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Background Rapid increase in COVID-19 suspected cases has rendered disease diagnosis challenging, mainly depending upon RT-qPCR. Reliable, rapid, and cost-effective diagnostic assays that complement RT-qPCR should be introduced after thoroughly evaluating their performance upon various disease phases, viral load, and sample storage conditions. Objective We investigated the correlation of cycle threshold (Ct) value, which implies the viral load and infection phase, and the storage condition of the clinical specimen with the diagnosis of SARS-CoV-2 through our newly developed in-house rapid enzyme-linked immunosorbent assay (ELISA) system. Method Naso-oropharyngeal samples of 339 COVID-19 suspected cases were collected and evaluated through RT-qPCR that were stored up to 30 days in different conditions (i.e. −80°C, −20°C and initially at 4°C followed by −80°C). The clinical specimens were evaluated with our in-house ELISA system after finalizing the assay method through checkerboard assay and minimizing the signal/noise ratio. Result The ELISA system showed the highest sensitivity (92.9%) for samples with Ct ≤30 and preserving at −80°C temperature. The sensitivity reduced proportionally with increasing Ct value and preserving temperature. However, the specificity ranged between 98.3% and 100%. Conclusion The results indicate the necessity of early infection phase diagnosis and lower temperature preservation of samples to perform rapid antigen ELISA tests.

Bijon Kumar Sil,1 Mohd Raeed Jamiruddin,2 Md Ahsanul Haq,1 Mohib Ullah Khondoker,3 Nowshin Jahan,1 Shahad Saif Khandker,1 Tamanna Ali,1 Mumtarin Jannat Oishee,1 Taku Kaitsuka,4 Masayasu Mie,5 Kazuhito Tomizawa,6 Eiry Kobatake,5 Mainul Haque,7 Nihad Adnan8 1Gonoshasthaya-RNA Molecular Diagnostic and Research Center, Dhaka, 1205, Bangladesh; 2Department of Pharmacy, BRAC University, Dhaka, 1212, Bangladesh; 3Gonoshasthaya Samaj Vittik Medical College, Savar, Dhaka, 1344, Bangladesh; 4School of Pharmacy, International University of Health and Welfare, Okawa, Fukuoka, 831-8501, Japan; 5School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Kanagawa, 226-8502, Japan; 6Department of Molecular Physiology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, 860-0811, Japan; 7The Unit of Pharmacology, Faculty of Medicine and Defence Health Universiti Pertahanan, Nasional Malaysia (National Defence University of Malaysia), Kuala Lumpur, 57000, Malaysia; 8Department of Microbiology, Jahangirnagar University, Savar, Dhaka, 1342, BangladeshCorrespondence: Nihad AdnanDepartment of Microbiology, Jahangirnagar University, Savar, Dhaka, 1342, BangladeshTel +8801705709910Email nihad@juniv.eduMainul HaqueThe Unit of Pharmacology, Faculty of Medicine and Defence Health, Universiti Pertahanan, Nasional Malaysia (National Defence University of Malaysia), Kem Perdana Sungai Besi, Kuala Lumpur, 57000, MalaysiaTel +60109265543Email runurono@gmail.comBackground: Serological tests detecting severe acute respiratory syndrome coronavirus− 2 (SARS-CoV-2) are widely used in seroprevalence studies and evaluating the efficacy of the vaccination program. Some of the widely used serological testing techniques are enzyme-linked immune-sorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and lateral flow immunoassay (LFIA). However, these tests are plagued with low sensitivity or specificity, time-consuming, labor-intensive, and expensive. We developed a serological test implementing flow-through dot-blot assay (FT-DBA) for SARS-CoV-2 specific IgG detection, which provides enhanced sensitivity and specificity while being quick to perform and easy to use.Methods: SARS-CoV-2 antigens were immobilized on nitrocellulose membrane to capture human IgG, which was then detected with anti-human IgG conjugated gold nanoparticle (hIgG-AuNP). A total of 181 samples were analyzed in-house. Within which 35 were further evaluated in US FDA-approved CLIA Elecsys SARS-CoV-2 assay. The positive panel consisted of RT-qPCR positive samples from patients with both < 14 days and > 14 days from the onset of clinical symptoms. The negative panel contained samples collected from the pre-pandemic era dengue patients and healthy donors during the pandemic. Moreover, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of FT-DBA were evaluated against RT-qPCR positive sera. However, the overall efficacies were assessed with sera that seroconverted against either nucleocapsid (NCP) or receptor-binding domain (RBD).Results: In-house ELISA selected a total of 81 true seropositive and 100 seronegative samples. The sensitivity of samples with < 14 days using FT-DBA was 94.7%, increasing to 100% for samples > 14 days. The overall detection sensitivity and specificity were 98.8% and 98%, respectively, whereas the overall PPV and NPV were 99.6% and 99%. Moreover, comparative analysis between in-house ELISA assays and FT-DBA revealed clinical agreement of Cohen’s Kappa value of 0.944. The FT-DBA showed sensitivity and specificity of 100% when compared with commercial CLIA kits.Conclusion: The assay can confirm past SARS-CoV-2 infection with high accuracy within 2 minutes compared to commercial CLIA or in-house ELISA. It can help track SARS-CoV-2 disease progression, population screening, and vaccination response. The ease of use of the assay without requiring any instruments while being semi-quantitative provides the avenue of its implementation in remote areas around the globe, where conventional serodiagnosis is not feasible.Keywords: COVID-19, SARS-CoV-2, dot-blot immunoassay, AuNP, gold nanoparticle, serosurveillance, nucleocapsid, receptor binding domain

Background Vaccination with the Oxford-AstraZeneca COVID-19 vaccine (AZD1222) initially started in the UK and quickly implemented around the Globe, including Bangladesh. Up to date, more than nine million doses administrated to the Bangladeshi public. Method Herein, we studied the antibody response to the first dose of AZD1222 in 86 Bangladeshi individuals using in-house ELISA kits. Study subjects were categorized into two groups, convalescent and uninfected, based on prior infection history and SARS-CoV-2 nucleocapsid-IgG profiles. Results All the convalescent individuals presented elevated spike-1-IgG compared to 90% of uninfected ones after the first dose. Day >28 post-vaccination, the convalescent group showed six times higher antibody titer than the uninfected ones. The most elevated antibody titers for the former and later group were found at Day 14 and Days >28 post-vaccination, respectively. The spike-1-IgA titer showed a similar pattern as spike-1-IgG, although in a low-titer. In contrast, the IgM titer did not show any significant change in either group. Conclusion High antibody titer in the convalescent group, signify the importance of the first dose among the uninfected group. This study advocates the integration of antibody tests in vaccination programs in the healthcare system for maximizing benefit.