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Analysis of the cause of low positive rate of new coronavirus pneumonia with nucleic acid test
Since the occurrence of the new type of coronavirus pneumonia (NCP) in 2019, the number of infections has risen rapidly, and the epidemic has been effectively controlled with the unremitting efforts of medical staff. As of 24:00 on February 18, 2020, according to reports from 31 provinces (autonomous regions, municipalities) and the Xinjiang Production and Construction Corps, there are 57 805 confirmed cases (including 11,977 severe cases), and a total of 14 376 discharged patients have been cured. A total of 2 004 deaths have occurred, and 5 248 suspected cases exist. A total of 574,418 close contacts were tracked, and 135,881 close contacts were still in medical observation.
Viral nucleic acid detection based on real-time fluorescent reverse transcription-polymerase chain reaction (RT-PCR) method is an important means for clinical diagnosis. However, recent media and experts have reported that the positive rate of the new type of coronavirus nucleic acid is low, and some cases have been tested repeatedly. Positive results appeared after being negative. Pharyngeal swab specimens were negative several times, but positive results were finally detected in respiratory lavage fluid specimens. Computed tomography (CT) image screening had NCP characteristics but nucleic acid detection. Negative. After communicating with clinical molecular diagnostics experts, clinical laboratory medicine experts, clinical respiratory medicine experts, and clinical imaging medicine experts, the above problems are now analyzed.
First, the application of nucleic acid detection in the diagnosis of NCP patients
NCP is an acute infectious disease caused by 2019 novel coronavirus (2019-nCoV) infection, and the gold standard for diagnosis is to find the presence of 2019-nCoV in patients. Nucleic acid detection is the detection of the presence of certain specific nucleic acid sequences in the 2019-nCoV gene, so there is no doubt that it is considered as a gold standard for laboratory diagnosis from a methodological perspective, and CT has important value as a clinical imaging screening index, but whether it is from Early diagnosis, differential diagnosis, exclusion diagnosis, or final diagnosis all have certain limitations, and they cannot replace the etiology diagnosis [1]. In fact, any disease itself has the concept of hierarchical diagnosis. In the diagnosis of infectious diseases, clinical manifestations, epidemiological history, imaging, etiology and other indicators all play an important role, but the pathogenic diagnosis is definitely the most accurate diagnosis. . Of course, while we value the important value of nucleic acid detection in the diagnosis of viral infections, we must also consider objectively due to its methodological characteristics, disease development process, specimen collection, specimen preservation and transportation, nucleic acid extraction, amplification system, personnel operations, and other factors. Can cause false negatives or false positives in the final test result.
About 2019-nCoV detection methods
At present, the real-time fluorescence RT-PCR method [2,3,4] is widely used in clinical laboratories or center for disease control and prevention (CDC) laboratories. This detection method is mature and reliable, and It has been widely used in routine inspection and scientific research experiments [5]. The method is to amplify a specific nucleic acid sequence in a specimen. In theory, the number of nucleic acids after each amplification is multiplied. After more than 40 amplifications, the number of nucleic acids is sufficient to be detected by conventional methods such as fluorescence. . If you question the reliability of the 2019-nCoV nucleic acid detection method currently used in the laboratory, it may be because it does not know much about the principle and clinical application of the method. The real-time fluorescent RT-PCR method is used to transport the collected specimens to the laboratory, and then receive and process the specimens (sometimes inactivation is required for safety reasons, which is important for biosafety, but some experts have suggested that it may affect the detection efficiency) , Viral nucleic acid extraction, nucleic acid amplification, data processing and reporting, generally takes about 4 hours or more. How to improve the nucleic acid extraction and amplification process and shorten the entire detection time is an urgent problem to be solved. At present, some manufacturers' detection reagents do not directly detect 2019-nCoV nucleic acids, but antibodies produced by the body after the virus enters the body (indirect detection method). It takes a period of time for the virus to enter the body to produce specific antibodies [6], so the detection Antibodies to viruses must lag behind the detection of viral nucleic acids.
Therefore, it is not difficult to understand that despite the advantages of convenience and speed, indirect virus detection methods have their application limitations compared to nucleic acid detection. How to effectively combine indirect virus antibody (antigen) detection methods with direct viral nucleic acid detection methods. Utilizing their respective advantages, evaluation and verification are urgently needed. Recently, some experts have recommended more sensitive digital PCR methods, RT-PCR capillary electrophoresis methods that can detect more viral gene loci, high-throughput nucleic acid mass spectrometry methods, and more reliable gene sequencing methods, but these advanced frontiers There are three problems with the detection technology: first, the time is too long, everyone thinks that the real-time fluorescent RT-PCR is 4 to 6 hours long, some other methods use more than 10 hours, or even 24 hours or longer; second Most of the routine tests in the laboratory use real-time fluorescent RT-PCR methods. If you switch to a new method, the laboratory will have to re-purchase equipment and retrain personnel, which is too expensive. Third, the detection process is relatively complicated. The quality of testing personnel is high, and the quality of testing is not easy to control. In the future, how to improve the front-end detection technology with ease and ease of use and widely apply it in clinical detection needs to become the focus of attention.
Third, 2019-nCoV nucleic acid test has a low positive rate of NCP patients
(A) the course and condition of the patient
The onset of most NCP patients will go through a period from asymptomatic to mild symptoms after infection, until they are cured; a small number of patients will progress to the appearance of severe symptoms [7]. The amount of virus in patients with different courses and conditions may be different (there is already a virus infection, but the virus is not collected at the relevant site or the amount of virus is too small to be detected by the existing methods). Therefore, for patients with a high clinical likelihood of NCP, samples should be continuously collected for nucleic acid testing [8] (this may be one of the main reasons for clinical patients with multiple negative tests and late positive results). In fact, any pathogenic test cannot make a negative diagnosis based on only one negative result, nor can it miss a clinically suspected NCP patient based on a negative nucleic acid test, especially in high-incidence areas and during large epidemics.
(II) Specimen collection, transportation and preservation
1. Specimen collection site:
Many experts have concluded from practical experience that nasopharyngeal swab specimens have a higher positive rate of nucleic acid detection than oropharyngeal swabs, and sputum and alveolar lavage fluid specimens collected from the lower respiratory tract have higher positive rates than the upper respiratory tract Specimens collected from swabs (this is also the reason why a patient's throat swab specimen that was widely commented for two days was negative for three nucleic acid tests, and the patient received an alveolar lavage fluid sample for nucleic acid positive during the rescue process after admission). However, the collection of sputum and alveolar lavage fluid samples from the lower respiratory tract is too difficult, which may cause the patient to splatter and increase the risk of infection of the collection operator. It is generally not recommended (tracheotomy and ventilator rescue for patients). The recently released "Diagnosis and Treatment of Pneumonitis with New Coronavirus Infection (Trial Fifth Revised Version)" [9] has updated the specimen collection "pharyngeal swab" to "nasopharyngeal swab". In order to improve the positive test rate, it is recommended to collect multiple specimens from the same patient and combine them for testing. For example, oropharyngeal swabs and nasopharyngeal swabs are used to collect specimens at the same time, and then placed in the same collection tube for examination. For suspected patients with gastrointestinal symptoms, stool or anal swabs can be collected for testing.
2. Specimen collection method:
The accuracy of the sampling depends on the individual operator. If the specimen collector does not operate normally, it will also cause sampling errors. The pre-2019-nCoV nucleic acid detection specimens were mainly oropharyngeal swabs. When collecting, let the patient open his mouth and use a special cotton swab or a small brush to scrape his throat to collect virus-containing specimens [4]. If there are not many samples of throat swabs from medical staff, and the response of the patient is relatively large when scraping the specimen, it often leads to the wrong location of the scraped specimen, or insufficient strength and time, and no specimen with virus or scraped specimen is scraped. The number of virus-bearing specimens is small, and the negative result of the final test will inevitably increase.
3． Specimen transportation and preservation:
2019-nCoV is an RNA virus, which can be easily degraded by endogenous RNase released from exogenous or cell destruction from reagents, consumables, etc. [10], which affects the final detection efficiency. Strictly speaking, specimens should be sent to laboratories (CDC, hospital laboratory, third-party laboratories) promptly after collection, and testing should be completed as soon as possible. Prolonged storage of specimens at room temperature may also be the cause of false negative results in the final test [2]. Although anti-virus nucleic acid degradation sample collection tubes can be used, currently such products are small in number, high in cost, and the actual application effect needs further evaluation. It is recommended that the specimens be sent for inspection immediately after collection (4 h). If for some reason the specimen cannot be submitted for inspection or timely detection after collection, it should be stored at 4 ? C (24 h). Specimens that cannot be detected within 24 hours are recommended to be stored below 70 ? C and avoid repeated freeze-thaw cycles [8].
(Three) nucleic acid detection reagents
1. Nucleic acid extraction:
As mentioned earlier, the 2019-nCoV nucleic acid detection requires the extraction of nucleic acids from the collected specimens. At present, the more commonly used methods for extracting nucleic acids from the laboratory are manual extraction and automatic extraction of nucleic acids. The manual method is difficult to standardize due to personnel and other factors; the fully automatic nucleic acid extraction instrument method requires the use of a matching nucleic acid extraction instrument and corresponding reagents throughout the process, and the entire operation is easy to standardize. Compared with the manual method, the automatic nucleic acid extraction instrument can avoid some errors that are prone to manual operations, and has less impact on the detection results [11,12]. The control of different extraction reagents and manual extraction processes may differ in the amount and quality of nucleic acids that are finally extracted, and the amount and quality of nucleic acids extracted from specimens are essential for the next step of nucleic acid amplification in the detection method. Thereby directly affect the final test results.
2. Nucleic acid amplification:
As the research and development of reagents of different manufacturers is based on the specific sequences of 2019-nCoV nucleic acid OFR1ab, N, E and other genes (amplification reagents of different manufacturers can be divided into 1, 2, or 3 target gene loci Detection reagents), and reagents containing different target gene loci have different detection sensitivities and specificities, and differences in components and quality of reagents such as enzymes and metal ions may also affect amplification efficiency and final detection results [13] .
3． Development and application of reagents:
As the 2019-nCoV that caused this epidemic is a new type of virus, and the epidemic is sudden, reagent manufacturers can only carry out research and development of reagents based on the limited publicly available viral nucleic acid research data. The relevant national authorities first recommended some manufacturers' testing products [ 4], and later approved the application for registration of some manufacturers' testing reagents. Depending on the manufacturer of the reagent, the quality of the nucleic acid test kits varies, which will also affect the test results. At present, there are more than 100 manufacturers carrying out R & D and production of 2019-nCoV testing reagents. Under normal circumstances, a clinical testing reagent product needs to pass a series of clinical verification and evaluation from R & D to application. However, due to the special nature of the epidemic, the time is urgent. The developed reagents are too late to complete the routine process, especially the verification of a certain number of clinical patient specimens. Some manufacturers can only use 2019-nCoV target RNA sequences to simulate viruses in real specimens by means of phage protein shell encapsulation. Therefore, for some highly suspected clinical cases, or cases where the test results are difficult to determine, it is recommended to use more than two reagents for testing and verification.
(Four) nucleic acid amplification equipment
At present, the types of instruments (PCR instruments or nucleic acid amplification instruments) used in 2019-nCoV nucleic acid detection in different laboratories are different, the prices are different, and the accuracy of different equipments for controlling different temperatures during the detection process and the sensitivity to fluorescent signal detection And other differences, which may lead to differences in detection results [3,4].
(V) Nucleic acid mutation
Since 2019-nCoV is a single-stranded positive-strand RNA virus, it has a large molecular weight and is susceptible to mutation [13]. With the extension of the epidemic, mutations in the nucleic acid sequence of gene loci may occur during human-to-human transmission. At present, the research on 2019-nCoV is not extensive enough, and the mutation frequency and hotspots of the virus genes are not clear. If the mutation site is in the primer binding region of nucleic acid amplification, false negative results may occur. However, there are currently no theoretical reports about the impact of viral mutation detection results, and during the development of the kit, amplification of multiple nucleic acid regions can also effectively avoid the impact of nucleic acid mutation on the detection results.
The above is some analysis of the recent media and experts reflecting the possible reasons for the low positive rate of the 2019-nCoV nucleic acid test. From this, it can be understood that these or other problems caused by various reasons in the current 2019-nCoV nucleic acid test are real, and they are also inevitable. We cannot universally doubt or even deny the current laboratory 2019-nCoV nucleic acid test results because of these problems. At present, most of the 2019-nCoV nucleic acid tests are carried out in tertiary hospitals or CDC secondary laboratories.The laboratory management level, the quality of technical staff, the advanced level of inspection equipment, and the quality control measures of the inspection process are generally high. And each laboratory is continuously improving and perfecting the 2019-nCoV nucleic acid detection method through reviewing and summarizing work or exchanging experiences between laboratories.
Recently, the Chinese Medical Association Laboratory Medicine Branch also organized relevant experts to discuss the 2019-nCoV nucleic acid detection, and introduced the 2019-nCoV nucleic acid detection-related expert consensus [4]. At the same time, some new genetic testing platforms are also constantly being developed. With the application of gene sequencing, digital PCR, RT-PCR capillary electrophoresis and other technologies, the problems in 2019-nCoV nucleic acid detection will be continuously improved.
