key: cord-333239-nj5dma98 authors: Ducrest, P. J. title: Development and evaluation of a new IgM/IgG rapid diagnostic test for SARS-CoV-2 date: 2020-10-13 journal: nan DOI: 10.1101/2020.10.09.20209866 sha: doc_id: 333239 cord_uid: nj5dma98 There is an urgent need in rapid diagnostic test (RDT) to detect antibodies against SARS-CoV-2. We have developed a rapid and simple point-of-care lateral flow immunoassay (LFIA) detecting IgM and IgG against SARS-CoV-2 in 10 minutes. The aim of this study is to evaluate the diagnostic performance of this RDT. RT-PCR positive plasma samples (n=35) for SARS-CoV-2 and 97 negative control samples were studied. Diagnostic performance of IgG/IgM RDT was assessed using both gold standard RT-PCR and Electro-chemiluminescence immunoassay (ECLIA) Elecsys Anti-SARS-CoV-2 total Ig. Overall, RDT sensitivity was 100% (95% confidence interval [95%CI]: 88-100%) and specificity 93% (95% CI: 85-97%). This IgG/IgM RDT done in plasma displays a high diagnostic accuracy for SARS-CoV-2 IgG/IgM in high COVID-19 prevalence settings. Its use could be considered in the absence of routine diagnostic serology facilities for samples collected between 10 and 180 days after symptoms onset. The seroconversion sequentially appeared for total antibody, IgM and then IgG, with a median time of 11, 12 and 14 days after onset. The majority of antibodies are produced against the most abundant protein of the virus, which is the Nucleocapsid protein (NP). Therefore, tests that detect antibodies to NP would be the most sensitive. However, the receptor-binding domain of Spike protein (RBD-S) is the host attachment protein, and antibodies to RBD-S would be more specific and are expected to be neutralizing. Therefore, according to authors, using one or both antigens would result in high sensitivity 5 . Moreover, the source of protein (prokaryotic vs eukaryotic) as well as the region of the protein (RBD, full-length) and the tag used (His tag or Fc Tag) is a major concern in the diagnostic performance 6 . The aim of this study is to develop and evaluate the performance of a new IgM/IgG rapid diagnostic test (RDT) based on lateral flow assay (LFA) technology, in a high COVID-19 prevalence setting using gold standard RT-PCR as well as an Electro-chemiluminescence immunoassay (ECLIA) Elecsys® Anti-SARS-CoV-2 total Ig (Roche, Switzerland). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209866 doi: medRxiv preprint Anti-human IgM was purchased from Hybridoma Reagent Laboratory Inc. (Baltimore, USA) and The pouched test was opened immediately before use. To perform the test, 10 µL of plasma sample was pipetted into the sample area followed by adding 2-3 drops (60-70 µL) of running buffer (PBS + 1% tween 20 + 1% BSA). The result is interpreted after 10 minutes. Three detection lines are on the stip. The control (C) line appears when sample has flowed through the strip. The presence of anti-SARS-CoV-2 IgM and/or anti-SARS-CoV-2 IgG will be indicated by a red line in the M and/or G region. If only the control line (C) appear, the sample does not contain IgM nor IgG antibodies. If the control line does not appear, the test is invalid, and the test should be repeated (figure 1). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209866 doi: medRxiv preprint plasma samples (n=97), supplied by AbBaltis (Kent, UK) with a collection date before 2018, were used as negative control group. Elecsys® Anti-SARS-CoV-2 total Ig (Roche, Switzerland) uses the nucleocapsid protein of SARS-CoV-2 as antigen. Total Ig was analyzed according to the manufacturer's instructions. The assays were run on Cobas e 601 (Roche, Switzerland) according to the manufacturer's protocol. Positivity is defined by manufacturer as cut-off index (COI) ≥ 1.0. Vassarstats online tool (www.vassarstats.net) was used to calculate sensitivity (SE), specificity (SP), positive and negative predictive values (PPV, NPV), 95% confidence intervals, median, and Interquartile range (IQR); while significance (p-values) was calculated using Mann-Whitney U test for categorical variables. Statistical significance was defined as p < 0.05. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 13, 2020. . The baseline demographic characteristics of patients samples were as follows: the 35 RT-PCR confirmed COVID-19 samples from patients were older (median 50 years old, IQR 34.5-60.5) compared to the healthy plasma samples patients (n=97) (median 33.5 years old, IQR 19-43; p<0.0001). The proportion of females in COVID-19 cohort was 54% (n=19) and 56% (n=54) in healthy control group Among COVID-19 samples, the median delay between symptoms onset and sampling was 21 days (IQR 16-32 days), but not less than 10 days. The longest DPS for one single sample was 180 days. All RT-PCR tests were done in nasopharyngeal secretions. The median CT value was 25.6 (IQR 20.05-29.15). IgG/IgM RDT diagnostic specificity against negative control group with a sampling date before 2018 (n=97) is shown in Table 1 is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 13, 2020. IgG/IgM RDT diagnostic sensitivity against RT-PCR confirmed samples (n=35) is shown in Table 1 . IgG/IgM RDT accuracy against Electro-chemiluminescence immunoassay (ECLIA), Elecsys® Anti-SARS-CoV-2 total Ig (Roche, Switzerland) (n=23) is shown in Table 1 . Both methods revealed similar results in all samples. These resulted in an IgG/IgM RDT sensitivity (SE) of 100% (95% CI: 68-100%), while the PPV was 61% (95% CI: 36-82%) and the NPV 100% (95% CI: 95-100). is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 13, 2020. The key finding of the present evaluation study, using an unmatched case-control design including 73.5% of negative control samples, is that the diagnostic accuracy of IgG/IgM RDT on plasma samples when compared to RT-PCR or ECLIA is the same and displayed a SE of 100%, a SP of 93% and a NPV of 100%. When compared to ECLIA, positive predictive value is of 61% and 83% when compared to RT-PCR due to prevalence. Such performances indicate that this RDT could be fit for purpose in clinical settings where a high prevalence of COVID-19 prevails, especially in situations where ECLIA are not available, or cannot be reliably used. Diagnostic performances in low prevalence populations still needs to be determined and larger populations need to be tested. In addition, it is clear that serological assay for SARS-CoV-2 antibodies need to be performed at least 10 to 15 days after symptoms onset 7, 8 . Finally, here we used plasma and the test was performed in a laboratory environment; we may expect different results in reallife at patients' bed and using capillary blood. The second notable finding of this study lies in the fact that IgG and IgM seropositivity is still present 180 days after symptoms onset even with normal antibody decline 9 . To our knowledge, it is the first time that SARS-CoV-2 IgG and IgM seropositivity is demonstrated with RDT 180 DPS. This study indicates also that SARS-CoV-2 IgG/IgM are present constantly and with enough concentration to be detectable with RDT, from 15 days to at least 180 days post symptoms. This finding applies to the this RDT only and must not be applied to other RDTs currently available. It is also necessary to quantitatively determine the level of IgG/IgM 180 days after symptoms onset to confirm this finding obtained with a qualitative assay. is the author/funder, who has granted medRxiv a license to display the preprint in (which was not certified by peer review) preprint The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20209866 doi: medRxiv preprint In addition, the test provided clear results, without indeterminate or invalid results (no Control line). There are several limitations to this study. First, we present here the results of a method evaluation study and not a seroprevalence study. Therefore, the PPV obtained here (based on a 26.5% proportion of cases defined as laboratory confirmed SARS-CoV-2 by RT-PCR) will probably be lower in a low prevalence setting. Nevertheless, our results also showed that when targeting a population 10 days after symptoms onset, sensitivity and NPV reach high diagnostic performances (100%). Although not fully established here, we observed that the RDT results were consistent with the other automated serological test (ECLIA) with similar analytical sensitivity. Another limitation of this validation study lies in its limited sample size leading to broad 95% confidence intervals, requiring confirmation of these data at a larger scale. Finally, our present conclusions only apply to this RDT, and must not be applied to any other RDTs currently available. In conclusion, this RDT is not meant to replace a SARS-CoV-2 RT-PCR diagnostic test in the first week of the disease, but could be a reliable option for assessing the SARS-CoV-2 serology in moderate to high COVID-19 prevalence settings, especially in situations where automated ECLIA or ELISA are not available, with samples collected at least 10 days after symptoms onset and up to 180 days after the onset of symptoms. Further investigations in low prevalence situations and using capillary blood are necessary. PJD is the CEO of GaDia SA, the developer and manufacturer of this RDT. This study was funded by GaDia SA. COVID-19 (Novel Coronavirus 2019) -recent trends Pneumonia outbreak associated with a new coronavirus of probable bat origin Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease Interpreting Diagnostic Tests for SARS-CoV-2 Serological detection of 2019-nCoV respond to the epidemic: A useful complement to nucleic acid testing Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: an observational cohort study Antibody responses to SARS-CoV-2 in patients with COVID-19 Longitudinal evaluation and decline of antibody responses in SARS-CoV-2 infection