Three questions Meixin crown detection: how to operate? Why detection? Why can’t you check it out?

As of March 16, there were five confirmed cases of new coronavirus in Ohio. But according to Amy Acton, Ohio’s health secretary, this is just a fraction of the tip of the dangerous iceberg. “We know that the fact that the community is spreading suggests that at least 1 percent of the population of Ohio may be carriers of the virus,” Acton said at a news conference Thursday. In other words, acton’s guess suggests that there are 117,000 people infected in Ohio alone.

Three questions Meixin crown detection: how to operate? Why detection? Why can't you check it out?

Product: Sina Technology

Compilation: Muir

Who’s sick? How serious is the illness? How fast does the disease spread? In the United States, there is no satisfactory answer, for a simple reason to elaborate: there are not enough kits.

How did Acton know that? In theory, she didn’t know. She can only infer from the limited data available to scientists on the pandemic. Official figures show a total of 130,000 confirmed cases worldwide. So the potential six-figure number of cases in Ohio is appalling, not to mention the number of potential cases in other U.S. states.

Whether this figure is correct or not is what public health workers are eager to know. Who’s sick? How serious is the illness? How fast does the disease spread? Only in the United States, there is no satisfactory answer, the reason is simple and percep, the reason is very much: 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 of determining whether the person sitting in front of them is a carrier of the new coronavirus. The detection technique is actually there, and it is not very complicated. It’s just that the kit isn’t enough. Now, four months after the outbreak, two weeks after the first “community-borne” case struck in the United States, doctors and health workers are still unable to test for the new coronavirus. (That may eventually change.) On March 13, the U.S. Food and Drug Administration (FDA) approved commercial testing by both companies. The two companies are Swiss Roche Pharmaceuticals and medical device manufacturer Thermo Seiol. The two companies’ extensive experience in medical testing and sales means that providing 200 kits on the fly is not a pipe dream. )

The American Enterprise Institute has a project to estimate the current total testing capability in the United States. The project believes the total testing capacity should be more than 22,000 a day — about twice the number of daily tests in South Korea. Such a large gap limits scientists’ understanding of disease and their research into treatments. The U.S. may be on a very scary infection curve, with an exponential increase in the number of people infected. A large proportion of Americans are likely to develop critically ill patients and will need hospital treatment, and some of them are likely to die. In January or February, none of us started to do enough testing to prevent micro-duds; The reasons are both scientific and political, and the solution of future problems also requires a combination of science and politics.

Broadly speaking, there are two phases to respond to infectious disease outbreaks: containment and mitigation. The containment phase begins at the beginning of the case discovery. Public health staff who oversee the investigation need to check, identify people who may have been in contact with a confirmed patient, notify them and provide them with isolation or treatment to reduce further transmission. But here’s the problem: if it’s impossible to detect if a contact person is infected, then health workers can’t effectively stop further transmission.

In the second phase, the disease has spread, the public health system has been unable to investigate and deal with individual cases, and people need to know the dangers they are about to face. But if large numbers of people cannot be tested, it is not possible to work out how many patients need hospitalization or how many are likely to die. These data are shown by region. In response to this new coronavirus outbreak, according to the depth of testing in each country and the data they actually published, the fatality rate ranged from 1% to 15%. The factors that make the difference range from the overall health of the population to the national health care system. It would be nice to know which disease death rate is more accurate.

As the epidemic has risen from an individual to a population level, mitigation measures such as social distance, cancellation of large-scale events, school closures and notification of people working from home are not only designed to reduce the number of people infected, but also to “smooth the curve”, the epidemiologist explains. The curve represents the number of patients in a given time, and the ideal state at the moment is to try to reduce the “tsunami”-like curve to a “one-piece” shape, which, in addition to the necessary reduction in the total number of infected people, lengthens the time interval. That’s because U.S. hospitals have limited staff, equipment and space: an average of 2.9 beds per 1,000 people. That’s not as good as Italy’s, but better than Iran, where health systems are being tested. In addition, it is also to develop treatment options, to buy more time.

How new coronavirus detection works

In testing new viruses such as SARS-Cov-2, the first wave of diagnostics almost all relied on two techniques that were not very modern but very important.

The first technique, called polymerase chain reaction (PCR), is a DNA amplification technique, commonly used in the laboratory, to amplify the target DNA in a short period of time for analysis. Kary Mullis invented polymerase chain reactions in 1983, using heating and cooling cycles to replicate large quantities of DNA from rare samples. Mullis won the 1993 Nobel Prize in Chemistry. Combined with fluorescent dyes that can be labeled with DNA, PCR allows scientists to know the exact amount of DNA. PCR technology can be used to effectively determine the presence of pathogens, whether in the host body or left on the surface of an object.

However, because the genomes of viruses such as SARS-CoV-2 are made up of single-stranded RNA, scientists need to turn the virus’s genome into double-stranded DNA before testing it. At this point, they need reverse transcriptase to help. Combining these two techniques is the reverse transcription polymerase chain reaction (RT-PCR).

Currently, RT-PCR is the only way to detect whether a person is infected with a new coronavirus. Other tests fail to distinguish between neo-coronaviruses and influenza viruses, let alone from more than a dozen other viruses that cause respiratory diseases at this time of year. “RT-PCR is a standard and reliable technique commonly used in microbial laboratories that can be used quickly in clinical testing and is probably the fastest-growing test,” said Louis Mansky, director of the Institute of Molecular Virology at the University of Minnesota. “

The problem is that until other tests are developed and approved, all new coronavirus tests can only be performed in the lab by specially trained technicians. Testing requires PCR equipment and trained equipment operators, which means that new coronavirus testing cannot be performed in clinics or patients’ homes. But Mansky also says that because of its role in biology, many university and hospital research laboratories are equipped with the necessary equipment and personnel. Only in the United States can clinical samples be processed in laboratories accredited by the Federal Health Insurance and Medicaid Services Center. This so-called certification process takes months. Kalorama Information is a market research firm specializing in medical diagnostics. Bruce Carlson, the company’s head of publishing, said: “We have a very cumbersome regulatory system for medical testing, both for misstatement and for misreporting. “

If the reagents are complete, the results can be produced in one day. But a shortage of reagents and transportation logistics can lead to a test that takes days or even weeks to produce results. (In fact, this problem already exists, and we will go into more detail later.) Let’s go through the normal process:

The first step is to collect samples. Paramedics wipe the patient’s nasal cavity or back throat with a sterile swab to collect substances that have been stopped in the lungs before. At present, it is widely believed that the virus replicates in the lungs. Once collected, the swabs are sealed and placed in a refrigerated container and sent to the testing laboratory. The temperature of the sample must be kept between 1.7 and 4.4 degrees C. In addition, if the sample is not processed within four days of collection, the sample needs to be placed in a freezer or disposed of.

Once the sample is sent to the lab, the first step is to isolate the RNA from the sample. Other substances in the sample — human cells, proteins, enzymes, etc. — can destroy the virus’s genetic code. This step is called RNA extraction. Manual extraction of RNA requires the addition of chemicals and the use of centrifugal separation to extract rna. Some large biochemical suppliers provide kits with everything they need for RNA isolation. There are also automated machines that can complete RNA separation.

When RNA is isolated, reverse transcriptase is added to the inside, turning single-stranded RNA into double-stranded DNA. Next, the treated DNA, along with nucleotides, DNA polymerases, and a small piece of DNA synthesis fragments (i.e. “primers”) is placed in the test tube. The role of these primers is to discover and bind specific fragments of the virus genome. In short, if all goes well, they should only identify and amplify the viral’s genetic material, ignoring all the other “impurities” in the sample, such as human or bacterial DNA.

It’s all in tact in the PCR instrument. The PCR instrument controls the temperature cycle. When the test tube is heated, the double helix structure of THE DNA is split into two single chains, with one side of each single chain exposed. The test tube is then cooled, at which point the primer is paired to the bare side of the single chain. DNA polymerases then use primers as the starting point for DNA replication, starting to build complementary chains based on the structure of the single strands. Five minutes later, a complete DNA double-strand is replicated. After 30 to 40 cycles, a DNA structure can be expanded to millions, enough for scientists to test it.

The testing process is this: during the PCR amplification phase, scientists will add fluorescent dyes to the test tube. These dyes only glow when exposed to DNA and can be used well to mark it. As the amount of DNA increases, so does the intensity of fluorescence. Special photometers inside the PCR instrument can read these fluorescence maps to determine which samples have viruses and which do not. “If there is a coronavirus in a sample, its RNA is transcribed into DNA, which in turn replicates amplified, and the final fluorescent signal tells us whether the test results are positive or negative,” Mansky said.

Protocol development

With regard to RT-PCR, it is important that it is not a test for a particular virus; And the specific “recipes” that scientists use to get reliable results — which RNA extraction kit is used, which PCR instrument, and which primers — vary. These specific “recipes” are what we call “agreements”.

When new diseases occur, such as this time, universities, national research institutions and public health organizations (such as the National Centers for Disease Control and Prevention) tend to develop RT-PCR protocols in the first place. They have biosafety labs that can handle deadly new pathogens, including the ability to grow pathogens, a critical step in verifying the effectiveness of various tests. Once the agency has a preliminary test, they can send it to local public health laboratories and hospitals. Eventually, if the outbreak continues to spread, commercial laboratories and diagnostic companies will produce their own test kits.

Since January, Chinese researchers have been the first to release the first genome-wide sequence of SARS-CoV-2. Shortly thereafter, organizations around the world began designing, testing and publishing RT-PCR protocols that had been used to detect new coronaviruses. The World Health Organization, as an agency that provides resources to testing laboratories, has been updating the protocol list, as well as best practice guidelines.

Among them is the agreement developed for the U.S. Centers for Disease Control and Prevention. The CDC’s testing includes four sets of primers. The first two groups of primers, called “N1” and “N2”, are unique lying areas of the SARS-CoV-2 genome that are related to proteins that wrap and protect viral genetic material. The third group of primers targets common genes of sars-like virus genus. The fourth (and final) primer was targeted at the human gene and could be used as positive quality control. In theory, each test should identify 100% of the human gene target; Some DNA that is not related to SARS-CoV-2 theoretically does not react with the first three primers, and there is a corresponding negative control statement for this kit. The U.S. Centers for Disease Control and Prevention began using the protocol in January to test people with a history of travel in Wuhan for virus testing.

On February 4, two weeks after the CDC detected the first new coronavirus patient in the United States, the FDA issued an emergency mandate to directly allow the virus to be tested in accredited public health laboratories across the country, across the usual regulatory channel. The CDC packages four sets of primers, along with associated fluorescent samples, into a kit that can handle 700 to 800 samples. Starting February 5, the CDC officially began sending 200 kits to 115 qualified public health center laboratories across the country through the department’s own distribution agency, International Resources.

However, that’s where it starts to run.

There are loopholes, chaos, red tape and fueling

Once the kits were delivered to state laboratories, local scientists began to verify the results. This involves the use of a kit to detect known samples to ensure that THE kit correctly detects SARS-CoV-2 without incorrectly marking uninfected samples. But many labs have all sorts of problems. In some laboratories, negative controls do not work, while in others, those that should have produced positive results are not known.

On February 12, the CDC announced that the problem was with reagents. The third group of primers of the labeled coronavirus common gene did not work properly. CDC officials told the lab not to rush, the new reagent, Freshippo, was delivered. As a result, a few weeks later, only a handful of laboratories in a few states are capable of detecting new coronaviruses. Elsewhere, health authorities can only send samples of suspected patients directly to the CDC for testing. What’s more, the CDC’s testing standards were harsh: only patients with obvious symptoms and a history of travel to China were eligible for testing. In response, a New York Times report says this means that many infected people are not spotized in time for the virus’s spread to the United States.

While the U.S. Centers for Disease Control and Prevention is working to send more kits, other countries affected by the outbreak are adopting other PROTOCOLs published by WHO. By contrast, as of February 25, only 12 laboratories in five U.S. states, with the exception of the CDC, were able to detect new coronaviruses. Only 426 people in the U.S. were tested at the time, according to a web archive on the CDC’s website. (As of March 2, the agency will no longer release the number of tests.) Meanwhile, 15,000 people are reported to be tested daily in South Korea – the equivalent of the total number tested so far in the United States.

In principle, any qualified laboratory in the United States should be able to detect viruses, including hospitals and commercial laboratories, as long as it is fully equipped. All they need to do is use any agreement issued by WHO to order primers and start testing for new coronaviruses. But in fact, because of the existence of various newly released red tape, they can do nothing, can only wait for the CDC to allocate more kits.

Why is this so? That starts with January. At the time, the federal health agency declared the new coronavirus disease a public health emergency, followed by a series of rules that required all tests to be reviewed by the FDA’s “emergency license” process. These rules raise the bar for kit development and independent laboratory testing of viruses. In non-emergency situations, the FDA does not typically intervene in such tests. But under emergency rules, any lab that wants to test for a new coronavirus must conduct a series of validation studies and submit the data to the FDA for review. However, a validation study would first require a virus sample, but without the CDC, it would be easy to get a virus sample.

In order to maintain consistency, the FDA has chosen to open emergency permits only to CDC kits to ensure uniform oversight of state, county and municipal health departments. “The government’s detection strategy is very narrow. In other words, even if the kit is effective, the testing capacity will not improve significantly in a short period of time,” said Joshua Sharfstein, a health policy researcher at the Johns Hopkins School of Public Health. 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 test for new coronaviruses using kits from the CDC. They can’t order their own primers and fluorescence reagents, even if they buy them exactly like the ones used by the CDC. To make matters worse, when the CDC’s kits were defective, the lower labs had no choice but to wait.

At this point, no one other than the government knows why the United States does not use the testing methods developed for WHO. This test method has been used in more than 120 countries. A WHO spokesman declined to answer questions, and U.S. lawmakers such as Alex Azar, the health and human services secretary, shied away from answering the question. Predictably, bureaucracy is a big factor – it can take a long, long time to get approval in the United States. Some suspect that politics is on the wrong foot. Dan Diamond, a health policy reporter, said in a statement that President Trump intends to delay testing efficiency because the high number of confirmed cases could affect his re-election plans.

On February 29th, as the public health disaster intensified, so did the pressure to improve testing capabilities. The FDA now has to change its regulations to allow qualified clinical laboratories to develop and use their own kits to detect new coronaviruses without prior approval. “The west coast community has been a wake-up call for cases of community transmission,” Schaffstein said. By the end of the week, the FDA will have to let go. “

Under the new policy, labs are still required to submit data to the FDA for review. But now, after internally validating the test, they have two weeks to prepare the data that needs to be submitted to the FDA. During this time, testing of patient samples can also be synchronized.

After the agreement was relaxed, two large commercial diagnostic laboratories, Quest Diagnostics and LabCorp, began testing thousands of samples a day, according to data compiled by the American Enterprise Institute. A Quest Diagnostics spokesman also said the company expects to be able to test tens of thousands of samples a week by the end of March. Universities and hospitals are also taking action.

However, the regulatory problem is now solved, but logistics has presented a new challenge. As more and more labs open their online applications, almost every step of the RT-PCR testing is stuck. Eric Blank, chief project officer of the Public Health Laboratory Association, says the biggest problem right now is the RNA extraction kit. “Everyone is snapping up these auxiliary materials, and we know from member labs that a lot of the materials are out of order now. “

The RNA extraction kits recommended by the CDC’s testing protocol are supplied by Qiagen. But it was reported on March 10th that the kits were now in a state of waiting to be replenished as the world has accelerated testing for the new coronavirus over the past few weeks. Automated RNA extraction devices and RT-PCR instruments are also reported to be out of stock. The CDC also has requirements for devices used to detect new coronaviruses, and not all labs have approved equipment. Laboratories that lack approved equipment are now looking for ways to procure equipment that meets the requirements.

Qiagen executives also said in an email that they are hiring new workers, expanding production capacity at European factories, working three shifts a day and stepping up production seven days a week. The FDA recently further relaxed regulations 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 diagnose new coronavirus patients in a timely manner. “There is no quick fix to this,” Blank said. It can take weeks to solve the supply chain problem, that is, for a short period of time, testing will remain in short supply. “

Testing technology under development

More kits can certainly improve detection capabilities. Ultimately, new detection techniques may also help.

At least 20 companies have announced plans to develop “molecular point-of-care” testing. These tests are automated systems that allow front-line health care workers to get test results in half an hour without waiting for days or more. “You can take the patient’s nasal swabs or pharynx, put them in the container, put the container in the device, and press the button. After 30 minutes, you get a diagnosis report, and that’s their goal,” Carlson said.

Cepheid is a company that makes such tests and whose products have been used in the diagnosis of other diseases. At present, the detection tool of the new coronavirus is already under development. Similar detection tools developed by Coyote Bioscience have been deployed in China. There is even a test under development that can detect both new coronaviruses and influenza viruses.

Start-ups such as Sherlock Biosciences and Mammoth Biosciences are also considering using DIAGNOSTIC methods based on CRISPR technology to detect new coronaviruses. These tests utilize CRISPR’s programmable gene search capabilities to produce results in less than an hour without the need for cumbersome laboratory instruments. Mammoth published a preprinted article on March 10, referring to a testing technology developed by the company for SARS-CoV-2 that can be used in conjunction with test paper. At present, the company is in the process of further verification of the test results.

Another very marketable way is to look for viral antibodies, so-called serum tests, from the patient’s blood. This method is naturally good, because in addition to detecting the new coronavirus, it can also tell you whether the other person has been infected with the virus, but now has been cured. Martin Hibberd, an infectious disease researcher at the London School of Hygiene and Tropical Medicine who has been involved in the development of sars virus, said: “The faster the test, the more cases are confirmed, but even the best tests can’t be missed.” The exact rate of missed is not known until we have conducted a thorough test of this type of test. “

Serum testing may also be cheaper than PCR-based testing and is more suitable for automated and high-throughput testing. Serodetection may not be as accurate as molecular testing, but on the other hand, serum tests previously used to detect SARS virus can identify SARS-CoV-2 viruses, Hibberd said. It remains to be seen whether this will be useful in the development of new testing methods.

What does this have to do with the people who eat melons?

The different detection capabilities in this place right now mean that if you’re not feeling well, whether you’re going to be tested for a new coronavirus depends largely on where you live. Reports say some hospitals do not offer new coronavirus tests because they don’t have enough masks to protect health care workers who collect patient samples. Other hospitals have taken quantitative tests, i.e. only for patients with severe cases to be tested for new coronavirus, while others with milder symptoms are recommended for home treatment. Currently, each state, county, and part of the city has its own policy on who can be tested. “Our system is not keeping up with current needs,” Anthony Fauci, director of the National Institute of Allergy and Infectious Diseases, said at a congressional hearing last week. “

At a White House news conference friday, President Trump tried to salvage that failure. He declared a “national emergency” in the light of the new coronavirus outbreak, which would allow states and localities to respond flexibly to funding and regulatory issues. The president also announced a public-private partnership with several major stores and pharmacies across the country, including Wal-Mart and CVS, to allow them to leave some parking spaces as “free-of-the-vehicle” access. Working with testing companies such as Roche Pharmaceuticals, Quest and LabCorp means “we will be able to complete millions of tests in a very, very, very short period of time,” President Trump said. Vice President Mike Pence said they expected to eventually conduct 15,000 to 20,000 tests a day.

In addition, a new detection system will soon be available. Pence said he will release a new site in two days’ time. The site, which is said to have been developed by Google, allows people to enter their symptoms and then, if certain conditions are met, they can receive information about the location of a nearby car park that offers a free-to-drop test. Good, but it seems that symptoms must still be developed to test for the new coronavirus, which does not solve the broader epidemiological problem. Google later denied that Verilli, the medical and health company owned by Alphabet, was developing similar technology, but Verily did plan to test a small d-test program for medical workers in the Bay Area.

After the press conference, how to help solve the problem of testing and treatment costs is still pending. Also, if Google’s website is really online, then how to protect people’s personal information, test positive, and how to deal with it?

And in the meantime, as ordinary people, all we can do is keep our distance from others and avoid large parties. If you can work from home, don’t go anywhere to work from home. If you find yourself in contact with a confirmed patient, self-isolation at home for 14 days. If you feel unwell, please also self-isolation at home for 14 days. If the symptoms are not severe, there is no need to go to the hospital. Wash your hands frequently and soap for at least 20 seconds. If you can, give your cell phone and other supplies are also frequently disinfected. Don’t touch your face. Finally, keep an eye out for your neighbors, who have elderly people and patients in their homes, who may need help.

This pandemic affects each and every one of us, and all we can do is do everything we can to help those around us who need help.