Long-term validation of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for the rapid detection of SARS-CoV-2 from March 2020 to October 2021 in Central Africa, Gabon.

BACKGROUND: Despite the development of several methods for diagnosing COVID-19, long-term validation of such methods remains limited. In the early phase of the COVID-19 pandemic, we developed a rapid and sensitive diagnostic method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) methodology, which is suitable for point-of-care application or for use in resource-limited settings to detect SARS-CoV-2. To assess the applicability of the RT-LAMP assay technique to resource-limited regions, such as rural areas in Africa, and to verify the usability of the method against various SARS-CoV-2 variants, the method was validated using clinical samples collected longitudinally during the pandemic. METHODOLOGY/PRINCIPAL FINDINGS: First, the sensitivity of the RT-LAMP assay for detecting 10 SARS-CoV-2 variants was evaluated using viral RNA samples extracted from cell culture with a portable battery-supported device, resulting in the successful detection of 20-50 copies of the viral genome within 15 min, regardless of the variant. COVID-19 positive samples collected in Gabon between March 2020 and October 2021 were used to evaluate the sensitivity of the assay and to calculate the copy number of the SARS-CoV-2 genome. More than 292 copies of the viral genome were detected with 100% probability within 15 min in almost all tests. CONCLUSIONS: This long-term validation study clearly demonstrated the applicability of the RT-LAMP assay for the clinical diagnosis of COVID-19 in resource-limited settings of Africa, such as rural areas in Gabon. The results show the potential of the assay as a promising COVID-19 diagnostic method, especially in rural and remote regions located far from the official diagnosis facilities in urban or semi-urban areas.

Unique Evolution of SARS-CoV-2 in the Second Large Cruise Ship Cluster in Japan.

In the initial phase of the novel coronavirus disease (COVID-19) pandemic, a large-scale cluster on the cruise ship Diamond Princess (DP) emerged in Japan. Genetic analysis of the DP strains has provided important information for elucidating the possible transmission process of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on a cruise ship. However, genome-based analyses of SARS-CoV-2 detected in large-scale cruise ship clusters other than the DP cluster have rarely been reported. In the present study, whole-genome sequences of 94 SARS-CoV-2 strains detected in the second large cruise ship cluster, which emerged on the Costa Atlantica (CA) in Japan, were characterized to understand the evolution of the virus in a crowded and confined place. Phylogenetic and haplotype network analysis indicated that the CA strains were derived from a common ancestral strain introduced on the CA cruise ship and spread in a superspreading event-like manner, resulting in several mutations that might have affected viral characteristics, including the P681H substitution in the spike protein. Moreover, there were significant genetic distances between CA strains and other strains isolated in different environments, such as cities under lockdown. These results provide new insights into the unique evolution patterns of SARS-CoV-2 in the CA cruise ship cluster.

Co-infection of SARS-CoV-2 and influenza virus causes more severe and prolonged pneumonia in hamsters.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently a serious public health concern worldwide. Notably, co-infection with other pathogens may worsen the severity of COVID-19 symptoms and increase fatality. Here, we show that co-infection with influenza A virus (IAV) causes more severe body weight loss and more severe and prolonged pneumonia in SARS-CoV-2-infected hamsters. Each virus can efficiently spread in the lungs without interference by the other. However, in immunohistochemical analyses, SARS-CoV-2 and IAV were not detected at the same sites in the respiratory organs of co-infected hamsters, suggesting that either the two viruses may have different cell tropisms in vivo or each virus may inhibit the infection and/or growth of the other within a cell or adjacent areas in the organs. Furthermore, a significant increase in IL-6 was detected in the sera of hamsters co-infected with SARS-CoV-2 and IAV at 7 and 10 days post-infection, suggesting that IL-6 may be involved in the increased severity of pneumonia. Our results strongly suggest that IAV co-infection with SARS-CoV-2 can have serious health risks and increased caution should be applied in such cases.

Development and evaluation of a rapid and simple diagnostic assay for COVID-19 based on loop-mediated isothermal amplification.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly pathogenic novel coronavirus that has caused a worldwide outbreak. Here we describe a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay that uses a portable device for efficient detection of SARS-CoV-2. This RT-LAMP assay specifically detected SARS-CoV-2 without cross-reacting with the most closely related human coronavirus, SARS-CoV. Clinical evaluation of nasal swab samples from suspected SARS-CoV-2 pneumonia (COVID-19) patients showed that the assay could detect over 23.7 copies within 15 min with a 100% probability. Since the RT-LAMP assay can be performed with a portable battery-supported device, it is a rapid, simple, and sensitive diagnostic assay for COVID-19 that can be available at point-of-care. We also developed the RT-LAMP assay without the RNA extraction step-Direct RT-LAMP, which could detect more than 1.43 x 103 copies within 15 min with a 100% probability in clinical evaluation test. Although the Direct RT-LAMP assay was less sensitive than the standard RT-LAMP, the Direct RT-LAMP assay can be available as the rapid first screening of COVID-19 in poorly equipped areas, such as rural areas in developing countries.