key: cord-0761797-edt4mism
authors: Oliveira, B. A.; Oliveira, L. C. d.; Oliveira, F. M. d.; Pereira, G. M.; Souza, R. M. d.; Manuli, E. R.; Marchini, F. K.; Espinoza, E. P. S.; Park, M.; Taniguchi, L.; Mendes, P. V.; Franco, L. A. M.; Nastri, A. C.; Oliveira, M. S. d.; Vieira Junior, J. M.; Kallas, E. G.; Levin, A. S.; Sabino, E. C.; Costa, S. F.
title: EVALUATION OF ELEVEN IMMUNOCHROMATOGRAPHIC ASSAYS FOR SARS-CoV-2 DETECTION: INVESTIGATING DENGUE CROSS-REACTION
date: 2020-10-13
journal: nan
DOI: 10.1101/2020.10.09.20210039
sha: 7535a0fd8bb6608752100b6d43b339b2e71f1ce9
doc_id: 761797
cord_uid: edt4mism

Background: COVID-19 disease (Coronavirus disease 2019) caused by SARS-CoV-2 (Severe acute respiratory syndrome coronavirus 2) is widespread worldwide, affecting more than 11 million people globally (July 6th, 2020). Diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and low cost alternative for monitoring the spread of COVID-19 in the population. Methods: Here we evaluate the sensitivity and specificity of eleven different immunochromatografic tests in 98 serum samples from confirmed cases of COVID-19 through RT-PCR and 100 negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using 20 serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests. Results: Our results demonstrated agreement between immunochromatographic assays and RT-PCR, especially after 10 days since the onset of symptoms. The evaluation of IgG and IgM antibodies combined demonstrated a strong level of agreement (0.85) of IC assays and RT-PCR. It was observed cross-reactivity between Dengue and COVID-19 using four different IC assays for COVID-19 diagnosis. The specificity of IC assays to detected COVID-19 IgM antibodies using Dengue serum samples varied from 80% to 85%; the specificity of IgG detection was 100% and total antibody was 95%. Conclusions: We found high sensitivity, specificity and good agreement of IC assays, especially after 10 days onset of symptoms. However, we detected cross-reactivity between Dengue and COVID-19 mainly with IgM antibodies demonstrating the need for better studies about diagnostic techniques for these diseases.

Health Organization (July 6 th , 2020) [1] [2] [3] [4] [5] .

The COVID-19 diagnosis is based on clinical and epidemiological features, image exams and finally the analysis of nucleic acids through reverse-transcription polymerase chain reaction (RT-PCR), established as the gold standard for COVID-19 disease [1, 2, 6] .

However, the accuracy of this method depends on the viral load on the collection site and on time from symptom onset. It presents limitations such as incorrect collection and processing of samples, the need of expensive equipment and reagents, trained operators and the delay in releasing the results [1, 2, 6, 7] .

The immunochromatographic (IC) assay is a feasible and low-cost alternative for monitoring the spread of COVID-19 in the population in general and specially in low and middle-income countries which lack of structure. Contact-tracing, population survey including health workers, teachers and students on resuming classes are situations that may be useful to use serology since the sensitivity of RT-PCR in asymptomatic is low and ranges from 8 to 10% [1, 2] .

Likewise COVID-19, Dengue is also an emerging disease, especially in tropical and subtropical countries transmitted by Aedes mosquito, and both diseases are similar regarding clinical and laboratory features hampering the diagnosis [8] [9] [10] . Another matter is the temporal dynamic of both diseases in Brazil. The spread of Dengue usually increases between March and April due the rainy seasons, the same period when respiratory diseases are most common, and period in which the number of COVID-19 confirmed cases started to rise [10] .

Despite the high number of IC assays available, the readiness to perform the diagnosis and advantages involving its use, here we evaluate the sensitivity and specificity of eleven different IC assays in serum samples from confirmed cases of COVID-19 through RT-PCR and negative serum samples from blood donors collected in February 2019. Considering the endemic situation of Dengue in Brazil, we also evaluated the cross-reactivity with Dengue using serum samples from patients with confirmed diagnosis for Dengue collected in early 2019 through four different tests.

This is a prospective multicenter study of COVID-19 hospitalized patients at two Brazilian Hospitals: Hospital das Clínicas da Faculdade de Medicina da Universidade de São All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20210039 doi: medRxiv preprint Paulo (HC-FMUSP), a public teaching hospital with 2,000 beds; and Hospital Sírio-Libanes (HSL), a private 400-bed hospital. Both hospitals are located in Sao Paulo.

This study was approved by the Brazilian national ethics review board (CONEP), protocol number 30701920200000068.

The positive serum samples used in this study were collected in April at HSL and HC- perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20210039 doi: medRxiv preprint according to manufacturer instructions. In addition, 40 samples from COVID-19 confirmed cases were evaluated using PANBIO DENGUE IgM CAPTURE ELISA (Abbott, USAbatch 01P20E014) in which 31 of them were collected after 10 days of symptoms. We also evaluated the cross-reactivity using IC assays for detection of antibodies against Dengue in 33 samples of COVID-19 confirmed cases using ALERE DENGUE DUO-NS1 IgG and IgM (Abbott, USAbatch 11DDE008A-A and 11DDE007A-A).

The validity of tests was measured through sensitivity (true positive / true positive + false negative = %) and specificity (true negative / true negative + false positive = %) calculations [11] . The concordance analysis was performed by the Fleiss's Kappa method comparing the agreement between the gold standard (RT-PCR) and each one of the eleventested IC assays, the confidence interval was 95%.

Altogether 198 serum samples were collected for this study, among them, 98 samples were collected from confirmed COVID-19 patients through RT-PCR, 74 of these patients' samples were collected before the 10 th day since the onset of symptoms and 24 samples were collected after 10 days since the beginning of symptoms. In addition, as negative control were used 100 samples collected from blood donors on early 2019. The sensitivity and specificity as well as agreement ratio of eleven commercial qualitative IC assays were evaluated; the results were obtained through tests performed between May/2020 and July/2020.

A panel containing 74 positive samples and 100 negative samples was used to evaluate the sensibility of eleven IC tests. As summarized in table 2, among the tests, ten of them describe separately the detection of IgG and IgM antibodies and one of them Wondfo (China) evidence the presence of total immunoglobulins. The greater sensitivity for detection of only IgG antibody among ten IC tests was observed in TBG (Taiwan) IC test at 93.3%, followed by Ecotest (Brazil) and Lepu (China) IC test at 90%, Bioclin (Brazil) at 81%, MedTest (China) and Thermogenesis (China) presented equivalent results (80%), followed by MedNet (China), Advagen (Brazil) and Luxus (China) at 77.5%, 75.2% and 75% respectively. The Camtech (Singapore) presented the lower sensitivity among the tests at 70%.

The sensitivity for detection of only IgM antibody was also evaluated, the better result was achieve by MedTest (China) at 93.3%, followed by Ecotest (Brazil) and Lepu (China) at 90%. The Camtech (Singapore) presented 87.5% of sensitivity while TBG (Taiwan), Thermogenesis (China) and MedNet (China) achieved identical sensitivities of 80%. Luxus (China) sensibility was 72.5% followed by Bioclin (Brazil) at 61% and finally Advagen (Brazil) All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20210039 doi: medRxiv preprint at 35.5%. The evaluation of total immunoglobulins presented a sensitivity of 76.7% as observed in Wondfo test (China).

The assay specificity was evaluated according to table 2. In IgG IC assays the specificity was equal (100%) in all the tests except for MedNet (China) at 97.5%. In the evaluation of IgM detection the assays sensitivity was 100% for Ecotest (Brazil), Lepu perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. In the analyze of immunochrographic tests for COVID-19 diagnosis Advagen (Brazil) and Camtech (Singapore) reached 100% specificity for IgG antibody detection using 20 dengue samples while MedNet (China) achieved 95%. In IgM detection the specificity was 85% for Advagen (Brazil) and Camtech (Singapore), MedNet (China) reached 80% specificity while Wondfo specificity was 95% for detection of total antibodies as described in table 4.

In this study, we evaluated eleven IC colloidal-gold qualitative based assays and analyzed the quality measurements of each one of them. According to our results, most of IC assays provide high sensitivity and specificity compared to the gold standard RT-PCR, especially after 10 days since the onset of disease with an excellent agreement ratio compared to the gold standard using Wondfo test evaluating total antibodies against SARS-CoV-2.

The COVID-19 pandemic impacted the world health system in an unprecedented way. In Brazil the first reported case occurred on February 27 th , 2020, since them more than 1 million Brazilian people tested positive for COVID-19 [4, 12] . Besides COVID-19, Brazil also faces an endemic situation regarding to Dengue, and considering the importance of both diseases, here we evaluated the cross-reactivity of Dengue and COVID-19 using different tests.

According to our results, the specificity in detection of IgG antibodies was greater in comparison with IgM detection using IC assays for detection of COVID-19 in Dengue patient's samples. It is widely known that before the high-affinity response by IgG antibodies to pathogens the first defense provided by organism occurs through IgM molecules [13]. Our results suggest that the evaluation of total antibodies (IgG and IgM) is a useful tool to broaden the range of detection, enabling the evaluation of acute phase through IgM detection and convalescent phase by the presence of IgG. The evaluation of combined antibodies helps not All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20210039 doi: medRxiv preprint only the discrimination of Dengue and COVID-19 but also avoid false negative results for COVID-19 and contributes to the establishment of control measures. On the other hand, the use of IC assays that detect IgM may have cross-reaction with Dengue and lead to a false positive result that might impact on the clinical management of the patient.

The IC assay is based on the detection of antibodies secreted by B lymphocytes against the viral pathogens using serum, plasma or whole blood samples from patients and a specific buffer on an immunochromatographic cassette [1, 2, 14, 15] . The cassette is composed by plastic backing involving the strip where reaction occurs, sample pad for sample addition, conjugated pad containing antigens bound to colloidal nanoparticles, absorbent pad for waste reservoir, and the nitrocellulose membrane in which by capillary attraction the antigenantibody reaction occurs, evidenced through a colored band (red color for colloidal-gold nanoparticles and blue color for colloidal selenium nanoparticles) in about 15 to 20 minutes [1, 2, 14, 15] .

Immunochromatographic is an easy and affordable method for diagnosis enabling prompt results in large scale. According to our results, the agreement ratio for detection of only IgG antibodies before the 10º day of symptom using IC technique in comparison with RT-PCR was moderate in the majority of used tests (50%), followed by strong agreement (40%) and weak agreement (10%). In contrast, for only IgM detection the agreement ratio between IC and RT-PCR was strong in the majority of tests (40%) followed by a moderate (30%), weak (20%) and minimal (10%) agreement. The evaluation of total antibodies using Wondfo test demonstrated better results using samples collected after the 10º day of symptoms (almost perfect agreement) than before this period (moderate agreement) compared to RT-PCR, the increase in sensitivity was also observed using MedNet test in samples after 10 days since the onset of symptoms.

In the literature, it is possible to find the detection of immunoglobulin on the 5 th day since the onset of disease. However, this detection is greater from the 8 th day on [15] . Our results are in accordance with literature, demonstrating an increase in sensitivity levels especially using the combined assessment of IgG and IgM antibodies.

Our study has limitations such as sample size; however, to our knowledge it was the first study that analyzed several SARS-CoV-2 IC tests and cross-reaction with Dengue. Thus, our findings can be useful in countries with high prevalence of Dengue to alert to the possible cross-reactivity of the SARS-CoV-2 IC assays, mainly IgM, with Dengue antibodies.

We found in general high sensitivity and specificity and good agreement of IC assays, especially after 10 days since the onset of symptoms. Our results also evidenced the importance in evaluating total immunoglobulins to increase sensitivity and specificity of IC assays. On the other hand, we detected cross-reactivity between Dengue and COVID-19 demonstrating the need for better studies and improvements in diagnostic techniques for these diseases.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 13, 2020. . https://doi.org/10.1101/2020.10.09.20210039 doi: medRxiv preprint

In this study, a small number of samples was tested due to the availability of tests that we had at the beginning of COVID-19 in Brazil. Studies with more samples are needed to ensure validity of the results. Both authors contributed equally in the development of the study.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in 

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