngeal swab specimens, the positive testing rate of throat washing was much higher than that of swabs.
Xiao AT, Tong YX, Zhang S. False-negative of RT-PCR and prolonged nucleic acid conversion in COVID-19: Rather than recurrence. J Med Virol. 2020 Apr 9. PubMed: https://pubmed.gov/32270882. Full-text: https://doi.org/10.1002/jmv.25855
Negative does not mean absolutely negative. Among 70 COVID-19 patients, 15 (21%) experienced a “turn positive” of SARS-CoV-2 PCR after two consecutive negative results (up to 45 days after symptom onset).
Xu K, Chen Y, Yuan J, et al. Factors associated with prolonged viral RNA shedding in patients with COVID-19. Clin Infect Dis. 2020 Apr 9. PubMed: https://pubmed.gov/32271376. Full-text: https://doi.org/10.1093/cid/ciaa351
In a cohort of 113 symptomatic patients from two hospitals outside Wuhan, the median duration of SARS-CoV-2 RNA detection was 17 days (IQR, 13-22 days) as measured from illness onset. Male sex, delayed hospital admission, and invasive mechanical ventilation were independent risk factors for prolonged SARS-CoV-2 RNA shedding.
Okba NMA, Muller MA, Li W, et al. Severe Acute Respiratory Syndrome Coronavirus 2-Specific Antibody Responses in Coronavirus Disease 2019 Patients. Emerg Infect Dis. 2020 Apr 8;26(7). PubMed: https://pubmed.gov/32267220. Full-text: https://doi.org/10.3201/eid2607.200841
Small study, demonstrating that most PCR-confirmed SARS-CoV-2-infected persons seroconverted by 2 weeks after disease onset. Sensitivity varied between the assays (IgA ELISA showed higher sensitivity). It remains crucial to calibrate and standardize assays developed by different laboratories by using well-defined standard references as part of diagnostic assay validation.
Yuan J, Kou S, Liang Y, Zeng J, Pan Y, Liu L. PCR Assays Turned Positive in 25 Discharged COVID-19 Patients. Clin Infect Dis. 2020 Apr 8. PubMed: https://pubmed.gov/32266381. Full-text: https://doi.org/10.1093/cid/ciaa398
Among 172 discharged COVID-19 patients, 25 (14.5%) had positive testing again 2 to 13 days after discharge, without aggravation of symptoms. Two negative RT-PCR tests 24 hours apart may not be sufficient for viral clearance evaluation, suggesting the need for additional measures to confirm illness resolution.
Hope MD, Raptis CA, Henry TS. Chest Computed Tomography for Detection of Coronavirus Disease 2019 (COVID-19): Don´t Rush the Science. Ann Intern Med. 2020 Apr 8. PubMed: https://pubmed.gov/32267912. Full-text: https://doi.org/10.7326/M20-1382
Can chest CT be used as a primary tool for detecting COVID-19 in epidemic areas? Some early studies from China said yes. The authors comment that this is a cautionary tale about the consequences of rushing the scientific review process: harsh criticism on faulty design, incomplete methods, biased patient cohorts, confounding and scant discussion, calling into question the broad conclusions that were made in these studies. Bottom line: CT should not be used to screen for or as a first-line test to diagnose COVID-19, all the more considering that performing CT safely is problematic.
Nair A, Rodrigues JCL, Hare S, et al. A British Society of Thoracic Imaging statement: considerations in designing local imaging diagnostic algorithms for the COVID-19 pandemic. Clin Radiol. 2020 May;75(5):329-334. PubMed: https://pubmed.gov/32265036. Full-text: https://doi.org/10.1016/j.crad.2020.03.008
Same issue. The British Society of Thoracic Imaging has explored different scenarios integrating CT into a diagnostic algorithm. Of note, the clinical value, even in the absence of PCR availability, remains unclear. Again: CT can help, but probably not as a tool for diagnosing COVID-19.
Chapman AR, Bularga A, Mills NL. High-Sensitivity Cardiac Troponin Can Be An Ally in the Fight Against COVID-19. Circulation. 2020 Apr 6. PubMed: https://pubmed.gov/32251612. Full-text: https://doi.org/10.1161/CIRCULATIONAHA.120.047008
Nice review on how to use and interpret troponin results in COVID-19 patients. According to the authors, clinicians must recognize that troponin is not a test for myocardial infarction, and it never was. No biomarker has ever had the ability to detect acute atherothrombotic occlusion in a coronary artery. Elevations of cardiac troponin can inform the diagnosis of a number of cardiac conditions related to COVID-19.
Pan Y, Long L, Zhang D, et al. Potential false-negative nucleic acid testing results for Severe Acute Respiratory Syndrome Coronavirus 2 from thermal inactivation of samples with low viral loads. Clin Chem. 2020 Apr 4. PubMed: https://pubmed.gov/32246822. Full-text: https://doi.org/10.1093/clinchem/hvaa091
Don’t put your swabs in the sun! In this small study, all samples were inactivated by incubation in a water bath at 56˚ for 30 minutes. Of note, 7/15 specimens with low virus levels converted into false negative. Longer storage also caused false negative results in a few cases.
Yuan M, Wu NC, Zhu X, et al. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science. 2020 Apr 3. PubMed: https://pubmed.gov/32245784. Full-text: https://doi.org/10.1126/science.abb7269
Insights into antibody recognition and how SARS-CoV-2 can be targeted by the humoral response, revealing a conserved epitope shared between SARS-CoV and SARS-CoV-2. This epitope could be used for vaccines and the development of cross-protective antibodies.
Pan Y, Long L, Zhang D, et al. Potential false-negative nucleic acid testing results for Severe Acute Respiratory Syndrome Coronavirus 2 from thermal inactivation of samples with low viral loads. Clin Chem. 2020 Apr 4. PubMed: https://pubmed.gov/32246822. Full-text: https://doi.org/10.1093/clinchem/hvaa091
Don’t put your swabs in the sun! In this small study, all samples were inactivated by incubation in a water bath at 56˚ for 30 minutes. 7/15 specimens with low virus levels converted to false negative. Longer storage could also cause false-negative results.
Vetrugno L, Bove T, Orso D, et al. Our Italian Experience Using Lung Ultrasound for Identification, Grading and Serial Follow-up of Severity of Lung Involvement for Management of Patients with COVID-19. Echocardiography. 2020 Apr 1. PubMed: https://pubmed.gov/32239532. Full-text: https://doi.org/10.1111/echo.14664
Experience from Italy with lung ultrasound as a bedside tool to improve evaluation of lung involvement, and also reduce the use of chest x-rays and CT. A point scoring system is employed by region and ultrasound pattern.
Brief report about current knowledge and development on antibody testing.
Qiu L, Liu X, Xiao M, et al. SARS-CoV-2 is not detectable in the vaginal fluid of women with severe COVID-19 infection. Clin Infect Dis 2020, April 2, ciaa375, full-text: https://doi.org/10.1093/cid/ciaa375
Is the virus everywhere? No. Not in the vaginal fluid (of 10 women with severe COVID-19).
Saito M, Adachi E, Yamayoshi S, et al. Gargle lavage as a safe and sensitive alternative to swab samples to diagnose COVID-19: a case report in Japan. Clinical Infectious Diseases 2020, April 2, ciaa377, https://doi.org/10.1093/cid/ciaa377
Case report of a patient who did not produce sputum. Gargle lavage testing was sensitive. If confirmed by larger studies, this can be done by patients themselves without putting healthcare professionals at increased risk.
Wölfel R, Corman VM, Guggemos W. et al. Virological assessment of hospitalized patients with COVID-2019. Nature 2020, April 1. Full-text: https://doi.org/10.1038/s41586-020-2196-x ll (Outstanding)
Important work, showing active virus replication in upper respiratory tract tissues (in contrast to SARS). In a detailed virological analysis of nine cases, pharyngeal virus shedding was very high during the first week of symptoms (peak at 7.11 × 108 RNA copies per throat swab, day 4), more than 1000 times higher than seen with SARS-CoV. Infectious virus was readily isolated from throat- and lung-derived samples, but not from stool samples, in spite of high virus RNA concentration. Blood and urine never yielded virus. Shedding of viral RNA from sputum continued after the end of symptoms.
Chen C, Gao G, Xu Y, et al. SARS-CoV-2-Positive Sputum and Feces After Conversion of Pharyngeal Samples in Patients With COVID-19. Ann Intern Med. 2020 Jun 16;172(12):832-834. PubMed: https://pubmed.gov/32227141. Full-text: https://doi.org/10.7326/M20-0991
Among 133 patients, 22 patients who had positive RT-qPCR results for SARS–CoV-2 in the sputum or feces (up to 39 and 13 days, respectively) after pharyngeal swabs became negative. Although uncontrolled, this study raises concern about whether patients with negative pharyngeal swabs are truly virus-free, or sampling of additional body sites is needed.
Yu F, Yan L, Wang N, et al. Quantitative Detection and Viral Load Analysis of SARS-CoV-2 in Infected Patients. Clin Infect Dis. 2020 Mar 28. PubMed: https://pubmed.gov/32221523. Fulltext: https://doi.org/10.1093/cid/ciaa345 ll (Outstanding)
Is sputum sufficient for diagnosis? In a total of 323 samples from 76 pts, the average viral load in sputum (17429 copies/test) was significantly higher than in throat swabs (2552) and nasal swabs (651). Viral load was also higher in the early and progressive stages than in the recovery stage. If these data are confirmed, collection of specimen would be much easier.
Zhao J, Yuan Q, Wang H, et al. Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin Infect Dis. 2020 Mar 28. PubMed: https://pubmed.gov/32221519. Fulltext: https://doi.org/10.1093/cid/ciaa344
More on antibody response. Among 173 patients, the seroconversion rate (median time) for Ab, IgM and IgG was 93.1% (11 days), 82.7% (12 days) and 64.7% (14 days), respectively. A higher titer of Ab was independently associated with a worse clinical classification.