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      Evaluation of rapid diagnosis of novel coronavirus disease (COVID-19) using loop-mediated isothermal amplification

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          Highlights

          • RT-LAMP test kits have good utility.

          • Detection sensitivity of 1.0 × 10 1 copies/μL can be obtained within 35 min.

          • RT-LAMP can be used as a point-of-care test because it is judged by turbidity under natural light.

          Abstract

          With the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is an urgent need for more rapid and simple detection technologies at the forefront of medical care worldwide.

          In this study, we evaluated the effectiveness of the Loopamp® 2019-SARSCoV-2 Detection Reagent Kit, which uses loop-mediated isothermal amplification (LAMP) technology. In this protocol, cDNA is synthesized from SARS-CoV-2 RNA using reverse transcriptase, followed by DNA amplification under isothermal conditions in one step. The RT-LAMP test kit amplified the targeted RNA of a SARS-CoV-2 isolate with a detection limit of 1.0 × 101 copies/μL, which was comparable to the detection sensitivity of quantitative reverse transcription PCR (RT-qPCR).

          Comparison with the results of RT-qPCR for 76 nasopharyngeal swab samples from patients with suspected COVID-19 showed a sensitivity of 100% and a specificity of 97.6%. In the 24 RNA specimens derived from febrile Japanese patients with or without influenza A, no amplification was observed using RT-LAMP. RT-LAMP could be a simple and easy-to-use diagnostic tool for the detection of SARS-CoV-2.

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          Most cited references7

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          Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China

          Summary Background A recent cluster of pneumonia cases in Wuhan, China, was caused by a novel betacoronavirus, the 2019 novel coronavirus (2019-nCoV). We report the epidemiological, clinical, laboratory, and radiological characteristics and treatment and clinical outcomes of these patients. Methods All patients with suspected 2019-nCoV were admitted to a designated hospital in Wuhan. We prospectively collected and analysed data on patients with laboratory-confirmed 2019-nCoV infection by real-time RT-PCR and next-generation sequencing. Data were obtained with standardised data collection forms shared by WHO and the International Severe Acute Respiratory and Emerging Infection Consortium from electronic medical records. Researchers also directly communicated with patients or their families to ascertain epidemiological and symptom data. Outcomes were also compared between patients who had been admitted to the intensive care unit (ICU) and those who had not. Findings By Jan 2, 2020, 41 admitted hospital patients had been identified as having laboratory-confirmed 2019-nCoV infection. Most of the infected patients were men (30 [73%] of 41); less than half had underlying diseases (13 [32%]), including diabetes (eight [20%]), hypertension (six [15%]), and cardiovascular disease (six [15%]). Median age was 49·0 years (IQR 41·0–58·0). 27 (66%) of 41 patients had been exposed to Huanan seafood market. One family cluster was found. Common symptoms at onset of illness were fever (40 [98%] of 41 patients), cough (31 [76%]), and myalgia or fatigue (18 [44%]); less common symptoms were sputum production (11 [28%] of 39), headache (three [8%] of 38), haemoptysis (two [5%] of 39), and diarrhoea (one [3%] of 38). Dyspnoea developed in 22 (55%) of 40 patients (median time from illness onset to dyspnoea 8·0 days [IQR 5·0–13·0]). 26 (63%) of 41 patients had lymphopenia. All 41 patients had pneumonia with abnormal findings on chest CT. Complications included acute respiratory distress syndrome (12 [29%]), RNAaemia (six [15%]), acute cardiac injury (five [12%]) and secondary infection (four [10%]). 13 (32%) patients were admitted to an ICU and six (15%) died. Compared with non-ICU patients, ICU patients had higher plasma levels of IL2, IL7, IL10, GSCF, IP10, MCP1, MIP1A, and TNFα. Interpretation The 2019-nCoV infection caused clusters of severe respiratory illness similar to severe acute respiratory syndrome coronavirus and was associated with ICU admission and high mortality. Major gaps in our knowledge of the origin, epidemiology, duration of human transmission, and clinical spectrum of disease need fulfilment by future studies. Funding Ministry of Science and Technology, Chinese Academy of Medical Sciences, National Natural Science Foundation of China, and Beijing Municipal Science and Technology Commission.
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            Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus–Infected Pneumonia

            Abstract Background The initial cases of novel coronavirus (2019-nCoV)–infected pneumonia (NCIP) occurred in Wuhan, Hubei Province, China, in December 2019 and January 2020. We analyzed data on the first 425 confirmed cases in Wuhan to determine the epidemiologic characteristics of NCIP. Methods We collected information on demographic characteristics, exposure history, and illness timelines of laboratory-confirmed cases of NCIP that had been reported by January 22, 2020. We described characteristics of the cases and estimated the key epidemiologic time-delay distributions. In the early period of exponential growth, we estimated the epidemic doubling time and the basic reproductive number. Results Among the first 425 patients with confirmed NCIP, the median age was 59 years and 56% were male. The majority of cases (55%) with onset before January 1, 2020, were linked to the Huanan Seafood Wholesale Market, as compared with 8.6% of the subsequent cases. The mean incubation period was 5.2 days (95% confidence interval [CI], 4.1 to 7.0), with the 95th percentile of the distribution at 12.5 days. In its early stages, the epidemic doubled in size every 7.4 days. With a mean serial interval of 7.5 days (95% CI, 5.3 to 19), the basic reproductive number was estimated to be 2.2 (95% CI, 1.4 to 3.9). Conclusions On the basis of this information, there is evidence that human-to-human transmission has occurred among close contacts since the middle of December 2019. Considerable efforts to reduce transmission will be required to control outbreaks if similar dynamics apply elsewhere. Measures to prevent or reduce transmission should be implemented in populations at risk. (Funded by the Ministry of Science and Technology of China and others.)
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              Reasons for healthcare workers becoming infected with novel coronavirus disease 2019 (COVID-19) in China

              Sir, The outbreak of novel coronavirus disease 2019 (COVID-19) in mainland China has been declared as a public health emergency (PHE) by the World Health Organization (WHO) [1]. Globally, until February 28th, 2020, there have been reported 83,774 confirmed cases and 2867 deaths [2]. During the periods of outbreak of COVID-19 or other infectious diseases, implementation of infection prevention and control (IPC) is of great importance in healthcare settings, especially regarding personal protection of healthcare workers [3,4]. In order to contain the outbreak of COVID-19 in mainland China, the National Health Commission of the People's Republic of China (NHCPRC) has so far dispatched medical support teams (41,600 healthcare workers from 30 provinces and municipalities) to assist with medical treatment in Wuhan and Hubei provinces [5]. A survey by the Health Commission of Guangdong Province released information on the distribution of 2431 healthcare workers in the Guangdong medical support teams [6]. Nurses (∼60%) were the predominant healthcare workers in the teams, followed by clinicians (∼30%). Half of clinicians with job titles were deputy chief physician, and 25% specialized in respiratory and critical medicine [6]. It is worth mentioning that 5.8% (140/2431) healthcare workers worked on the outbreak of severe acute respiratory syndrome in 2003 [6]. Recently, Wu et al. have reported the problems relating to COVID-19 IPC in healthcare settings, highlighting the personal protection of healthcare workers [7]. However, at a press conference of the WHO–China Joint Mission on COVID-19, NHCPRC reported that up until February 24th 2055 healthcare workers (community/hospital-acquired not to be defined) had been confirmed infected with COVID-19, with 22 (1.1%) deaths [8]. Ninety percent of infected healthcare workers were from Hubei province, and most cases happened in late January. It is worth mentioning that the proportion of healthcare workers infected by COVID-19 (2.7%, 95% CI: 2.6–2.8) was significantly lower compared with healthcare workers infected by SARS (21.1%, 95% CI: 20.2–22.0). Therefore, the director of the National Hospital Infection Management and Quality Control Centre summarized some reasons for such a high number of infected healthcare workers during the beginning of the emergency outbreak [9]. First, inadequate personal protection of healthcare workers at the beginning of the epidemic was a central issue. In fact, they did not understand the pathogen well; and their awareness of personal protection was not strong enough. Therefore, the front-line healthcare workers did not implement the effective personal protection before conducting the treatment. Second, long-time exposure to large numbers of infected patients directly increased the risk of infection for healthcare workers. Also, pressure of treatment, work intensity, and lack of rest indirectly increased the probability of infection for healthcare workers. Third, shortage of personal protective equipment (PPE) was also a serious problem. First-level emergency responses have been initiated in various parts of the country, which has led to a rapid increase in the demand for PPE. This circumstance increased the risk of infection for healthcare workers due to lack of sufficient PPE. Fourth, the front-line healthcare workers (except infectious disease physicians) received inadequate training for IPC, leaving them with a lack of knowledge of IPC for respiratory-borne infectious diseases. After initiation of emergency responses, healthcare workers have not had enough time for systematic training and practice. Professional supervision and guidance, as well as monitoring mechanisms, were lacking. This situation further amplified the risk of infection for healthcare workers. Finally, international communities, especially in other low- and middle-income countries with potential COVID-19 outbreaks, should learn early how to protect their healthcare workers. Furthermore, the COVID-19 confirmed cases have been reported to have surged in South Korea, Japan, Italy, and Iran in the past few days [2]. The increase in awareness of personal protection, sufficient PPE, and proper preparedness and response would play an important role in lowering the risk of infection for healthcare workers. Conflict of interest statement None declared. Funding sources None.
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                Author and article information

                Contributors
                Journal
                J Clin Virol
                J. Clin. Virol
                Journal of Clinical Virology
                Elsevier B.V.
                1386-6532
                1873-5967
                21 May 2020
                21 May 2020
                : 104446
                Affiliations
                [a ]Department of Clinical Laboratory, Saitama Medical University Hospital, Saitama, Japan
                [b ]Department of Infectious Disease and Infection Control, Saitama Medical University, Saitama, Japan
                [c ]Department of Laboratory Medicine, Saitama Medical University, Saitama, Japan
                Author notes
                [* ]Corresponding author at: Saitama Medical University, 38 Morohongo, Moroyama-machi, Iruma-gun, Saitama, 350-0495, Japan. t_maeda@ 123456saitama-med.ac.jp
                Article
                S1386-6532(20)30188-8 104446
                10.1016/j.jcv.2020.104446
                7241399
                32512376
                9cf7817e-8e21-4839-b958-3dec437fa199
                © 2020 Elsevier B.V. All rights reserved.

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                : 25 April 2020
                : 8 May 2020
                : 14 May 2020
                Categories
                Article

                Microbiology & Virology
                sars-cov-2,covid-19,lamp,rt-qpcr
                Microbiology & Virology
                sars-cov-2, covid-19, lamp, rt-qpcr

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