On February 3, 2020, Zhang Xiaochun, deputy director of the imaging department of Zhongnan Hospital of Wuhan University, sent a circle of friends: “Don’t be superstitious for nucleic acid detection, CT imaging is strongly recommended as the main basis of current 2019-nCoV pneumonia”, and said that it is “the first-line Shout out from the imaging doctor! “
Her remarks subsequently attracted much attention.
At the moment when the inflection point of the epidemic cannot be accurately predicted, if the only means of diagnosis cannot be guaranteed, there will be wrong and missed tests. The direct consequence will be that many people who have negative nucleic acid tests but still carry the virus will not be isolated, and they will become a new Source of infection.
A typical case is a female infection case in Tianjin. After fever on January 21, a total of three nucleic acid tests were negative before and after, and only positive after the fourth test on February 1.
Why do false negatives occur frequently in the kit test?
In this regard, many experts explained to China News Weekly that the test results of the kit are not only related to the quality of the kit, but also to the characteristics of the new crown virus, sampling locations, sampling volumes, transportation and storage links, and laboratory testing conditions and conditions. The operation of personnel is composed of various reasons and is very complicated, but the most critical factor is related to the particularity of the virus.
Errors in every link may cause inaccurate results
A researcher at a national key laboratory specializing in nucleic acid detection told China News Weekly that due to the special nature of the new crown virus, different sampling locations have a great impact on the detection rate.
He pointed out that the best sampling site is the lungs because the virus has the highest concentration in the lungs. However, if you want to take alveolar lavage fluid, the operation is complicated, equipment and various catheters are needed, and it is very harmful to people. Large, so only for critically ill patients on the ventilator; followed by sputum, but this time pneumonia is not like previous flu, many patients have a dry cough without sputum. Therefore, the most common and simplest sampling method is a throat swab, and the pharyngeal coronavirus is the least, so it will miss the test.
Another expert who specializes in nucleic acid detection in the disease control system pointed out to China News Weekly that studies have shown that the new crown virus replicates very slowly in human epithelial cells. Therefore, the base of the virus in the pharynx and even sputum They are all very low, which makes testing more difficult.
“Some patients are positive only after several tests, which is normal. Because the patient may secrete a small amount of virus in the early stage, and the virus may increase in the later stage as the disease progresses,” said the researcher said.
Besides, whether the sampling is accurate and the key parts are taken will also affect the sampling results. Taking a throat swab as an example, although it is a very simple operation, you only need to take a swab on the pharynx, but the weight and amount of each medical staff are different. Especially in Wuhan, it is very difficult for medical staff wearing heavy protective clothing to achieve accurate sampling under the condition that the average daily detection amount is overloaded.
In addition to sampling, in every link from sample transport to laboratory testing, errors in each link may cause inaccurate test results.
Because viral RNA is easily degraded and highly sensitive, cold-chain transportation must be used during sample delivery and maintained at minus 20 degrees. Once frozen and thawed more than four times, samples will be damaged. Previously, Shanghai Zhejiang Biotechnology Co., Ltd., one of the first six manufacturers of new crown virus kits approved by the State Drug Administration, told the media that because the kits required a storage environment of minus 20 degrees Celsius, only a few were cold. Chain-capable logistics companies can carry it. A little carelessness during transportation may affect the final test results of the kit.
The aforementioned researchers
Also, there are high thresholds for testing with kits, and the conditions in the laboratory are also very demanding. The aforementioned researchers told China News Weekly that, in theory, PCR (that is, polymerase chain reaction) testing must be performed in a P3 laboratory with biosafety protection level three. Relaxed standards also allow P2 laboratories to perform tests, but the protection means must reach P3, such as wearing level 3 protective clothing, and testers are required to operate in biological safety cabinets.
He pointed out that viral gene amplification is very easy to cross-contaminate, so the laboratory should be partitioned, and samples should be extracted, reacted, and reagents deployed in three independent rooms. The pressure gradient between the rooms forms the pressure of the room in the previous step. It should be larger than the latter step to ensure that the amplified molecules do not flow backward, otherwise, the reagents are easily contaminated and cause false positives. “This is also the most headache of the laboratory at present.”
This puts high demands on the operating standards of the inspectors. The aforementioned disease control system experts pointed out that many problems will be encountered during the sample detection process, such as sometimes the amplification curve does not come out, and it is difficult for inexperienced testers to judge whether the quality of the kit is a problem or an operational problem. “This is not something that can be trained in a day or two. It requires a lot of experience.”
Chinese CDC delegated testing authority
Since the Chinese CDC delegated testing authority to the top three hospitals in various cities on January 26, a total of 41 hospitals in Hubei Province have obtained the qualification for testing. Currently, it has expanded to 66. But in general, hospitals only have P2 laboratories, and even if they are temporarily put in place to ensure protection, the lack of professional testing staff is still a problem.
According to a report released by the official website of the Hubei Clinical Laboratory Center in January last year, the number of participants in the 16th training course for technicians of the PCR laboratory in Hubei was 268, up from 220 in the previous year.
Liu Weiyong, Department of Laboratory Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology admitted in an interview with the media that inspectors must have certain qualifications and experience, and not everyone can do it. “Insufficient staff in the laboratory, we are still those who used to be about a dozen. In addition to testing, now we have to manually organize and report a lot of patient information.”
A technical expert from Chongqing Zhongyuan Huiji Biotechnology Co., Ltd., a manufacturer of the New Crown Virus Kit, told China News Weekly that the current detection mode includes manual monitoring and automatic instrument detection. Among them, manual testing requires that the testing personnel have a relevant professional background and be proficient in operation. “The testing process is like looking at a pathological slice. It is difficult for short-term surprise training personnel to be competent for testing.”
Hubei Provincial Health and Health Committee
According to Liu Yingzi, director of the Hubei Provincial Health and Health Committee, as of February 4, 97 laboratories in Hubei Province could provide PCR testing, including 18 disease control centers at provincial and municipal levels, 66 hospitals, and 13 The third-party testing agency can achieve an average daily testing volume of 6,000.
Specific to Wuhan, as of now, a total of 31 institutions in Wuhan have been conducting nucleic acid testing at the same time, and the single-day nucleic acid sample detection capacity has been increased from the initial 200 to more than 4,000 daily. According to Wuhan City’s new coronavirus-infected pneumonia epidemic prevention and control headquarters, the highest capability of nucleic acid detection in Wuhan can currently reach up to 6000 to 8,000 copies per day.
According to the deployment of the headquarters, the nucleic acid testing of all suspected cases in Wuhan will be completed within two days from February 5 to 7.
At the press conference of the Hubei Provincial Government on February 6, Zhang Hongxing, director of the Wuhan Municipal Health and Health Committee, said that the current nucleic acid detection reagents can meet the needs, but there are still some gaps in sampling tubes, protective facilities, and sampling personnel.
The aforementioned disease control experts pointed out that at present, whether it is the increase of the capacity of the kit or the layout of the laboratory, the detection conditions have improved. The next step is more critical on how to strictly operate and improve the actual detection capacity.
He said, “You continue to decentralize the testing power to the hospital, depending on whether it has this condition and does not have this ability. It can’t meet the numbers alone, but it can’t meet the real first-line needs, whether the production capacity is met and whether it can Healing patients is two different things. “
CT image or kit test?
On February 5th, the National Health and Medical Commission released the “Pneumonitis Diagnosis and Treatment Program for New Coronavirus Infection (Trial Fifth Edition)”. One of the main changes is to distinguish the diagnostic classification criteria of Hubei Province and other provinces.
In Hubei Province, the original standard of suspected cases was split. The original “three old strips”: fever, leukocytes or lymphopenia, and CT images of the lungs were divided into two. As long as the first two are met, they can be regarded as “ “Suspect” is equivalent to relaxing the suspect standard. However, at the same time, the third lung CT manifestation was regarded as “clinical diagnosis”, which is a new type of diagnosis in Hubei. The diagnostic criteria have not changed, and the kit is still positive and the gene sequencing is homologous to the new crown virus.
This put an end to the debate between CT and kits. Zhang Xiaochun later responded by writing that instead of negating the results of nucleic acid testing, he believed that as the ultimate method of testing, he is still limited by production and sampling methods. Wuhan cannot rely on nucleic acid testing to screen patients at this stage and cut off the source of infection. Prevention and control effect.
It can be seen that she previously advocated CT testing mainly for prevention and control purposes. In that intensely vocal circle of friends, she wrote: CT should be started as soon as possible if anyone has been in contact with the suspected case. As long as the CT is positive, it should be concentrated in the hotel, hotel, or school and should not be allowed to be asymptomatic or nucleic acid negative Family watching.
Types of pneumonia, such as influenza
However, many experts have told China Newsweek that the imaging performance of CT of the New Crono Virus is similar to that of many other viral types of pneumonia, such as influenza, respiratory syncytial virus, and adenovirus infection, and it is difficult to make a differential diagnosis. Therefore, at the current level of technology, the final diagnosis depends on the kit nucleic acid detection.
Therefore, how to improve the accuracy of the kit becomes the key. In this regard, experts suggest that, in addition to sampling, transportation, and testing to be as standard as possible in operation, you can also take “double-yang testing”, that is, use two companies’ kits for testing, or multiple tests to minimize leaks.
At present, in some conditional hospitals, this more cautious testing method has begun to be adopted. A fever doctor at a tertiary hospital in Beijing told China News Weekly that in their hospital, all suspected patients must undergo a “double-yang test” before the diagnosis can be confirmed.
A doctor at a designated hospital in Wuhan also said that to improve the accuracy, the hospital would reconfirm the “double sun test”. According to the latest news from Wuhan City’s new coronavirus-infected pneumonia epidemic prevention and control headquarters, as of February 5, the second round of testing of suspected patients in Wuhan hospitals has been completed.
Emergency approval, the process from years to days
On February 3, Tian Yulong, a member of the Party Leadership Group and chief engineer of the Ministry of Industry and Information Technology, said at the State Council’s launch conference that as of February 1, the daily average kit production capacity of each company reached 773,000, equivalent to 40 of suspected patients. Times and this is only “sixty to seventy percent of the maximum capacity.”
Several kit manufacturers told China News Weekly that the gap between the supply and demand of kits quickly disappeared, thanks to the “green approval channel” launched by the State Administration of Drug Administration.
On January 22, the day when Wuhan established the epidemic prevention and control headquarters, 17 companies underwent an on-site emergency response convened by the State Drug Administration. Earlier, they received a notice that due to the severe epidemic situation of new crown pneumonia, first-line hospitals needed a large number of kits for testing in the short term, and the State Drug Administration decided to open a fast-track approval channel for special events.
A person in charge of the company participating in the defense at the time told China News Weekly that although the notification was rushed, the relevant materials must be prepared and similar to those submitted in the past when applying for such in vitro diagnostic reagents, mainly including review materials, Raw material, production process and product description, registration inspection of 3 consecutive production batches, etc. Many experts at the scene served as judges and voted after the company reported in detail. In the end, 7 companies entered the emergency approval channel.
On January 26, the State Drug Administration issued an announcement saying that four companies passed the emergency approval and the entire approval process took only four days.
New Coronavirus Kit
Since then, a total of six new coronavirus kit manufacturers have been approved for listing on January 28 and January 31, respectively: Shanghai Zhejiang, Shengxiang Biological, Shanghai Jeno, Shanghai Berger Medical, Zhongshan University Daan Gene and BGI. Pfizer is still in the process of approval.
This speed was called “unprecedented speed” by the aforementioned researchers. He said, taking the Ebola virus as an example, in the state of emergency at that time, it was approved one month after the clinical completion.
The New Coronavirus Kit, a reagent related to the detection of pathogenic pathogen antigens, antibodies, and nucleic acids, belongs to the third category of in vitro diagnostic reagents according to national regulations, which is the highest level of review in medical devices. The review by the State Food and Drug Administration has made the review process cumbersome and lengthy.
Under normal circumstances, the approval process takes at least 2 to 3 years. If it is not successful, it will sometimes be delayed to 5 years, and various materials, such as clinical data, are often required to be submitted. Previously, the stability study of the kit required at least one year, that is, it was placed in an environment of minus 20 degrees Celsius for at least one year, and it was placed for at least 14 months.
Drug Administration states
An official notice from the State Administration of Drug Administration states that although this is a special event, the approval is conducted by the principles of “unified command, early intervention, on-the-spot review, and scientific approval”, without omitting processes and lowering standards.
State Drug Administration assigned two approval consultants
The general manager of one of the first batch of approved enterprises told China News Weekly that the State Drug Administration assigned two approval consultants to each company, and the two parties jointly established a WeChat group to communicate the approval issues promptly within the group, almost in seconds. She still remembers a detail. One night at three o’clock, the other person suddenly asked in the group. Are you ready for this information? “They work hard day and night, and they want to get the company approved as soon as possible,” she said.
But the aforementioned researchers pointed out that from a few years to a few days, it is impossible to reduce the standard. According to him, the first batch of audits allowed clinical trials to be waived, which lowered the quality threshold of the kit to a certain extent. However, the researcher also acknowledged that under the current emergency, it would be impossible to copy the old regulations.
Declaration of a kit Product
According to previous regulations, the declaration of a kit product must be submitted to the medical records of a total of 500 persons from three clinically qualified medical institutions and must be signed and sealed by three units. Among them, whether to obtain the consent of the other party before using the kit for these 500 patients, which involves ethical issues, the complete plan must be submitted to the ethics committee of the medical institution for review and submitted with the medical records after approval. “This aspect of light ethics will not come in half a year,” he said.
To make up for this loophole, in the remarks column of the first batch of “Medical Device Registration Certificates”, the State Food and Drug Administration has stated that this product is only an auxiliary diagnosis and emergency reserve for pneumonia infected by New Coronavirus, which is valid for one year. Renewal of registration is required to submit clinical data. Previously, such documents were generally valid for five years.
Huang Gaojian, the chairman of Shenzhen Precon Biotech Co., Ltd. Dongguan, who is participating in the second critical review, told China News Weekly that in the second batch of applications, the requirements are higher than the first batch, and clinical trials cannot be waived. data. However, it is still lower than the conventional requirements. There is no clear requirement. The three medical institutions must be clinically qualified, which is equivalent to relaxing the scope of obtaining clinical data and does not require ethical review.
Drug Administration devolved
According to him, in the second batch of audits, the State Drug Administration devolved the preliminary screening authority to the provinces, and the provincial drug supervision bureaus organized the preliminary screening, and after passing, the province reported to the state. The pace of the whole process is also very fast, and the next day after receiving the notice from the province, he will reply. He is currently awaiting the results of national approval.
According to his understanding, there were more than 100 enterprises declared in the second batch, and there were nearly 30 in Guangdong Province alone.
Behind high capacity
Many people in the industry pointed out to China News Weekly that due to the low technical threshold of PCR detection, many biotechnology companies across the country have joined the wave of production of detection kits. This is the second batch of companies that have reported a surge in the application. It can be seen from the number that for this relative niche industry, new entrants are constantly flowing in, and there are many small companies without much experience.
Fluorescent PCR technology
The aforementioned researchers pointed out that at present, the nucleic acid detection kits being developed by various companies use fluorescent PCR technology. This technology was proposed by American scientist Kelly Mullis as early as 1983 and was subsequently applied to many fields such as virus detection, forensic appraisal, and archeology. After several versions of optimization, the technology itself has become very mature and a Complete process system.
The principle of the PCR technology is to reverse-transcribe an RNA fragment of the new crown virus into double-stranded DNA and then use DNA polymerase to amplify it. The original viral genetic information is extremely small, and it is not easy to find. It is easier to identify after amplification and then captured by a fluorescent probe. When the amplified virus concentration reaches a critical value, a fluorescent signal is generated. This means that the sample was detected as carrying the coronavirus.
This technology has been used in previous tests for viruses such as Ebola, H1N1 influenza and the Middle East respiratory syndrome. In other words, the method and technology of the kit detection is already a fixed and mature system, as long as different virus genes are applied, the technical threshold is not high.
Therefore, on January 10, after the country announced the whole genome sequence of the New Coronavirus, from January 13 to 14, many companies have announced the development of nucleic acid detection kits. It took only two days for Zhongyuan Biological to develop a new coronavirus nucleic acid detection kit. A person in charge of the company pointed out to China News Weekly that because Zhongyuan Biological has a lot of technology accumulation in the development of in vitro diagnostic reagents, Developing a new coronavirus nucleic acid detection kit is like “add one more dish”.
Several interviewed kit manufacturers told China News Weekly that their kit development took only two or three days.
Although the threshold is not high, in the view of the aforementioned disease control experts, the selection of RNA fragments (target sequences), the design of primers and probes, and the optimization of a complete set of industrial processes require enterprises to have at least five years of accumulation and precipitation. Otherwise, although the basic technical method is PCR, the quality of the kit is still uneven.
Sensitivity, and Repeatability.
He pointed out that there are three main indicators for measuring the quality of the kit, specificity, sensitivity, and repeatability. The specificity means that this kit can only detect neocrown virus, not other viruses, such as influenza or adenovirus. A high sensitivity will reduce the probability of missed detection and high detection rate. Strong repeatability means good stability. Therefore, the most important of the three indicators is sensitivity, which is also the core of the quality difference of various kits.
He said that as long as the technology of these existing companies is approved by the State Drug Administration, that is, at the technical standard level, it is tested by the China Food and Drug Inspection Institute (hereinafter referred to as the China Inspection Institute), and the specificity can generally be guaranteed. Above 99%, but the sensitivity is different. “The accreditation standards of the Chinese People’s Procuratorate are a minimum threshold.
There are several steps above this threshold. Some are higher and others are lower. Different sensitivities have a great impact on the accuracy of the test. It is very sensitive, and viruses with very low concentrations can also be detected, and there will be no missed detection. Some are not very sensitive, and false negatives will appear. “
With the development of the epidemic, the requirements for the quality standards of detection reagents are not only fast, but also to ensure high sensitivity. However, the reality is that the R & D and production of related testing reagents lack a uniform and clear industry standard.
According to the aforementioned technical experts of Zhongyuan Biotechnology, although industry standards have long existed in the diagnostic reagent industry, the standards have not refined the restrictions on categories in different products.
The new coronavirus is newly discovered
The new coronavirus is newly discovered, and it is unrealistic for the country to dominate the standards for a new crown pneumonia reagent. In his view, some companies in the industry are unavoidably hyped by kits. During the epidemic protection process, the quality of the products of the relevant testing reagent manufacturers was uneven.