Produced by: Sina Technology
Compiled by: Muir
Who is sick? How serious is it? How fast does the disease spread? In the United States, there is no satisfactory answer for the simple reason that there are not enough kits.
As of March 16, there were 5 confirmed cases of new coronavirus in Ohio. But Amy Acton, the director of Ohio Health Bureau, thinks that this is only a small part of the dangerous iceberg. "We know that the fact of community communication shows that at least 1% of the population in Ohio may be carriers of the virus," Acton said at a news conference on Thursday, "while the total population of our state is 11.7 million." In other words, according to Acton's speculation, there are 117000 infected people in Ohio alone.
How did Acton know this? Theoretically, she doesn't know. She can only infer from the limited data on the pandemic currently available to scientists. Official data show that there are 130000 confirmed cases worldwide. Therefore, the potential six digit cases in Ohio are really shocking, not to mention the potential cases in other states of the United States.
Whether this data is correct or not is what public health workers are eager to know. Who is sick? How serious is it? How fast does the disease spread? But in the United States, there is no satisfactory answer. The reason is simple and terrible: there are not enough kits. Unlike influenza, or other previous coronaviruses such as SARS or MERS, or sexually transmitted diseases, or other infections, health workers have no way to judge whether the person sitting in front of them is a new coronavirus carrier. In fact, there are some detection technologies, and they are not very complicated. Only the kit is not enough. At present, four months have passed since the outbreak of the epidemic, and two weeks have passed since the first case of "community transmission" was found in the United States. Doctors and health workers are still unable to test everyone for COVID-19. (This situation may eventually change. On March 13, the US Food and Drug Administration (FDA) approved the two companies to conduct commercial testing. The two companies are Swiss Roche Pharmaceuticals And medical device manufacturers Thermo Fisher 。 The rich experience of these two companies in medical testing and sales means that it is not impossible to provide 200 kits in real time.)
The American Enterprise Institute has a project to estimate the current total detection capacity of the United States. The project believes that the total inspection capacity should be more than 22000 times a day - about twice the number of daily inspections in South Korea. Such a big gap limits scientists' understanding of diseases and their research on treatment measures. The United States may usher in a very terrible infection curve, with the number of infected people growing exponentially. A large number of Americans may develop into severe patients and need to be hospitalized, and some of them are likely to die. In January or February, no one of us started to carry out enough tests to prevent the gradual change; Now, there is still not enough detection to find out the seriousness of the future. There are both scientific and political reasons for this, and the solution of problems in the future also requires both science and politics.
Broadly speaking, the response to the outbreak of infectious diseases includes two stages: containment and mitigation. The containment phase begins at the beginning of case detection. The public health staff responsible for supervision need to check one by one, identify the people who may have contact with the confirmed patients, inform them and isolate or treat them to reduce further transmission. But there is a problem here: if it is impossible to detect whether the contact person is infected, then health workers will not be able to effectively prevent further transmission.
In the second stage, the disease has spread, and the public health system has been unable to investigate and deal with individual cases. People need to know the dangers they will face. However, if a large number of people cannot be detected, people will not be able to calculate how many patients need hospitalization or how many patients may die. These data will be displayed by region. For this new coronavirus epidemic, according to the depth of detection in various countries and their actual published data, the case fatality rate ranges from 1% to 15%. There are various factors that cause differences, ranging from the overall health status of the population to the national health care system. It would be great if we could know which mortality rate is more accurate.
As the epidemic prevention has risen from the individual level to the whole population level, various mitigation measures, such as maintaining social distance, canceling large-scale activities, closing schools and notifying people to work at home, are not only to reduce the number of infected people, but also to "smooth the curve" - epidemiologists explained. This curve represents the number of patients in a given time. The most ideal state at present is to reduce the "tsunami" curve to a "ripple" shape as far as possible. In addition to reducing the total number of infected people, it is necessary to lengthen the time interval. This is because American hospitals have limited personnel, equipment and space: 2.9 beds per 1000 people on average. This data is not as good as that of Italy, but better than that of Iran. The medical systems of both countries are undergoing severe tests. In addition, it is also to gain more time for research and development of treatment plans.
Working principle of new coronavirus detection
When testing new viruses such as SARS Cov-2, the first wave of diagnosis almost all relied on two very important technologies, although not very modern.
The first technology is called polymerase chain reaction (PCR), which is a DNA amplification technology, usually used in the laboratory, and can amplify target DNA in a short time for analysis and research. In 1983, Kary Mullis invented the polymerase chain reaction, which uses heating and cooling cycles to replicate a large number of DNA with few samples. For this reason, Morris won the 1993 Nobel Prize in Chemistry. In combination with the fluorescent stain of labeled DNA, PCR can let scientists know the specific amount of DNA. PCR technology can be used to effectively judge the presence of pathogens (whether in the host or on the surface of objects).
However, since the genome of SARS CoV-2 and other viruses is composed of single stranded RNA, scientists need to transform the virus genome into double stranded DNA before detection. At this time, they need the help of reverse transcriptase. The combination of these two technologies is reverse transcription polymerase chain reaction (RT-PCR).
At present, RT-PCR is the only method to detect whether a person is infected with COVID-19. Other detection methods are unable to distinguish COVID-19 from influenza virus, let alone COVID-19 from more than a dozen other viruses that cause respiratory diseases at this time of year. Louis Mansky, director of the Institute of Molecular Virology at the University of Minnesota, said: "(RT-PCR) is a standard and reliable technology commonly used in microbiology laboratories. It can be quickly applied to clinical tests, and is probably the fastest developed detection method."
However, the problem is that until other detection methods are developed and approved, all new coronavirus detection can only be carried out in the laboratory by specially trained technicians. Testing requires PCR equipment and trained equipment operators, which means that NCV testing cannot be carried out in clinics or patients' homes. However, Mansky also said that because PCR plays an important role in the biological community, many research laboratories in universities and hospitals are equipped with necessary equipment and personnel. Only in the United States can clinical samples be processed by laboratories certified by the Federal Health Insurance and Medicaid Service. This so-called certification process takes several months. Kalorama Information is a market research company specializing in medical diagnosis. Bruce Carlson, the publishing director of the company, said: "We have a very cumbersome regulatory system for medical testing, and we are worried about both false and false reports."
If the reagents are complete, the results can be obtained in one day. However, reagent shortage and transportation logistics will cause a test to take days or even weeks to produce results. (In fact, this problem already exists, and we will discuss it later.) Next, let's go through the normal process:
The first step is to collect samples. The medical staff wiped the nasal cavity or the back of the throat of the patient with a sterile swab in order to collect the material that had stayed in the lungs before. At present, it is generally believed that the virus replicates in the lungs. After the collection, the swabs are sealed, packed in refrigerated containers and sent to the testing laboratory. The storage temperature of samples must be between 1.7 ℃ and 4.4 ℃. In addition, if the samples are not processed within four days after collection, they need to be put into the freezer or discarded.
After the sample is sent to the laboratory, the first step is to isolate RNA from the sample. Other substances in the sample - human cells, proteins, enzymes, etc. will destroy the genetic code of the virus. This step is called RNA extraction. To extract RNA manually, chemical substances need to be added, and then RNA is separated by centrifugation. Some large biochemical suppliers will provide kits that contain everything needed for RNA separation. There are also automated machines that can perform RNA separation.
After RNA is separated, reverse transcriptase is added to convert single stranded RNA into double stranded DNA. Next, put the processed DNA, together with nucleotide, DNA polymerase and a short DNA synthesis segment (namely "primer"), into the test tube. The role of these primers is to discover and combine specific segments of the virus genome. In short, if everything is normal, they should only recognize and amplify the genetic material of the virus, ignoring all other "impurities" in the sample, such as human or bacterial DNA.
All these are complete in the PCR instrument. The PCR instrument can control the temperature cycle. When the test tube is heated, the double helix structure of DNA splits into two single strands, and one side of each single strand is exposed. Then, the test tube was cooled, and the primer was paired to the exposed side of the single strand. Then, DNA polymerase uses primers as the starting point of DNA replication, and starts to construct complementary strands according to the structure of single strands. Five minutes later, a complete double strand of DNA is duplicated. After 30 to 40 cycles, one DNA structure can be expanded to millions, enough for scientists to detect it.
The detection process is as follows: In the PCR amplification phase, scientists will add fluorescent dye to the test tube. These dyes will only glow when they encounter DNA, and can be used to mark DNA well. With the increase of the amount of DNA, the fluorescence intensity also increases. The special light measuring instrument inside the PCR instrument can read these fluorescence images to determine which samples have viruses and which have no viruses. "If there is a coronavirus in a sample, its RNA will be transcribed into DNA, and then replicated and amplified. Finally, the fluorescent signal will tell us whether the test result is positive or negative," Mansky said.
Protocol development
The important thing about RT-PCR is that it is not a test for a certain virus; It is a method used to identify specific gene sequences, which is widely used in academic, commercial and public health laboratories around the world. Moreover, the specific "formula" scientists use to obtain reliable results - which RNA extraction kit to use, which PCR instrument, and which primers - also varies. These specific "formulas" are called "agreements".
When there are new diseases, such as the new coronavirus disease this time, universities, national research institutions and public health organizations (such as the National Center for Disease Control and Prevention, etc.) often formulate RT-PCR agreements at the first time. They have biosafety laboratories that can handle deadly new pathogens (including the ability to cultivate pathogens), which is a key step to verify the effectiveness of various tests. Once the institutions have a preliminary test, they can transfer the test to local public health laboratories and hospitals. Finally, if the epidemic continues to spread, commercial laboratories and diagnostic companies will also produce their own test kits.
Since January this year, Chinese researchers have taken the lead in releasing the first full genome sequence of SARS CoV-2. Shortly thereafter, organizations around the world began to design, test and publish RT-PCR protocols that had been used to detect new coronaviruses. As an agency providing resources to testing laboratories, the World Health Organization has been updating the list of protocols and best practice guidelines.
Among them is the agreement developed by the US Centers for Disease Control and Prevention. The detection of the United States Centers for Disease Control and Prevention includes four sets of primers. The first two sets of primers are called "N1" and "N2" respectively, targeting the unique coding region of the SARS CoV-2 genome, which is related to the protein that wraps and protects the viral genetic material. The third set of primers targeted at the common genes of SARS like virus genus. The fourth group (and the last group) of primers targeted at human genes can be used for positive quality control. In theory, every test should 100% identify the human gene target; If not, it indicates that there is a problem in the detection. Some DNA unrelated to SARS CoV-2 will not react with the first three primers in theory, and the kit also has corresponding negative control instructions. In January, the United States Centers for Disease Control and Prevention began to use this protocol to detect viruses in people with travel history in Wuhan.
On February 4, two weeks after the U.S. Centers for Disease Control and Prevention detected the first case of COVID-19 in the United States, FDA issued an emergency authorization to directly allow national certified public health laboratories to detect the virus, bypassing the usual regulatory channels. CDC will pack four groups of primers, together with relevant fluorescent samples, into a kit, and each kit can handle 700 to 800 samples. From February 5, through the department's own distribution agency, International Agent Resource, CDC officially began to send 200 kits to 115 public health center laboratories qualified for testing.
However, this is where things start to drift.
It is full of loopholes and chaos, adding fuel to the red tape
After the kit was delivered to the state laboratories, local scientists began to verify the test results. It involves using the kit to detect known samples to ensure that the kit can correctly detect SARS-CoV-2 without mistakenly labeling uninfected samples. But many laboratories have encountered various problems. In some laboratories, the negative control does not work; In other laboratories, the test results of those that should have been positive are unknown.
On February 12, the CDC announced that the problem was reagent. The third set of primers that should have marked the common gene of coronavirus did not work properly. CDC officials informed the laboratory, "Don't worry, the new kit will be delivered immediately.". As a result, weeks later, only a few sporadic laboratories in a few states were able to detect COVID-19. In other places, health departments can only send samples of suspected patients directly to the CDC for testing. Unfortunately, at that time, the testing standards of the CDC were very strict: only patients with obvious symptoms and a history of travel in China were eligible for testing. In this regard, a report in the New York Times said that this meant that many infected people could not be found in a timely manner at the critical early stage when the virus spread to the United States.
While the United States Centers for Disease Control and Prevention tried to send more kits, other countries affected by the epidemic began to adopt other agreements announced by WHO. By contrast, until February 25, only 12 laboratories in five states in the United States were able to detect COVID-19, except for the Centers for Disease Control and Prevention. According to the website archive of the CDC, only 426 people in the United States were tested at that time. (Since March 2, the agency will not release the number of people tested.) At the same time, it is reported that the number of people tested in South Korea every day is 15000 - equivalent to the total number of people tested in the United States so far.
In principle, as long as the equipment is complete, any qualified laboratory in the United States should be able to detect viruses, including hospitals and commercial laboratories. All they need to do is use any agreement issued by WHO to order primers, and then start new coronavirus detection. But in fact, due to the existence of various new red tape, they can do nothing but wait for the CDC to allocate more kits.
Why is this? This will start with the meeting in January. At that time, the federal medical and health institutions declared COVID-19 as a public health emergency, and then triggered a series of rules, in which the package required that all tests must be reviewed by the FDA's "emergency use permit" procedure. These rules raise the threshold for kit development and independent laboratory detection of viruses. In non emergency situations, FDA usually does not interfere with such testing. However, according to the emergency rules, any laboratory that wants to detect COVID-19 must first conduct a series of validation studies and submit the data to FDA for review. However, virus samples are required for validation research; But it is not easy to obtain virus samples without CDC.
In order to maintain consistency, FDA has chosen to open emergency permits only to CDC kits, in order to ensure unified supervision of health departments in states, counties and cities. "The detection strategy adopted by the government is very narrow. In other words, even if the kit is effective, the detection ability will not be significantly improved in a short time," said Joshua Sharfstein, a health policy researcher at Johns Hopkins School of Public Health, "What the government means is that we can only use the kit developed by the CDC. Moreover, this kit can only be packaged and distributed by the CDC, and can only be tested by state laboratories."
As a result, all laboratories in the United States can only use the kit sent by the CDC to detect COVID-19. They cannot order their own primers and fluorescent reagents, even if the primers and fluorescent reagents they buy are the same as those used by CDC. To make matters worse, when the CDC kit was defective, the lower level laboratory had no choice but to wait.
At this moment, no one except the government knows why the United States does not use the detection method developed for WHO. This detection method has been used in more than 120 countries. A WHO spokesman refused to answer the question, and US lawmakers such as the Minister of Health and Human Services, Alex Azar, have always avoided answering the question. It is conceivable that bureaucracy plays a major role - it may take a long time for the test to be approved in the United States. Others suspect that political factors are at fault. Dan Diamond, a health policy reporter, said in a program that President Trump intended to delay the efficiency of detection because too many confirmed cases would affect his re-election plan.
On February 29, with the public health disaster becoming more and more serious, the pressure to improve the detection ability is also increasing. At this time, FDA had to change its regulatory provisions to allow qualified clinical laboratories to develop and use their own kits to detect COVID-19 without prior approval. Shafstein said: "The emergence of community transmitted cases on the west coast has alarmed people. By the end of the week, the FDA can only relax."
Under the new policy, the laboratory still needs to submit the data to FDA for review. But now, after internal verification and testing, they can have two weeks to prepare the data to be submitted to FDA. During this period, the detection of patient samples can also be carried out synchronously.
According to the data compiled by the American Enterprise Institute, after the relaxation of the agreement, Quest Diagnostics and LabCorp, two large commercial diagnostic laboratories, have begun to test thousands of samples every day. A spokesman for Quest Diagnostics also said that it was expected that by the end of March, the company could test tens of thousands of samples every week. Universities and hospitals have also started to take action.
However, the problem of supervision has now been solved, but logistics has put forward new challenges. When more and more laboratories open online applications, almost every step of RT-PCR testing will encounter bottlenecks. Eric Blank, the chief program officer of the Public Health Laboratory Association, said that the biggest problem at present was the RNA extraction kit. "Everyone is snapping up these auxiliary materials. We have learned from member laboratories that many materials are now unavailable."
The RNA extraction kit recommended by the CDC's detection protocol is supplied by Qiagen. However, some media reported on March 10 that these kits are currently in the state of being replenished due to the accelerated detection of COVID-19 in the world in the past few weeks. It is reported that automated RNA extraction equipment and RT-PCR instruments are also out of stock. CDC also has requirements for equipment used to detect COVID-19, and not all laboratories have approved equipment. Those laboratories that lack approved equipment are now trying to purchase qualified equipment.
Qiagen executives also said in the email that they were recruiting new employees, expanding the production capacity of European factories, taking three shifts every day, and stepping up production seven days a week. The FDA has recently further relaxed its regulation to allow laboratories to use kits supplied by Roche Pharmaceuticals, another European manufacturer. However, the current shortage of supplies is expected to further affect the ability of the United States to timely diagnose patients with COVID-19. Blanc said: "There is no quick solution to this problem. It may take several weeks to solve the supply chain problem, that is, in a short time, the supply of testing will always be in short supply."
Testing technology under development
More kits can certainly improve detection capability. Eventually, new detection technologies may also help.
At least 20 companies have announced plans to develop "molecular point of care" testing methods. These detection methods are mainly automated systems, which can enable front-line medical staff to obtain detection results within half an hour without waiting for several days or even longer. "You can collect a patient's nasal swab or throat swab, put it into a container, put the container into the device, and press the button. After 30 minutes, you can get the diagnosis report, which is their goal," Carlson said.
Cepheid is a company that produces such detection tools, and their products have been used for diagnosis of other diseases. At present, the detection tool of COVID-19 has been under development. Similar detection tools developed by Coyote Bioscience have been deployed in China. There is even a detection method under development that can simultaneously detect COVID-19 and influenza virus.
Startups such as Sherlock Biosciences and Mammoth Biosciences are also considering using CRISPR based diagnostic methods to detect COVID-19. These tests use CRISPR's programmable gene search function, and can get results in an hour without tedious laboratory instruments. Mammoth released a preprint article on March 10, referring to a detection technology developed by the company for SARS-CoV-2, which can be used together with the detection test paper. At present, the company is further verifying the test results.
Another very marketable method is to find virus antibodies from patients' blood, which is called serum testing. This method is naturally good, because in addition to detecting COVID-19, it can also tell you whether the other party has ever been infected with the virus, but it has now been cured. Martin Hibberd, an infectious disease researcher at the London School of Hygiene and Tropical Medicine, was once involved in the detection and research of SARS virus. Only after we have fully tested this type of detection method can we know the exact rate of missed diagnosis. "
Serum detection may be cheaper than PCR based detection and more suitable for automation and high pass Quantity detection. Hibbard said that the accuracy of serum tests may not be as good as molecular tests, but on the other hand, serum tests previously used to detect SARS virus can recognize SARS CoV-2 virus. It remains to be seen whether this is useful for developing new detection methods.
What does this have to do with melon eating people?
At present, the different detection capabilities in different places mean that if you feel uncomfortable, whether you can accept the detection of COVID-19 depends largely on where you live. It is reported that some hospitals do not provide COVID-19 testing because they do not have enough masks to protect the medical staff who collect patient samples. Other hospitals adopt quantitative testing, that is, only severe patients are tested for COVID-19, and others with mild symptoms are recommended to be treated at home. At present, each state, county and some cities have their own policies to specify who can accept the test. "Our system can't keep up with the current demand," Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases, said at the congressional hearing last week. "This is a failure that we must admit."
At the White House press conference on Friday, President Trump tried to remedy this failure. He announced that, in view of the COVID-19 epidemic, the country has entered a "national emergency" so that states and regions can flexibly respond to funding and regulatory issues. The President also announced that Wal-Mart We have established public-private partnerships with a number of large stores and pharmacies across the country, including CVS, allowing them to reserve some parking spaces as "drive in detection" channels. The cooperation with Roche Pharmaceutical, Quest, LabCorp and other testing companies means that "we will be able to complete millions of tests in a very, very short time," President Trump said. Vice President Mike Pence said that they expected to conduct 15000 to 20000 tests every day.
In addition, a new detection system will be launched soon. Burns said that in two days, he will release the online time of the new website. It is said that this website is Google Development allows people to input their own symptoms, and then if certain conditions are met, they can receive the location information of nearby parking lots that provide drive in detection services. Good is good, but it seems that you must have symptoms to test for COVID-19, which does not solve the broader epidemiological problem. Later, Google also denied that the company or Verily, a medical and health company under Alphabet, was developing similar technologies, but Verily did plan to test a small d pilot project for medical workers in the Bay Area.
After the press conference, how to help solve the problem of testing and treatment costs remains unresolved. Also, if Google's website is really online, how to protect people's personal information, and how to deal with it after the test result is positive?
During this period, as ordinary people, what we can do is to keep a distance from people and avoid attending large gatherings. If you can work from home, don't go anywhere. If you find yourself in contact with a confirmed patient, please isolate yourself at home for 14 days. If you feel unwell, please isolate yourself at home for 14 days. If the symptoms are not serious, there is no need to go to the hospital. Wash your hands frequently with soap for at least 20 seconds. If possible, disinfect your mobile phone and other supplies frequently. Don't touch your face. Finally, pay attention to your neighbors. If there are elderly and sick people in the family, they may need help.
This pandemic affects every one of us. What we can do is to try our best to help those around us who need help.
Authors: Megan Molteni, Adam Rogers
Original link: https://www.wired.com/story/everything-you-need-to-know-about-coronavirus-testing/
