key: cord-0697670-r8btwnxl authors: Mao, Xiao-Dong; Li, Taiping; Xu, Zhirong; Liu, Kangsheng title: Pathogenesis of COVID-19 and the quality control of nucleic acid detection date: 2021-01-01 journal: Biochem Biophys Res Commun DOI: 10.1016/j.bbrc.2020.12.094 sha: bead54db7d6046e675b6884fcf93c5e951a50b37 doc_id: 697670 cord_uid: r8btwnxl The new coronavirus pneumonia (COVID-19) epidemic spread rapidly throughout the world. Considering the strong infectivity and clustering of COVID-19, early detection of infectious cases is of great significance to control the epidemic. Nucleic acid testing (NAT) plays an important role in rapid laboratory diagnosis, treatment assessment, epidemic prevention and control of COVID-19. However, since COVID-19 is caused by a new emerging virus and NAT for COVID-19 has not been clinically applied before, false negative results inconsistent with clinical diagnosis have appeared in clinical practice. Therefore, it is urgent to improve the sensitivity of NAT for COVID-19. This study aimed to summarize the current situation and prospect of NAT based on the latest findings on COVID-19 infection. Also, the quality control of sample collection was discussed. Hopefully, this study could help to improve the effectiveness of NAT for COVID-19. generate false negative results that are inconsistent with clinical diagnosis. Recently, it has been reported that a confirmed case in China-Japan Friendship Hospital in China was tested negative in the previous three NATs, and another case in Hangzhou was reported to be negative for six times before the 7th NAT, which makes the clinicians to question the effectiveness of NAT for COVID-19 [5] . According to the "Biosafety Guidelines for Laboratory Testing of issued by the Laboratory Medicine Branch of the Chinese Medical Association [3] , and "Risk Communication and Community Engagement (RCCE) Readiness and Response to issued by the World Health Organization (WHO) [6] combined with the latest research results and findings of COVID-19, the current situation and influencing factors of NAT were discussed [7] . Meanwhile, the quality control of NAT sample collection was investigated to improve the effectiveness of NAT. Structurally, SARS-CoV-2 belongs to the family of coronaviruses and is genetically similar (over 85% similarity) to bat coronaviruses [8] . SARS-CoV-2 shares 78% and 50% genetic sequence with the viruses of SARS and MERS, respectively [9] . It is generally considered that Rhinolophus sinicus bat is the natural reservoir of SARS-CoV-2. A latest research proposed that pangolins may be the intermediate host that introduces COVID-19 to humans [10] . A novel study suggested that COVID-19 might spread through aerosol transmission under certain circumstances. Very recently, detection of SARS-CoV-2 in stool samples indicated the possibility of fecal-oral transmission [11] . So far, no evidence has supported COVID-19 infection is caused by contaminated food, but the possibility of aerosol or contact transmission via fecal excretion cannot be excluded [12] . The urinary system may also be a potential transmission route for COVID-19 infection [13] . ACE2, the SARS-CoV-2 receptor, has been demonstrated to be lowly expressed in various cell types of the human maternal-fetal interface [14] , indicating that there might be no potential susceptible cell subpopulation of SARS-CoV-2 in the maternal-fetal interface and thus SARS-CoV-2 infection could not cause vertical transmission from mother to fetus. In addition, experimental evidence has proven that COVID-19 cannot be transmitted J o u r n a l P r e -p r o o f 4 through skin contact. Pathological lesions in the lung are the major characteristics of COVID-19, and diarrhea is uncommon, suggesting that lung is the target organ of COVID-19 [15] . The entry of SARS-CoV-2 into host cells requires the binding of S protein and ACE2 on the cell membrane [16] . During viral replication, amplification and release, the body defense response is initiated. Most of COVID-19 infected patients can be cured by adjuvant therapy for virus clearance and inflammatory damage repairing through autoimmune function. However, over-activated inflammatory response and envelope protein (E), membrane protein (M) and nucleocapsid protein (N). [19] . Comparing with other coronavirus nucleic acid sequences, ORF1ab fragment and N gene are specific genes of coronavirus and type. Specific primers were designed for these two genes to detect SARS-CoV-2, and S gene was added to some reagents to improve detection sensitivity. Compared with gene sequencing, the detection time is relatively short, the operation is convenient, and the specificity and repeatability are better [20] . Chu et al. [21] showed that the sensitivity of N gene detection was higher. Corman et al. [22] optimized the detection of SARS-CoV-2 and established a RT-PCR detection process. Positive case diagnosis requires two positive targets in the same specimen, or two positive targets in the same specimen, or two positive targets in the same specimen. So far, the approved reagent has not achieved desired performance verification, resulting in (II) Inflammatory cytokine storm may influence the result of NAT Clinical diagnosis is required for cases with typical pulmonary symptoms but negative NAT results in regions where COVID-19 is endemic, so that missed diagnosis can be significantly avoided [25] . It is also suggested that patients with severe symptoms receive deep sputum or BALF test. [27] . At present, most COVID-19 NAT kits are designed to detect either three gene fragments (ORF1ab gene, core-shell protein gene N fragment and enveloped protein gene E fragment), or two gene fragments (ORF1ab and core-shell protein gene N fragment) in the open reading frame. Because COVID-19 NAT kit only has negative control and positive control, the test results should be carefully interpreted. According to the recommendation of the National Health Commission of China, the results should be interpreted as follows [28] . In the open reading frame, for the Standardizing the transport and storage of specimens, the operation of clinical laboratories, and the interpretation of results are also key to the accuracy and reliability of test results. Improvement can be made in the training of competent laboratory personnel, the optimization of the laboratory quality management system, and proper apparition of the working zone. Recently, a small number of patients have been tested positive for COVID-19 again after being discharged from the hospital. A possible explanation could be that the antibodies produced in response to the original virus in the recovered patients might be ineffective against the mutated virus. Positive results may appear in the patient infected with the mutated virus [31] . Recently, an analysis of 103 genomes of SARS-CoV-2 from different countries and regions found 149 mutations, and evolved S and L (Orflab:T8517C,ORF8:C251T) subtypes, S and L subtypes whose mutation sites(generally located in N and S proteins) are not commonly used in NAT and antigen design [32] . Therefore, the pathogenesis, disease overview and course characteristics of the disease need to be further understood. Besides, the patients discharged from the hospital need a 14-day medical observation, during which follow-up, health monitoring and guidance should be carried out. Another cause of this condition could be the NAT kits, which may create problems in the following aspects: the selection of gene sequence, composition of reagents and sensitivity of the method [33] . After treatment, the virus in the patient's body is reduced, and the negative result will occur when the viral load in the tested sample is below the detection limit. However, this result J o u r n a l P r e -p r o o f does not mean that the virus in the body completely disappears because the virus may "rekindle" after treatment cessation. The COVID-19 NAT only tells the presence or absence of viral RNA, but cannot prove the viral activity or the transmissibility of the virus. In this sense, it is necessary to conduct virus culture on the clinical specimens to detect whether there exists a "live" virus. Nucleic acids, as the genetic material of the virus, can be exterminated after antiviral treatment, but the remaining RNA virus genetic material (DNA) pieces are still in the body, which cannot be discharged entirely from the body. In certain circumstances, they can be kept longer, and become the culprit for the "transient" positive result of NAT. As the patient's recovery progresses [34] , after the residual RNA fragments in the body being gradually exhausted, the patient's NAT result can turn negative. As SARS-CoV-2 is a new pathogen, common clinical methods, such as routine culture and serology, can hardly be applied to clinical detection of it. Therefore, viral NAT is an optimal method to diagnose, monitor, prevent and control COVID-19. But the pathological process and clinical mechanisms of the disease have not been fully elucidated. At present, most COVID-19 NAT kits in use have not undergone comprehensive clinical evaluation, and there are also problems in standardization of laboratory testing. In clinical practice, the operating procedures and quality control protocols should be strictly followed. When the NAT result is not consistent with the clinical manifestations, imaging detection (CT) and laboratory examination (NAT + virus specific antibody detection) should be combined for further diagnosis. Obtaining the gene sequence of COVID-19 and using it for effective detection is just the first step. More efforts are needed to explore the virus traceability, the viral transmission routes, the pathogenesis, and the effective targeted therapeutic drugs and vaccines. 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