key: cord-0757071-85b4lwh3 authors: Prazuck, T.; Colin, M.; Giache, S.; Gubavu, C.; Seve, A.; Rzepecki, V.; Chevereau-Choquet, M.; Kiani, C.; Rodi, V.; Lyonnet, E.; Courtellemont, L.; Guinard, J.; Pialoux, G.; Hocqueloux, L. title: Evaluation of performance of two SARS-CoV-2 Rapid whole-blood finger-stick IgM-IgGCombined Antibody Tests date: 2020-05-27 journal: nan DOI: 10.1101/2020.05.27.20112888 sha: 37cda5d8d37f0e8f060fd661274d2fb9df4dc145 doc_id: 757071 cord_uid: 85b4lwh3 Background The SARS-CoV-2 virus is responsible for the infectious respiratory disease called COVID-19 (COronaVIrus Disease). In response to the growing COVID-19 pandemic, Rapid Diagnostic Tests (RDTs) have been developed to detect specific antibodies, IgG and IgM, to SARS-CoV-2 virus in human whole blood. We conducted a real-life study to evaluate the performance of two RDTs, COVID-PRESTO and COVID-DUO, compared to the gold standard, RT-PCR. Methods RT-PCR testing of SARS-Cov-2 was performed from nasopharyngeal swab specimens collected in adult patients visiting the infectious disease department at the hospital (Orleans, France). Fingertip whole blood samples taken at different time points after onset of the disease were tested with RDTs. The specificity and sensitivity of the rapid test kits compared to test of reference (RT-PCR) were calculated. Results Among 381 patients with symptoms of COVID-19 who went to the hospital for a diagnostic, 143 patients were RT-PCR negative. Results of test with RDTs were all negative for these patients, indicating a specificity of 100% for both RDTs. In the RT-PCR positive subgroup (n=238), 133 patients were tested with COVID-PRESTO and 129 patients were tested with COVID-DUO (24 patients tested with both). The further the onset of symptoms was from the date of collection, the greater the sensitivity. The sensitivity of COVID-PRESTO test ranged from 10.00% for patients having experienced their 1st symptoms from 0 to 5 days ago to 100% in patients where symptoms had occurred more than 15 days before the date of tests. For COVID-DUO test, the sensitivity ranged from 35.71% [0-5 days] to 100% (> 15 days). Conclusion COVID-PRESTO and DUO RDTs turned out to be very specific (none false positive) and to be sensitive enough after 15 days from onset of symptom. These easy to use IgG/IgM combined test kits are the first ones allowing a screening with capillary blood sample, by typing from a finger prick. These rapid tests are particularly interesting for screening in low resource settings. At the end of 2019, a pneumonia of unknown cause detected in Wuhan, China was first 61 reported to the WHO Country Office in China. On January 9 th , 2020, the Chinese health 62 authorities and the World Health Organization (WHO) officially announced the discovery of a 63 novel coronavirus, first named 2019-nCoV, then officially termed SARS-CoV-2. This virus, 64 belonging to the coronavirus family, differs from the viruses SARS-CoV, responsible for the 65 SARS outbreak in 2003, and MERS-CoV, responsible for an ongoing outbreak that began in 66 2012 in the Middle East. 67 The SARS-CoV-2 virus is responsible for the infectious respiratory disease called COVID-19 68 (COronaVIrus Disease). This infection mainly results in pneumonia and upper/lower 69 respiratory tract infection. The symptoms of COVID-19 infection appear after an incubation 70 period of approximately 5.2 days [1] . The most common symptoms at onset of COVID-19 71 illness are fever, cough, and fatigue, but others include headache, sore throat, and even acute 72 respiratory distress syndrome, leading to respiratory failure. 73 Since the emergence of COVID-19 in China at the end of last year, the SARS-CoV-2 virus has 74 caused a large global outbreak and has become a major worldwide public health issue. The 75 WHO has declared this outbreak a global health emergency at the end of January 2020. On 76 April 12 th , 2020, the World Health Organization (WHO) announced that the total global deaths 77 from COVID-19 has surpassed 100 000. Globally, by April 28 th , 2020, 2,892,688 cases of 78 COVID-19 have been confirmed and 210,193 patients have died. An estimated 1.7 billion 79 people have been ordered to remain at home as governments take extreme measures to 80 protect their populations. 81 Due to the rapid spread and increasing number of COVID-19 cases caused by this new 82 coronavirus SARS-CoV-2, rapid and accurate detection of virus and/or disease is increasingly 83 vital to control the sources of infection and prevent the progression of the disease. 84 Besides the main priority, which is finding an efficient treatment, one of the most important 85 research questions targets the diagnosis of COVID-19. Currently, the real-time RT-PCR assay 86 is the gold-standard method to detect SARS- CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 27, 2020. . https://doi.org/10.1101/2020.05.27.20112888 doi: medRxiv preprint The study was approved by the local Ethics Committee on March 17 th 2020, and informed 103 consent was obtained from each participant. 104 The study population consisted of adult patients visiting the infectious disease department 106 (Centre Hospitalier Regional Orléans, France) from March, 18 th , 2020 to April 10 th , 2020. This 107 department receives patients whose symptoms, such as headache, fatigue, fever or 108 respiratory signs suggest a COVID infection, and for whom a diagnosis is requested. Date of 109 onset of symptoms as declared by the patient and age were collected at inclusion. According 110 to severity of disease, patients RT-PCR positive were either hospitalized in the infectious 111 diseases ward, only devoted to treat COVID-19 infected patients, or invited to have regular 112 medical visits in the outpatient consultation. Fingertip blood samples were performed at various 113 stages of the follow-up, even after clinical cure, in order to collect samples from convalescent 114 patients. 115 Nasopharyngeal (NP) swab specimens were collected from patients by trained surveillance 117 officers. A polyester-tipped flexible aluminum-shafted applicator (Microtest M4RT, Remel) was 118 inserted into one of the nostrils until resistance was felt at the nasopharynx, then rotated 180 119 degrees and withdrawn. After swabbing, the swab applicator was cut off, and each absorbent 120 swab was placed into a vial containing 3 mL of viral transport media. Vials were immediately 121 shipped via a triple packaging system to the virology unit located in the same building of the 122 hospital, then stored if necessary at 4°C for up to 24 hours until testing. 123 For whole blood samples taken at the fingertip, a lancet was used to prick the side of the 124 fingertip to let a large drop of suspended blood form. This blood sample was collected with a 125 10 µl capillary micropipette that filled automatically. The sample was then expelled by 126 squeezing the micropipette bulb to deposit the blood on the appropriate well of the test 127 cassette. Retesting was performed in a same patient only if the previous test was negative. 128 RT-PCR testing of SARS-CoV-2 was performed in Unit of Virology, CHR Orléans. Nucleic acid 130 extraction was performed with automated EZ1 (Qiagen The SARS-CoV-2 IgG/IgM antibody test kits, COVID-PRESTO ® and COVID-DUO ® , are 137 targeting on the antibodies specific to N-protein of SARS-CoV-2. They are manufactured and 138 marketed by AAZ-LMB. 139 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2020. . https://doi.org/10.1101/2020.05.27.20112888 doi: medRxiv preprint Tests were conducted at the site by clinical staff, physicians or nurses, according to 140 manufacturers' instructions. Health workers involved in the study received a two-hours training 141 session for each type of test prior to the beginning of the study. 142 Both COVID-PRESTO ® and COVID-DUO ® are lateral flow immune-chromatographic assays 143 (Figs 1 and 2) . These tests use anti-human IgM antibody (test line IgM), anti-human IgG 144 antibody (test line IgG) and rabbit IgG (control line C) immobilized on a nitrocellulose strip. The 145 Conjugate (recombinant COVID-19 antigens labeled with colloidal gold) is also integrated into 146 the strip. When a specimen is added to the sample well, followed by assay buffer, IgM and IgG 147 antibodies, if present, will bind to COVID-19 conjugates forming an antigen-antibodies 148 complex. This complex migrates through nitrocellulose membrane by capillary action. When the complex 150 meets the line of the corresponding immobilized antibody (anti-human IgM and/or anti-human 151 IgG), the complex is trapped, forming a burgundy colored band which confirms a reactive test 152 result. The result has to be read within 10 minutes by two independent operators. When the 153 control line is the only to be burgundy, the sample is negative. If the control line does not 154 appear, the test is invalid and should be repeated with a new cassette. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2020. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2020. . https://doi.org/10.1101/2020.05.27.20112888 doi: medRxiv preprint Overall, 381 patients with symptoms of COVID-19 who went to the hospital for a diagnostic, 173 were included in the study. 174 RT-PCR was performed in all patients: 62.47% were positive (n=238). Based on these results, 175 two sub-groups were defined: 143 patients with negative and 238 patients with positive RT-176 PCR results (Fig 3) . Interestingly, among patient with samples collected at two different times, an elderly woman, 193 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2020. When considering the distribution of IgM+ and IgG+ patterns among patients with a positive 208 RDT test, the IgM were the first antibodies to be detected and were systematically present in 209 the few positive patients with an onset of symptoms from 0 to 5 days ago (n=2 in COVID-210 PRESTO ® population; n=5 in COVID-DUO ® ). The IgM appeared first and stayed prevalent until 211 15 days after viral infection then IgG became more frequent (Figs 4 and 5) . 212 213 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2020. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2020. to evaluate the sensitivity of RDTs was only based on positive RT-PCR results, and not a mix 261 between syndromes, imagining findings and RNA detection. 262 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 27, 2020. . https://doi.org/10.1101/2020.05.27.20112888 doi: medRxiv preprint The results of the present study highlight two major points. Firstly, as for the assessed RDT, 263 the sensitivity of ELISA tests increases with the duration from symptom onset. Moreover, we 264 showed that direct antibody typing with whole blood is as sensitive as immunoassay performed 265 with serum in a retrospective way. Secondly, these diagnostic tests (either qualitative or 266 quantitative) can help to diagnose a past infection after elimination of the virus by the immune 267 system. Thus, combining RT-PCR and antibody detection allows to largely diagnose COVID-268 19 people regardless of the delay between infection and diagnosis. 269 Currently, the extent and the time kinetics of humoral response against SARS-CoV-2 are not 270 known. It is widely accepted that IgM is usually the first responded antibody providing the first 271 line of defense during viral infections, prior to the generation of adaptive, high affinity IgG 272 responses serving as the more robust long term immunity. We were not able to study the 273 humoral response at the individual level because too few patients could have been tested more 274 than once. At the population level, the patterns of IgM/IgG results obtained for positive tests 275 with COVID-DUO ® made it possible to perceive the switch between the first production of IgM 276 and the later onset of IgG. This coincided with our observations with the COVID-PRESTO ® . determine whether a person has already been infected by SARS-CoV-2. Serologic tests will 317 be needed to assess the response to vaccine candidates and to map levels of immunity in 318 communities. These rapid tests are particularly interesting for low resource settings such as at 319 the bedside or any other locations where lab tests are less obvious. 320 321 322 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 27, 2020. The authors report no conflicts of interest. The authors alone are responsible for the content 336 and the writing of the paper. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 27, 2020. . https://doi.org/10.1101/2020.05.27.20112888 doi: medRxiv preprint The epidemiology and pathogenesis of coronavirus disease 344 (COVID-19) outbreak Severe acute respiratory syndrome-related coronavirus : The species and its viruses -a 348 statement of the Coronavirus Study Group. Microbiology Advice on the use of point-of-care immunodiagnostic 351 tests for COVID-19. 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