On February 3, 2020, Zhang Xiaochun, deputy director of the imaging department of Wuhan University Zhongnan Hospital, issued a circle of friends: “Don’t be superstitious nucleic acid testing, strongly recommend CT imaging as the main basis for the current 2019-nCoV pneumonia”, and said it is “a front-line imaging doctor’s voice!” Her comments then attracted a lot of attention.
At a time when it is not yet possible to accurately predict the turning point of the epidemic, if the only means of diagnosis cannot be guaranteed, error seistesting and missed detection, the immediate consequence will be that many nucleic acid tests negative but still carry the virus are not isolated, they will become a new source of infection.
A typical case is a case of infection in a woman in Tianjin, after a fever on January 21, a total of three nucleic acid tests were negative, February 1 after the fourth test after the positive.
Why do kit tests frequently appear false negatives?
On this issue, a number of experts explained to China Newsweek that the test results of the kit are not only related to the quality of the kit, but also related to the characteristics of the new coronavirus itself, sampling site, sampling volume, transportation and storage links, as well as laboratory testing conditions and personnel operation, composed of a variety of reasons, very complex, but among the most critical factors, It’s related to the particularity of the virus.
Errors at each point can result in inaccurate results
A key laboratory researcher in a country specializing in nucleic acid testing told China Newsweek that due to the particularity of the new coronavirus, different sampling sites have a significant impact on detection rates.
He pointed out that the best sampling site is the lungs, because the virus is distributed in the largest concentration of the lungs, but to take alveoli irrigation liquid, complex operation, the need for instruments and various lead pipes, and the damage to people is very large, so only for the severe patients on the ventilator; Therefore, the most common and simplest sampling method is a pharynx swab, and the new coronavirus in the pharynx is the least, so it will cause a leak detection.
Another expert specializing in nucleic acid testing in the disease control system told China Newsweek that studies have shown that the new coronavirus replicates very slowly in human epithelial cells, so in the pharynx, even in the sputum, the virus’s base is very low, making it more difficult to detect.
“It’s normal for some patients to be positive several times. Because the amount of virus secreted in the early stages of the patient is very small, the later with the development of the disease, the virus increased, can be detected,” the researcher sat.
In addition, whether the sampling is accurate and the key parts are taken, it will also affect the sampling results. Take the swallow swab as an example, although it is a very simple operation, only need to take a cotton swab in the throat, but each medical staff stroke of the weight, how much is not the same. Especially in Wuhan, in the daily average detection of overload state, wearing heavy protective clothing medical staff to achieve accurate sampling, very difficult.
In addition to sampling, errors in each link, from sample transport ation to laboratory testing, can result in inaccurate test results.
Because the RNA of the virus is easy to degrade and highly sensitive, cold chain transport must be taken during the delivery process, maintained at minus 20 degrees, once frozen and thawed more than four times, the sample will be damaged. Previously, the first batch of the state drug administration approved by one of the six new coronavirus kit manufacturers, Shanghai JiangJiang Biotech Co., Ltd. has told the media that because the kit requires a storage environment of minus 20 degrees, only a few logistics enterprises with cold chain capacity can carry. A little attention during transport may affect the final test results of the kit.
Moreover, the use of kit testing has a high threshold, the laboratory conditions are also very demanding. The researchers told China Newsweek that in theory PCR (i.e. polymerase chain reaction) testing must be done in the P3 laboratory with biosecurity protection up to three levels, but because of the outbreak, in order to rapidly improve detection capabilities in the short term, the national standard will be relaxed, but also allow P2 laboratory testing, but the means of protection to reach P3, For example, wearing a level 3 protective suit, inspectors are required to operate in a biosecurity cabinet.
He pointed out that viral gene amplification is very easy to cross-contamination, so the laboratory to partition, sample extraction, reaction and deployment of reagents to be separated from each other in the room, the room formed a pressure difference gradient, the pressure of the room in the previous link is greater than the latter link, to ensure that the amplification of the molecules do not reverse, otherwise reagents are easy to be contaminated, With false positives, “this is one of the biggest headaches in the laboratory right now.”
This puts high demands on the operating specifications of the inspectors. The aforementioned CDC experts pointed out that the sample testing process will encounter many problems, such as sometimes the amplification curve did not come out, inexperienced testpersons are difficult to judge, is the kit quality problemor or operational problems. “It’s not a day or two to train, it takes a lot of experience. “
Since January 26th, when the CDC delegated testing authority to the sana-level hospitals in various cities, a total of 41 hospitals in Hubei Province have been qualified for inspection. At present, it has been expanded to 66. But the general hospital only P2 laboratory, even if temporarily on the horse, will be protected in place, the lack of professional testing personnel is still a problem.
A Hubei Provincial Clinical Inspection Center official website released in January last year, said that Hubei’s 16th PCR laboratory technical personnel on-the-job training course skilled in the number of 268 people, the previous year more than 220 people.
Liu Weiyong, a medical examiner affiliated with Tongji Medical College in Huazhong University of Science and Technology, admitted in an interview with the media that the inspectors must have certain qualifications and experience, not everyone can do. “The test section is not good enough, we are still the people before, about a dozen. In addition to testing, a large amount of patient information is now now being manually collated and reported. “
A technical expert from Chongqing Zhongyuan Huiji Biotechnology Co., Ltd., a manufacturer of new crown virus kits, told China Newsweek that the current testing model includes manual monitoring and automatic instrument testing. Among them, manual testing requires that testing personnel must have the relevant professional background, operation must also be skilled, “the testing process is like looking at pathological slices, short-term surprise training personnel are difficult to be competent for testing work.”
According to Liu Yingzi, director of the Hubei Provincial Health and Health Commission, as of February 4, the entire province of Hubei Province can provide PCR testing laboratories a total of 97, including the provincial and municipal CDC at all levels of 18, 66 hospitals, as well as 13 third-party testing institutions, the average daily testing capacity of up to 6000 people.
Specific to Wuhan, up to now, Wuhan City, a total of 31 institutions at the same time to carry out nucleic acid testing, nucleic acid single-day sample testing capacity from the initial 200, to more than 4000 copies per day. According to Wuhan City, the new coronavirus infection outbreak prevention and control command, Wuhan nucleic acid testing capacity, currently up to 6000 to 8000 copies of the day.
According to the deployment of the command, to February 5-7, two days to complete all suspected cases of the nucleic acid testing in Wuhan City.
At a press conference of the Hubei provincial government on February 6, Zhang Hongxing, director of wuhan’s health and health committee, said that the current nucleic acid testing reagents can meet the demand, but there is still a gap in sampling pipes, protective facilities, sampling personnel.
The aforementioned disease control experts pointed out that, at present, whether it is the kit to increase energy production, or laboratory layout, the testing conditions have been basically improved, the next key is, how to operate strictly, improve the actual detection capacity.
“You keep the testing authority going to the hospital to see if it has that condition, you don’t have the ability,” he said. It is not possible to satisfy the numbers alone, but not to meet the real demand on the front line, whether the capacity is met and whether the patient can be cured is two different things. “
CT imaging, or kit testing?
On February 5, the National Health and Reform Commission (NSC) released the Pneumonia Treatment Program for New Coronary Virus Infection (Trial 5th Edition). One of the most important changes is the distinction between diagnostic classification criteria in Hubei and other provinces.
In Hubei Province, the original suspected case standard was split, the original “old three”: fever, white blood cells or lymphocyte reduction, and lung CT imaging, is divided into two, as long as the first two can be considered “suspected”, equivalent to relaxing the suspected standard. But at the same time, the third lung CT performance as a “clinical diagnosis”, for Hubei’s new category of diagnosis. The diagnostic criteria have not changed, and the kit is still testing positive and gene sequencing is homologous with the new coronavirus.
This temporarily put an end to the ct and kit debate. Zhang Xiaochun later issued a reply that, not to deny the nucleic acid test results, but to think that it as the final means of testing, is still subject to production, sampling methods and other restrictions, Wuhan can not rely completely on nucleic acid testing at this stage to screen patients, to cut off the source of prevention and control effect.
It can be seen that she previously advocated CT testing, mainly for prevention and control purposes. In that heated circle of friends, she wrote: should immediately start as long as the suspected case has contact with ct, as long as CT positive near the concentration of isolation in hotels or schools, can not allow asymptomatic or nucleic acid-negative CT-positive family to stay.
However, a number of experts told China Newsweek that the imaging performance of the new coronavirus CT and many other viral pneumonia, such as influenza, respiratory syncytial virus, adenovirus infection and so on, is similar, it is difficult to make an differential diagnosis. Therefore, the current level of technology, the final diagnosis or rely on kit nucleic acid testing.
Therefore, how to improve the accuracy of the kit is the key. In this regard, experts suggest that, in addition to sampling, transportation and testing in the operation to be as standardized as possible, you can also take “double yang detection”, that is, with the two companies’ kits for testing, or multiple testing, to minimize the leakage rate.
At present, in some conditional hospitals, this more cautious approach to testing has begun. A fever clinic doctor at a Beijing Hospital told China Newsweek that in their hospital, all suspected patients have to undergo a “double-yang test” before they can be diagnosed.
A doctor at Wuhan Fixed Point Hospital also said that in order to improve accuracy, the hospital will reconfirm the “double yang test.” According to the latest news from the pneumonia epidemic prevention and control command of wuhan new coronavirus infection, as of February 5, the second test has been completed for suspected patients in Wuhan hospitals.
Emergency approval, process from years to days
On February 3, Tian Yulong, a member of the Party Group of the Ministry of Industry and Information Technology and chief engineer, said at a press conference of the State New Office that as of February 1, the average daily kit production capacity of enterprises had reached 773,000, equivalent to 40 times the number of suspected patients, and that this was only “60 percent of the maximum capacity”.
Several kit manufacturers told China Newsweek that the supply and demand gap in the kit quickly dissipated, thanks to the rapid introduction of the State Drug Administration “green approval channel.”
On January 22nd, the day the outbreak prevention and control command was established in Wuhan, 17 enterprises, convened by the State Drug Administration, conducted an on-site emergency response. Earlier, they received notice, because of the new coronapneumonia outbreak is serious, the first-line hospital in the short term need a large number of kits for testing, the State Drug Administration decided to open a fast-track approval channel, special case.
A company official involved in the defense at the time told China Newsweek that although the notice was in a hurry, the relevant materials should still be prepared, and the previous declaration of such in vitro diagnostic reagents submitted materials similar, including summary information, raw materials, production processes and product descriptions, three consecutive production batches of registration inspection. There are many experts on the scene as judges, after the detailed report of the enterprise to vote. In the end, seven companies entered the emergency approval channel.
On January 26, the State Drug Administration issued a notice saying that four companies had passed emergency approval and that the entire approval process would take only four days.
Since then, on January 28 and January 31 respectively, a total of six new coronavirus kit manufacturers have been approved for the market, namely, Shanghai Jiangjiang, Sanxiang Bio, Shanghai Jieno, Shanghai Berger Medical, Zhongshan University D’an Gene and Huada Gene. Phillip Bio is still in approval.
This speed, described by the aforementioned researchers as “unprecedentedly fast”. He said that in the case of the Ebola virus, in the state of emergency of the time, it was only a month after the completion of clinical approval.
In fact, the new coronavirus kit, which is related to the detection of pathogenic pathogen antigens, antibodies and nucleic acids, in accordance with national regulations, belongs to the third category of in vitro diagnostic reagents, that is, the highest level of examination in medical devices, needs to be reviewed by the State Food and Drug Administration, so the review process is cumbersome and lengthy.
Under normal circumstances, approval must be at least 2 to 3 years, if not smooth, sometimes delayed to 5 years, often require sintogup of various materials, such as clinical data. Previously, the stability study of the kit required at least one year, i.e. at least one year in an environment of minus 20 degrees, and actually for at least 14 months.
The official circular of the State Drug Administration states that although this is a special case, it is in accordance with the principle of “unified command, early intervention, on-the-go examination, scientific approval”, without saving the process and not lowering the standard.
The general manager of one of the first batch of approved enterprises told China Newsweek that the State Drug Administration assigned two approval and consulting teachers to each enterprise, and the two sides jointly built a WeChat group to communicate the approval in a timely manner within the group, almost every second. She remembers a detail, one night at three o’clock, the other side suddenly in the group asked, are you ready for this information? “They work around the clock, they work hard, and they really want to get the business approved as quickly as possible,” she said.
But the researchers point out that from a few years to a few days, it is impossible not to lower the standard. According to him, the first audits allowed for reductions in clinical trials, which to some extent lowered the quality threshold for the kit. But the researcher also admits that in the current state of emergency, it would not work if the old rules were copied.
According to the previous regulations, a kit product declaration must be submitted to three clinically qualified medical institutions a total of 500 people’s medical records data, and three units signed and sealed. Among them, whether to obtain the consent of the other party before the use of kits for these 500 patients, which involves ethical issues, must submit the complete program to the medical institution’s ethics committee for review, after approval and medical records data together. “The light ethics link doesn’t come in half a year, ” he said.
In order to fill this loophole, in the first batch of “medical device registration certificate” of the note column, the State Drug Administration has noted: this product is only the new coronavirus infection of pneumonia auxiliary diagnosis and emergency reserve, valid for 1 year. The clinical data should be replenished when continuing registration. Previously, such documents were generally valid for five years.
Huang Gaojian, chairman of Shenzhen Prikang Biotech Limited, who is participating in the second review, told China Newsweek that the second batch of filings required higher requirements than the first batch, could not be reduced from clinic, and that data from three institutions should be submitted. But it is still lower than the usual requirements, there is no explicit requirement that the three medical institutions must be clinically qualified, equivalent to relaxing the scope of access to clinical data, and do not require ethical review.
According to him, in the second batch of audit, the State Drug Administration to the primary screening authority delegated to the provinces, first by the provincial drug regulatory bureau to organize the initial screening, after the adoption of the province to the state report. The whole process rhythm is also very fast, received the provincial notice the next day to reply. He is currently awaiting the results of the country’s approval.
According to his understanding, the second batch of declared enterprises more than 100, Guangdong Province alone has nearly 30.
Behind the high capacity
A number of industry insiders to China Newsweek pointed out that because the technical threshold of PCR testing is not high, at present, a number of biotechnology companies across the country have joined the wave of testing kits, which from the second batch of declared surge in the number of enterprises can be seen, for this previously relatively small industry, the influx of new entrants, There is no shortage of small businesses with little experience.
The researchers pointed out that, at present, enterprises are developing and producing nucleic acid testing kits are basically using fluorescent PCR technology. The technology was proposed by American scientist Kelly Mullis in 1983 and later applied in a number of fields such as virus detection, forensic synoding and archaeology, and has since been optimized in several versions, and the technology itself is very mature and has a complete process system.
The principle of PCR technology is to reverse a fragment of RNA from a new coronavirus into double-stranded DNA, which is amplified by DNA polymerase. The original viral genetic information is extremely small, not easy to detect, amplifyed more easily identified, and then captured through a fluorescent probe, when the amplification of the virus concentration reaches a critical value, will produce a fluorescent signal. This means that the sample was found to carry the new coronavirus.
The technique has been used in previous tests for viruses such as Ebola, influenza A(H1N1) and MERS. That is to say, the method and technology of kit testing is already a fixed mature system, each time as long as the use of different virus genes on the line, the technical threshold is not high.
As a result, on January 10th, after the country announced the entire genome sequence of the new coronavirus, several companies announced on January 13-14 that they had developed nucleic acid testing kits. Zhongyuan Bio in the new coronavirus nucleic acid testing kit only took two days to develop, a person in charge of the company told China Newsweek, because of the in vitro diagnostic reagent research and development of Chinese meta-organisms have a lot of technical accumulation, the development of a new coronavirus nucleic acid testing kit is better than “add one more dish.”
Several of the kit manufacturers interviewed told China Newsweek that they only took two or three days to develop the kit.
Although the threshold is not high, in the aforementioned CDC experts, in the selection of RNA fragments (target sequence), primer and probe design, as well as a set of industrial process optimization, requires 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.
He points out that there are three main indicators for measuring the quality of the kit, specificity, sensitivity and repeatability. Good specificity means that the kit can only detect new coronaviruses, not other viruses, such as influenza or adenoviruses. High sensitivity, will reduce the probability of leakage, high detection rate. Strong repeatability means good stability. Therefore, the most important of the three indicators is sensitivity, which is at the heart of the differences in the quality of different kits.
He said that the existing technology of these companies, as long as it is approved through the State Drug Administration, that is, at the technical standard level through its subsidiary of the China Food and Drug Research Institute (THE Chinese Institute of Food and Drug Inspection) test, specificity can generally be guaranteed at more than 99%, but sensitivity varies. “The Chinese Academy of Inspection’s accreditation is a minimum threshold, after this threshold there are several steps above, some higher, some lower. Different sensitivity, the accuracy of the detection has a great impact, some are very sensitive, the concentration of low-concentration virus can also be detected, will not leak detection, some are not very sensitive, there will be false negative. “
With the development of the epidemic, the requirements for testing reagent quality standards are not only fast, but also require high sensitivity. The reality, however, is that there is a lack of uniform and clear industry standards for the development and production of relevant testing reagents.
According to the aforementioned technical experts, in the diagnostic reagent industry, although the industry standard already exists, but the standard for different products in the category of restrictions have not been refined. The new coronavirus is newly discovered, let the country to lead to do a new coronary pneumonia reagent standard, in the short term is not realistic. In his view, the current industry can not avoid some enterprises with the kit gimmick stoics for speculation. In this outbreak protection process, the relevant testing reagents between the products, the quality of the phenomenon is uneven.