What it Is, How its Done, What the Results Mean

Overview

What is a PCR test?

A polymerase chain reaction (PCR) test is performed to detect genetic material from a specific organism, such as a virus. The test detects the presence of a virus if you are infected at the time of the test. The test could also detect fragments of virus even after you are no longer infected.

What is a COVID-19 PCR test?

A PCR test for COVID-19 is a test used to diagnosis people who are currently infected with SARS-CoV-2, which is the coronavirus that causes COVID-19. The PCR test is the “gold standard” test for diagnosing COVID-19 because it’s the most accurate and reliable test.

Who should get tested for COVID-19?

Get tested:

• If you have symptoms of COVID-19.
• If you have been within six feet of someone for 15 minutes or more who has tested positive for Covid-19. (Note: some testing sites don’t offer testing if you’ve been exposed but don’t have symptoms.)

Test Details

How does a COVID-19 PCR test work?

There are three key steps to the COVID-19 PCR test: 1) sample collection, 2) extraction, and 3) PCR.

1. Sample collection is done using a swab to collect respiratory material found in your nose. A swab contains a soft tip on a long, flexible stick that is inserted into your nose. There are different types of nose swabs including nasal swabs that collect a sample immediately inside your nostrils and nasopharyngeal swabs that go further into the nasal cavity for collection. Either type of swab is sufficient for collecting material for the COVID-19 PCR test. After collection, the swab is sealed in a tube and then sent to a laboratory.
2. When a laboratory technologist receives the sample, they perform a process called extraction, which isolates genetic material from the sample including genetic material from any virus that may be present.
3. The PCR step then uses special chemicals and a PCR machine, called a thermal cycler, which cause a reaction to occur that makes millions of copies of a small portion of the SARS-CoV-2 virus’s genetic material. During this process, one of the chemicals produces a fluorescent light if SARS-CoV-2 is present in the sample. This fluorescent light is a “signal” that is detected by the PCR machine and special software is used to interpret the signal as a positive test result.

Results and Follow-Up

What do COVID-19 PCR test results mean?

A positive test result means that it is very likely that you have COVID-19. Most people have mild illness and can recover safely at home without medical care. Contact your healthcare provider if your symptoms get worse or if you have questions or concerns.

A negative test result means you probably didn’t have COVID-19 at the time you took your test. However, it is possible to be infected with SARS-CoV-2 but not have enough virus in your body to be detected by the test. For example, this may happen if you recently became infected but you don’t have symptoms, yet; or it could happen if you’ve had COVID-19 for more than a week before being tested. Keep in mind that a negative test doesn’t mean you are safe for any length of time. You can be exposed to COVID-19 after your test, get infected and spread the SARS-Cov-2 virus to others.

If your test is positive, talk with your healthcare provider, stay home and separate yourself from others. If your test is negative, continue to take steps to protect yourself and others from getting COVID-19. Read more about what to do if you test positive and ways to prevent getting infected with COVID-19.

How soon are results of a COVID-19 PCR test available?

You should receive the results of your test as early as 24 hours after sample collection, but sometime it can take a few days depending on long it takes the sample to reach the laboratory and how many other samples are in the queue to be tested.

What are the advantages of a COVID-19 PCR test?

The main advantages of COVID-19 PCR test are its accuracy and reliability. It is the most accurate test available for COVID-19 detection.

Are there downsides to a COVID-19 PCR test?

Because the test is able to detect very small amounts of virus material, it can continue to detect fragments of SARS-CoV-2 virus even after you’ve recovered from COVID-19 and are no longer contagious. So you may continue to test positive if you have had COVID-19 in the distant past, even though you can’t spread the SARS-CoV-2 virus to others.

Additional Details

How does the COVID-19 PCR test compare with other available COVID-19 tests?

Basically, there are two types of tests, diagnostic tests and antibody tests. Diagnostic tests tell you if you have an active (current) COVID-19 infection. Antibody tests tell you that you already had COVID-19.

Diagnostic tests:

• PCR test: This tests for the presence of the actual virus’s genetic material or its fragments as it breaks down. This is the most reliable and accurate test for detecting active infection.
• Antigen test: This test detects bits of proteins on the surface of the virus called antigens. Antigen tests are typically considered rapid, taking only 15 to 30 minutes but are less accurate than a PCR test. Rapid antigen tests are most accurate when used within a few days of the start of your symptoms, which is when the largest amount of virus is present in your body. Because this test is not as accurate as a PCR test, if an antigen test is negative, your healthcare provider may order a PCR test to confirm the negative test result.

Antibody test:

• Antibody (serology) test: This tests detects if you’ve had an immune response (antibodies) to the virus. This means that you’ve had the virus and your body (immune system, specifically antibodies) has mounted an attack to fight it. The test is detecting those antibodies. It typically takes about a week after being infected for enough antibodies to develop to be detected in your blood. For this reason, this test shouldn’t be used to diagnose an active infection.

How do I find out where to get tested for COVID-19?

If you have symptoms of COVID-19 or have been exposed to people who have symptoms or have tested positive, you may want to be tested. First, talk with your healthcare provider. They will review your symptoms in person or on a video appointment. The provider will place an order for a test and tell you where you can be tested. Keep in mind that if you’ve been exposed to the SARS-CoV-2 virus but don’t have symptoms, call the testing site first to make sure they will allow you to be tested.

You can also call or check the websites of your local hospitals in your health insurance network or check with community health centers or urgent care centers. The U.S. Department of Health and Human Services provides links to find community-based testing sites in your state. You can also check your state or local health department websites for the latest information on testing locations. The Centers for Disease Control provides links to these state and local health departments.

COVID-19 diagnostic testing – Mayo Clinic

Overview

COVID-19 diagnostic testing is done to find out if you’re currently infected with SARS-CoV-2, the virus that causes coronavirus disease 2019 (COVID-19).

The U.S. Food and Drug Administration (FDA) approved these types of tests for diagnosing a COVID-19 infection:

• PCR test. Also called a molecular test, this COVID-19 test detects genetic material of the virus using a lab technique called polymerase chain reaction (PCR). A fluid sample is collected by inserting a long nasal swab (nasopharyngeal swab) into your nostril and taking fluid from the back of your nose or by using a shorter nasal swab (mid-turbinate swab) to get a sample.

  In some cases, a long swab is inserted into the back of your throat (oropharyngeal swab), or you may spit into a tube to produce a saliva sample. Results may be available in minutes if analyzed onsite or a few days — or longer in locations with test processing delays — if sent to an outside lab. PCR tests are very accurate when properly performed by a health care professional, but the rapid test can miss some cases.

• Antigen test. This COVID-19 test detects certain proteins in the virus. Using a long nasal swab to get a fluid sample, some antigen tests can produce results in minutes. Others may be sent to a lab for analysis.

  A positive antigen test result is considered accurate when instructions are carefully followed, but there’s an increased chance of false-negative results — meaning it’s possible to be infected with the virus but have a negative result. Depending on the situation, the doctor may recommend a PCR test to confirm a negative antigen test result.

A PCR test called the Flu SC2 Multiplex Assay can detect any of three viruses at the same time: the COVID-19 virus, influenza A and influenza B (flu). Only a single sample is needed to check for all three viruses, and this could be helpful during the flu season. But a negative result does not rule out the possibility of any of these infections. So the diagnostic process may include more steps, depending on symptoms, possible exposures and your doctor’s clinical judgment.

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Why it’s done

In the U.S., a COVID-19 diagnostic test is needed if:

• You have COVID-19 symptoms, such as fever, cough, tiredness or shortness of breath
• You don’t have symptoms but you’ve had close contact with someone who tests positive for the COVID-19 virus or is suspected of having the virus. Close contact means you’ve been within 6 feet (2 meters) of a person who has COVID-19. But if you’ve tested positive for COVID-19 within the past three months, you don’t need to get tested. If you’ve been fully vaccinated and you’ve had close contact with someone who has the COVID-19 virus, get tested 3 to 5 days after you’ve had contact with them.
• You’ve participated in activities that increase your risk of COVID-19 and did not stay at least 6 feet away from others — examples include travel, large gatherings or crowded indoor settings.
• Your doctor or other health care professional or your public health department recommends a test

Certain groups are considered high priority for diagnostic testing. These include people with COVID-19 signs and symptoms who:

• Work in a health care facility or as first responders
• Live or work in long-term care facilities, such as nursing homes, or other places where people are housed closely together, such as prisons or shelters
• Are being cared for in a hospital

Other people may be given priority for testing depending on local health department guidelines for monitoring COVID-19 in individual communities.

Some people who are infected with the COVID-19 virus may be asymptomatic, meaning they don’t have any signs or symptoms. But they can still transmit the virus to others. In some areas of the U.S., testing is available to asymptomatic people. If people without symptoms have a positive test result, they should follow guidelines for self-isolation to help curb the spread of the virus.

The availability of COVID-19 diagnostic testing and where to get tested may vary depending on where you live and the recommendations of your local public health officials.

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Risks

There’s a chance that your COVID-19 diagnostic test could return a false-negative result. This means that the test didn’t detect the virus, even though you actually are infected with it. You risk unknowingly spreading the virus to others if you don’t take proper precautions, such as following social distancing guidelines and wearing a face mask when appropriate. There’s also a chance that a COVID-19 rapid antigen test can produce false-positive results — indicating an infection when actually there isn’t one — if instructions aren’t carefully followed.

The risk of false-negative or false-positive test results depends on the type and sensitivity of the COVID-19 diagnostic test, thoroughness of the sample collection, and accuracy of the lab analysis.

Be wary of any offers for at-home COVID-19 tests that the FDA has not cleared for use — they often give inaccurate results.

How you prepare

Whether or not you have symptoms, plan to wear a face mask to and from your doctor’s office or the testing center, and have anyone who comes with you wear one, too.

• If you think you may have COVID-19, call your doctor’s office or your local health department to review your symptoms and ask about testing before you go in, so staff can prepare for your visit, wearing personal protective equipment.
• If you have no symptoms but you’ve been in close contact with someone who has COVID-19, follow the testing advice of your doctor or public health department. Having a COVID-19 test 5 to 7 days after you were close to the person with COVID-19 is best. If you’re tested too soon, the test may not detect the virus.

If you think you may have COVID-19, call your doctor’s office to review your symptoms, if any, and ask about testing. Then your doctor and other staff can prepare for your visit, wear personal protective equipment, and give you instructions about where to go and how the test will be done. Plan to wear a face mask to and from the testing center, and have anyone who accompanies you wear one, too.

If you have no symptoms and have not knowingly been in contact with someone infected with the COVID-19 virus, but you want to get tested, ask your health care provider whether and where testing is available. Or you can call your state or local health department or visit their website for information on testing.

What you can expect

For a COVID-19 diagnostic test, a health care professional takes a sample of mucus from your nose or throat, or a sample of saliva. The sample needed for diagnostic testing may be collected at your doctor’s office, a health care facility or a drive-up testing center.

• Nose or throat swab.
  A long nasal swab (nasopharyngeal swab) is recommended, though a shorter nasal swab or throat swab is acceptable. Your doctor or other health care professional inserts a thin, flexible stick with cotton at the tip into your nose or brushes the swab along the back of your throat to collect a sample of mucus. This may be somewhat uncomfortable.

  For the nasal sample, swabbing may occur in both nostrils to collect enough mucus for the test. The swab remains in place briefly before being gently rotated as it’s pulled out. The sample gets sealed in a tube and sent to a lab for analysis.

• Saliva sample. Some locations offer saliva tests. While a saliva sample may be a bit less sensitive than a mucus sample that’s taken using a long nasal swab, a saliva test is easier to do and often less uncomfortable. You spit into a tube several times to provide a sample of your saliva to test. The tube is sealed before being sent to a lab for analysis.

If you have a productive cough, your doctor may collect a sputum sample, which contains secretions from the lungs, a part of the lower respiratory system. The virus is more concentrated in the nose and throat early in the course of the infection. But after more than five days of symptoms, the virus tends to be more concentrated in the lower respiratory system.

In addition to the COVID-19 diagnostic test, your doctor may also test for other respiratory conditions, such as influenza, that have similar symptoms and could explain your illness.

The FDA granted emergency use authorization for certain at-home COVID-19 test kits, including one that tests for both COVID-19 and the flu. Most of these tests require a doctor’s prescription. You collect your own sample of nasal fluid or saliva at home and then send it to a lab to be rapidly analyzed. One COVID-19 test provides fast results at home without sending the sample to a lab. And the FDA recently authorized an antigen test to buy over the counter with no prescription needed, though antigen tests are not considered as reliable as PCR tests.

The accuracy of each of these tests varies, so a negative test does not completely rule out having the COVID-19 virus. Only get an at-home test that’s authorized by the FDA or approved by your doctor or local health department.

Supporting Your Child During COVID-19 Nasal Swab Testing

The purpose of this video is to prepare children for a COVID-19 nasal swab test, to help ease some of their potential fear and anxiety. When children are prepared to take a medical test, they become more cooperative and compliant, which creates a positive coping experience for them. This video has been made to be watched by children as young as 4 years old.

Show transcript for video Supporting Your Child During COVID-19 Nasal Swab Testing

Jennifer Rodemeyer, Child Life Program Manager, Mayo Clinic: Hi, I’m Jennifer and I am a child life specialist at Mayo Clinic. My job is to help kids like you prepare for medical tests.

You may have heard there is a virus going around that can make people feel sick. A virus is a germ and it is so tiny you can’t even see it.

Some people who get this virus can have a fever or a cough and may feel achy and tired, while some people can have this virus and not feel sick at all. People may get this virus from touching things. That’s why it’s important to wash your hands often with soap and water. The virus also can spread through a cough or a sneeze. So it’s important to always cover your cough or sneeze.

Today, even though you may or may not be feeling sick, we will need to give you a test so we know how to best proceed with your medical care. This medical test will tell us if you have the virus.

When you go to take your test, the health care provider will wear special protective clothing. They wear this clothing to keep themselves and you safe from getting germs. They will wear a mask to cover their nose and mouth and a clear plastic shield to protect their eyes.

The most important thing you can do during your test is to sit perfectly still like a statue. To help make sure you don’t move, your parent or caregiver will help keep you still and calm during your test. The health care provider needs to touch the inside of the back of your nose with a long, skinny Q-tip. To do this, you need to hold your chin up, then the health care provider will put the Q-tip in your nose for a short time to collect a sample.

While this happens you may feel like you want to push the Q-tip away, but it’s really important to stay as still as possible so the health care provider can finish the test. The Q-tip will be in and out of your nose in a few seconds.

Some kids tell me that counting to 3 or taking a deep breath relaxes them before the test happens, and some tell me they like to hold on to their favorite stuffed animal or blanket. Maybe you have your own way to relax.

Remember that during the test, the most important thing to do is to keep your body perfectly still.

You may have many feelings seeing the health care provider wearing different clothing, but know this person is caring and wants to help you.

Thank you for helping us get this test done, so we know how to proceed with your medical care.

Results

Some facilities have rapid tests for COVID-19 diagnostic testing. In that case, you may get your results in less than an hour or on the same day that you’re tested. Other facilities may have to send the test sample to an outside lab for analysis. If they need to send out the sample, your results may not be available until a few days later.

Your COVID-19 diagnostic test result could be positive or negative.

• Positive result. This means you currently have an active infection with the virus that causes COVID-19. Take appropriate steps to care for yourself and avoid spreading the virus to others. You’ll need to self-isolate until: Your symptoms are improving, and it’s been 24 hours since you’ve had a fever, and at least 10 days have passed since your symptoms first appeared.

  If you have severe symptoms of COVID-19 or a health condition that lowers your ability to fight disease, your doctor may recommend that you stay in isolation longer. If you have a positive result but never developed symptoms, isolate for 10 days after the test.

• Negative result. This means that you likely weren’t infected with the COVID-19 virus. But a false-negative test result could happen depending on the timing and quality of the test sample.

  Even if you test negative, you could become infected in the future, so it’s important to follow guidelines for social distancing, face mask use and hand-washing to avoid potential spread. Your doctor may recommend repeat testing if you continue to have symptoms.

Contact tracing

If you test positive for the COVID-19 virus — or your doctor suspects that you have the virus but you don’t have test results yet — you may be asked to participate in contact tracing. Contact tracing plays a key role in limiting the spread of infectious diseases. The sooner contact tracing starts, the more effective it is in limiting virus spread.

To begin, you provide a list of people you had close contact with during the time you may have been contagious. Public health workers then get in touch with those close contacts to let them know about the exposure and their potential for being infected. Your identity is protected during this exchange of information.

The contact tracing team provides information on what close contacts can do to minimize the risk of spreading the virus. Steps may include getting a COVID-19 test, staying at home and away from others — called quarantine — after the exposure, learning about signs and symptoms, and taking other precautions.

Quarantine recommendations

If you’ve had close contact with someone who has COVID-19 and have been within 6 feet (2 meters) of the person, it’s best to stay at home and away from others (quarantine) for 14 days after the exposure to see if you develop COVID-19 symptoms. If you have had COVID-19 or been fully vaccinated in the past three months, you generally don’t need to quarantine if you have had a close contact with COVID-19.

Other options may include ending quarantine after 10 days if you don’t have symptoms and don’t get tested or ending quarantine after 7 days if you receive a negative test result and have no symptoms. But continue to watch for symptoms for the full 14 days.

Contact your doctor or local health department for advice on testing and quarantine recommendations.

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Aug. 03, 2021

Polymerase Chain Reaction in the Diagnosis and Management of Central Nervous System Infections | Infectious Diseases | JAMA Neurology

Polymerase chain reaction (PCR) is a broadly applied laboratory test for the diagnosis of a wide variety of central nervous system (CNS) diseases, including genetic and autoimmune diseases, malignant neoplasms, and infections.^1,2With its ability to detect minute amounts of DNA or RNA contained in tissues or fluids, PCR has improved the rapidity and accuracy of diagnosis, enhanced understanding of pathogenesis, and helped identify infectious causes for diseases previously considered idiopathic. In addition, PCR can be performed on a variety of tissues preserved in different ways—even archival specimens can be used to provide important epidemiological information. By making quick and precise diagnoses, appropriate treatments can be instituted, and unnecessary or invasive investigations can be avoided.

The power of PCR results from its ability to synthesize millions of copies of a specific gene segment in vitro, starting with one or only a few template copies, an amount undetectable by other methods. The PCR technique is illustrated in Figure 1. Once the sequence of the DNA segment of interest is identified (template), 2 synthetic oligonucleotides (primers) are chosen that have a sequence complementary to each strand of the DNA to be amplified. These primers are usually between 20 and 40 bases long. The first step requires denaturing the double-stranded DNA template by heating it to 92°C to 95°C. Once this occurs, the primers will anneal to their respective complementary template strands after cooling to 55°C to 70°C. To maximize primer-template annealing vs template-template reannealing, primers are supplied in great excess; thus, there are more primers available for binding than de novo DNA strands. Binding of primers to template is only the first step. In order to build a new DNA strand complementary to the template, 2 main ingredients are necessary: nucleotides (adenine, guanine, thymidine, cytosine) and the enzyme to catalyze their linkage (DNA polymerase). By supplying nucleotides in vast molar excess and by making use of a thermostable DNA polymerase (usually Taq
polymerase), a new complementary DNA sequence downstream of the annealed primer is assembled, resulting in 2 complete double-stranded DNA pairs. This completes the first cycle. Multiple cycles (usually 30-40) of denaturing, the annealing of new primers, and strand elongation ultimately yield more than 1 billion copies of the original DNA template in 2 to 3 hours. The entire reaction is completed within a self-contained thermal cycler using very small volumes (microliters) of reagents and sample.

When it is important to know not only that a gene (DNA) is present, but also that it is being expressed and transcribed into messenger RNA (mRNA), or when RNA viruses are the target of detection, a variant of PCR called reverse transcriptase PCR (RT-PCR) can be used. The general principle is identical to that outlined above, but instead of Taqpolymerase, an enzyme with reverse transcriptase activity is used that first reverse-transcribes the mRNA into complementary DNA. The complementary DNA then serves as the template from which the segment of interest is amplified. Situations in which this might be important include diseases caused by genetic defects with variable penetrance or viral infections in which both latent and lytic infection play a role in pathogenesis (eg, herpesviruses).

The amplification products of PCR can be detected using a variety of methods. The products can be loaded onto an ethidium bromide–stained agarose gel and then electrophoresed, allowing migration of the DNA product to its predicted location in the gel based on the size of the fragment generated (determined by the spacing of the 2 primers chosen). To ensure that these products are specific, however, it is necessary to perform Southern blot analysis. In this process, the target DNA is transferred and immobilized on a membrane, then tagged with a complementary probe to which a radioactive, fluorescent, or colorimetric marker is attached. An additional technique that can be used to increase specificity of products is termed nested PCR. In this technique, a second round of PCR is performed using primers internal to the original primers, thus identifying only the subset of amplification products that correspond to the target fragment. Nested PCR can also be used to increase the sensitivity of PCR. Perhaps the best standard for ensuring specificity is to sequence PCR products and compare the sequence with the known target gene sequence. However, PCR product sequencing is not typically performed as a routine procedure, and its use is largely limited to initial validation of PCR assays and as a research tool.

Synthesis of nonspecific products can result in false-positive PCR results if appropriate steps to confirm specificity are not taken. Mispriming may occur (primers binding to the wrong region of DNA), but this can be minimized by increasing the “stringency” of the reaction conditions and can be detected by failure to find amplified material using Southern blot analysis. False-positive PCR results can also occur because of contamination of reactions with nucleic acid either from products of prior reactions (carryover) or that is present in the laboratory environment. Experienced laboratories take extensive precautions to avoid sources of inadvertent contamination, including proper design of laboratory facilities and protocols for handling specimens. Negative controls are used in all reaction runs to detect inadvertent contamination.

Relevance to the practice of neurology

There are numerous areas in which PCR can be directly applied in clinical practice.²In this review, we will focus on the application of PCR to the diagnosis and management of infectious neurologic diseases.^2,3

Viruses are common causative agents in certain neurologic diseases (eg, meningitis, encephalitis, and myelitis). It is difficult, often impossible, to isolate many viruses by standard cell culture techniques. Even for viruses such as enteroviruses, which are generally more amenable to culture, a prolonged period may be required for accurate identification (often up to 8 days). False-negative cultures are common and may occur in 25% to 35% of specimens, and some enterovirus serotypes do not grow at all in cell culture. In the case of herpes simplex virus (HSV) encephalitis in adults, the virus is almost never detectable in cerebrospinal fluid (CSF) by culture. As a result of these problems, prior to the widespread use of CSF PCR testing in viral diagnosis, patients were often treated with unnecessary empiric antimicrobial therapy for prolonged periods and underwent invasive diagnostic procedures, such as brain biopsy. Serologic diagnosis of CNS viral infections is limited primarily by the time required to detect high levels of specific antibody production (often 2-3 weeks from the beginning of the disease) or by the ubiquitous nature of seropositivity to certain viruses (eg, HSV). Circumstantial evidence of viral infection, such as stool or respiratory excretion, may be helpful, but it does not necessarily correlate with active CNS disease (eg, the intermittent shedding of cytomegalovirus in urine or respiratory secretions and the prolonged shedding of enteroviruses both occur in asymptomatic individuals). Polymerase chain reaction is attractive because it circumvents most of these limitations. Table 1summarizes the broad range of CNS viral infections to which PCR is clinically applicable. As more specific antiviral compounds are identified that can be used therapeutically, accurate diagnosis will become increasingly important.

In addition to viral infections, PCR has also been used to diagnose bacterial, mycobacterial, rickettsial, and protozoal infections of the CNS (Table 2).

It is often difficult to accurately determine the sensitivity and specificity of PCR in CNS infections because in some situations the previously used standards for diagnoses appear to be less sensitive than PCR. Existing standards, such as viral CSF culture, are often both insensitive and slow. In some cases, definitive diagnosis was based on invasive procedures, such as brain biopsy, which may have been performed only in severely ill patients, potentially producing a skewed picture of the severity or nature of disease (eg, HSV encephalitis). Despite the extreme sensitivity of PCR, false-negative results do occur. In the case of most viral infections, the highest levels of nucleic acid are present acutely and typically decrease with treatment or over time. A delay in performing PCR may result in false negatives. Inhibitors of PCR may be present in body fluids or tissues and may lead to false-negative results. In CSF, the most commonly encountered inhibitors are heme products resulting from the breakdown of erythrocytes. Small numbers of red blood cells in CSF samples do not inhibit PCR, but this can be a problem with CSF samples that are grossly contaminated with blood. No inhibition has been noted in specimens with high levels of protein or high leukocyte counts. As opposed to other specimens, CSF tends to have low concentrations of endonucleases/exonucleases and proteins that could inhibit the action of the polymerase.

Polymerase chain reaction can be performed with as little as 30 µL of CSF. Polymerase chain reactions are best performed on fresh CSF specimens, although brief (days) storage of CSF at refrigerator temperature does not appear to significantly reduce diagnostic yield. Positive PCR results have been obtained from CSF specimens that have been frozen for years, but the decline in sensitivity with time has not been rigorously examined. In general, DNA appears to be more stable during storage than RNA, which is more susceptible to degradation. Finally, short courses (eg, a few days) of antimicrobial therapy do not appear to have a significant effect on PCR yields, which is a great advantage over culture techniques, in which diagnostic yields often drop precipitously following even brief periods of antimicrobial therapy.

Other clinical applications and relevance to the study of neuroscience

In addition to aiding in the diagnosis of infectious diseases, PCR can be used for a variety of other purposes. Polymerase chain reaction may facilitate the differential diagnosis between recurrent viral infection, which would be expected to be PCR-positive, and postinfectious immune-mediated disease, which is PCR-negative. For example, following antiviral therapy for HSV encephalitis, some patients show clinical relapse. A positive CSF HSV PCR result suggests that this is caused by recurrent viral infection and supports the reinstitution of antiviral therapy. A negative PCR result is consistent with a postinfectious immune-mediated process, and additional antiviral therapy is rarely of value. Polymerase chain reaction can be used to identify determinants of drug resistance by the sequencing of PCR-amplified genes encoding targets for antiviral therapy. For example, PCR amplification and sequencing of selected viral genes in patients with human immunodeficiency virus (HIV) infection may help identify mutations that correlate with resistance to specific antiretroviral therapies. As PCR techniques have become more sophisticated, it is now possible in many cases to accurately quantify the amount of nucleic acid that is present in the sample, rather than merely provide qualitative detection. Quantitative PCR may prove useful in monitoring the duration and adequacy of therapy or in providing prognostic information about illness. Qualitative PCR is being used with increasing frequency to study CNS infection caused by HIV and HSV. In the case of neonatal HSV encephalitis, qualitative PCR has led to changes in the recommended dosage and duration of acyclovir therapy and has resulted in more widespread use of adjunctive oral therapy following primary intravenous antiviral therapy. In the case of HIV infection, quantitation of viral load in CSF is being actively investigated as a potential marker of the risk for and severity of HIV-associated encephalopathy. Polymerase chain reaction may also help to identify a potential role for viruses or other microbial pathogens in neurologic and psychiatric diseases of uncertain causes, including schizophrenia, multiple sclerosis, Alzheimer disease, and other neurodegenerative diseases. One of the interesting results from initial studies in this area has been the frequency with which the nucleic acid corresponding to a variety of viral pathogens can be identified from brain tissue, even in patients without known neurological diseases. By contrast, CSF PCR results are almost invariably negative in asymptomatic individuals without known neurological diseases. In the case of CSF, positive PCR results almost invariably correlate with the presence of a clinically significant disease. The significance of positive PCR results in brain tissue needs to be interpreted with great caution until the nature, significance, and frequency of “normal brain genomic flora” identified by PCR are clarified.

Accepted for publication January 8, 1999.

This study was supported by Merit Review and Research Enhancement Award Program grants from the Department of Veterans Affairs, Washington, DC; by grant DAMD17-98-8614 from the US Army, Washington, DC; and by grant 5R01AG14071 from the National Institute on Aging, Bethesda, Md (Dr Tyler).

We thank Adriana Weinberg, MD, for her thoughtful comments.

Corresponding author: Kenneth L. Tyler, MD, Department of Neurology, B-182, University of Colorado Health Sciences Center, 4200 E Ninth Ave, Denver, CO 80262 (e-mail: [email protected]).

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What is the role of polymerase chain reaction (PCR) testing in the diagnosis of Lyme disease?

Author

John O Meyerhoff, MD Clinical Scholar in Rheumatology, Department of Medicine, Sinai Hospital of Baltimore

John O Meyerhoff, MD is a member of the following medical societies: American College of Physicians, American College of Rheumatology

Disclosure: Nothing to disclose.

Coauthor(s)

Russell W Steele, MD Clinical Professor, Tulane University School of Medicine; Staff Physician, Ochsner Clinic Foundation

Russell W Steele, MD is a member of the following medical societies: American Academy of Pediatrics, American Association of Immunologists, American Pediatric Society, American Society for Microbiology, Infectious Diseases Society of America, Louisiana State Medical Society, Pediatric Infectious Diseases Society, Society for Pediatric Research, Southern Medical Association

Disclosure: Nothing to disclose.

Gerald W Zaidman, MD Professor of Clinical Ophthalmology, New York Medical College; Chief of Cornea Service, Director, Department of Ophthalmology, Westchester Medical Center

Gerald W Zaidman, MD is a member of the following medical societies: American Academy of Ophthalmology, Association for Research in Vision and Ophthalmology, Medical Society of Virginia, American Uveitis Society, American College of Surgeons, American Medical Association, American Society of Cataract and Refractive Surgery, Medical Society of the State of New York, Phi Beta Kappa

Disclosure: Nothing to disclose.

Chief Editor

Herbert S Diamond, MD Visiting Professor of Medicine, Division of Rheumatology, State University of New York Downstate Medical Center; Chairman Emeritus, Department of Internal Medicine, Western Pennsylvania Hospital

Herbert S Diamond, MD is a member of the following medical societies: Alpha Omega Alpha, American College of Physicians, American College of Rheumatology, American Medical Association, Phi Beta Kappa

Disclosure: Nothing to disclose.

Acknowledgements

Stephen C Aronoff, MD Waldo E Nelson Chair and Professor, Department of Pediatrics, Temple University School of Medicine

Stephen C Aronoff, MD is a member of the following medical societies: Pediatric Infectious Diseases Society and Society for Pediatric Research

Disclosure: Nothing to disclose.

Richard G Bachur, MD Associate Professor of Pediatrics, Harvard Medical School; Associate Chief and Fellowship Director, Attending Physician, Division of Emergency Medicine, Children’s Hospital of Boston

Disclosure: Nothing to disclose.

Lawrence H Brent, MD Associate Professor of Medicine, Jefferson Medical College of Thomas Jefferson University; Chair, Program Director, Department of Medicine, Division of Rheumatology, Albert Einstein Medical Center

Lawrence H Brent, MD is a member of the following medical societies: American Association for the Advancement of Science, American Association of Immunologists, American College of Physicians, and American College of Rheumatology

Disclosure: Abbott Honoraria Speaking and teaching; Centocor Consulting fee Consulting; Genentech Grant/research funds Other; HGS/GSK Honoraria Speaking and teaching; Omnicare Consulting fee Consulting; Pfizer Honoraria Speaking and teaching; Roche Speaking and teaching; Savient Honoraria Speaking and teaching; UCB Honoraria Speaking and teaching

William E Caputo, MD Chief Resident Physician, Department of Emergency Medicine, Kings County Hospital

William E Caputo, MD is a member of the following medical societies: American College of Emergency Physicians, American Medical Association, and Emergency Medicine Residents Association

Disclosure: Nothing to disclose.

Eugene Y Cheng, MD, FCCM Consulting Staff, Department of Anesthesiology, The Permanente Medical Group

Disclosure: Nothing to disclose.

Dan Danzl, MD Chair, Professor, Department of Emergency Medicine, University of Louisville Hospital

Dan Danzl, MD is a member of the following medical societies: American Academy of Emergency Medicine, American College of Emergency Physicians, American Medical Association, Kentucky Medical Association, Society for Academic Emergency Medicine, and Wilderness Medical Society

Disclosure: Nothing to disclose.

Jonathan A Edlow, MD Associate Professor of Medicine, Department of Emergency Medicine, Harvard Medical School; Vice Chairman, Department of Emergency Medicine, Beth Israel Deaconess Medical Center

Jonathan A Edlow, MD is a member of the following medical societies: American College of Emergency Physicians and Society for Academic Emergency Medicine

Disclosure: Nothing to disclose.

Dirk M Elston, MD Director, Ackerman Academy of Dermatopathology, New York

Dirk M Elston, MD is a member of the following medical societies: American Academy of Dermatology

Disclosure: Nothing to disclose.

Kilbourn Gordon III, MD, FACEP Urgent Care Physician

Kilbourn Gordon III, MD, FACEP is a member of the following medical societies: American Academy of Ophthalmology and Wilderness Medical Society

Disclosure: Nothing to disclose.

Marvin Harper, MD Assistant Professor of Pediatrics, Departments of Emergency Medicine and Infectious Disease, Harvard Medical School; Director, Informatics Program, Children’s Hospital of Boston

Disclosure: Nothing to disclose.

Cindy R Hennen, RPh Assistant Director of Clinical Pharmacy Practice, Froedtert Hospital, Medical College of Wisconsin

Disclosure: Nothing to disclose.

Jon Mark Hirshon, MD, MPH Associate Professor, Department of Emergency Medicine, University of Maryland School of Medicine

Jon Mark Hirshon, MD, MPH is a member of the following medical societies: Alpha Omega Alpha, American Academy of Emergency Medicine, American College of Emergency Physicians, American Public Health Association, and Society for Academic Emergency Medicine

Disclosure: Nothing to disclose.

R Philip Kinkel, MD, FAAN Associate Professor of Neurology, Harvard Medical School; Director, Multiple Sclerosis Center, Beth Israel Deaconess Medical Center; Consultant Neurologist, Children’s Hospital of Boston

Disclosure: Nothing to disclose.

Kristine M Lohr, MD, MS Professor, Department of Internal Medicine, Center for the Advancement of Women’s Health and Division of Rheumatology, Director, Rheumatology Training Program, University of Kentucky College of Medicine

Kristine M Lohr, MD, MS is a member of the following medical societies: American College of Physicians and American College of Rheumatology

Disclosure: Nothing to disclose.

Larry I Lutwick, MD Professor of Medicine, State University of New York Downstate Medical School; Director, Infectious Diseases, Veterans Affairs New York Harbor Health Care System, Brooklyn Campus

Larry I Lutwick, MD is a member of the following medical societies: American College of Physicians and Infectious Diseases Society of America

Disclosure: Nothing to disclose.

Tarun Madappa, MD, MPH Attending Physician, Department of Pulmonary and Critical Care Medicine, Elkhart General Hospital

Tarun Madappa, MD, MPH is a member of the following medical societies: American College of Chest Physicians and American Thoracic Society

Disclosure: Nothing to disclose.

Augusto A Miravalle, MD Fellow, Department of Neurology, Beth Israel Deaconess Medical Center, Harvard Medical School

Augusto A Miravalle, MD is a member of the following medical societies: American Academy of Neurology

Disclosure: Nothing to disclose.

Christen M Mowad, MD Associate Professor, Department of Dermatology, Geisinger Medical Center

Christen M Mowad, MD is a member of the following medical societies: Alpha Omega Alpha, American Academy of Dermatology, and Phi Beta Kappa

Disclosure: Nothing to disclose.

Gary J Noel, MD Professor, Department of Pediatrics, Weill Cornell Medical College; Attending Pediatrician, New York-Presbyterian Hospital

Gary J Noel, MD is a member of the following medical societies: Pediatric Infectious Diseases Society

Disclosure: Nothing to disclose.

Wendy Peltier, MD Program Director, Assistant Professor, Department of Neurology, Medical College of Wisconsin

Disclosure: Nothing to disclose.

Julie L Puotinen, PharmD Clinical Coordinator of Pharmaceutical Services, Department of Pharmacy, Clinical Instructor, Saint Luke’s Medical Center

Disclosure: Nothing to disclose.

Karen L Roos, MD John and Nancy Nelson Professor of Neurology, Professor of Neurological Surgery, Department of Neurology, Indiana University School of Medicine

Karen L Roos, MD is a member of the following medical societies: American Academy of Neurology and American Neurological Association

Disclosure: Nothing to disclose.

Hampton Roy Sr, MD Associate Clinical Professor, Department of Ophthalmology, University of Arkansas for Medical Sciences

Hampton Roy Sr, MD is a member of the following medical societies: American Academy of Ophthalmology, American College of Surgeons, and Pan-American Association of Ophthalmology

Disclosure: Nothing to disclose.

Michael J Schneck, MD Associate Professor, Departments of Neurology and Neurosurgery, Stritch School of Medicine, Loyola University; Associate Director, Stroke Program, Director, Neurology Intensive Care Program, Medical Director, Neurosciences ICU, Loyola University Medical Center

Michael J Schneck, MD is a member of the following medical societies: American Academy of Neurology, American Society of Neuroimaging, Neurocritical Care Society, and Stroke Council of the American Heart Association

Disclosure: Boehringer-Ingelheim Honoraria Speaking and teaching; Sanofi/BMS Honoraria Speaking and teaching; Pfizer Honoraria Speaking and teaching; UCB Pharma Honoraria Speaking and teaching; Talecris Consulting fee Other; NMT Medical Grant/research funds Independent contractor; NIH Independent contractor; Sanofi Grant/research funds Independent contractor; Boehringer-Ingelheim Grant/research funds Independent contractor; Baxter Labs Consulting fee Consulting

Robert A Schwartz, MD, MPH Professor and Head, Dermatology, Professor of Pathology, Pediatrics, Medicine, and Preventive Medicine and Community Health, University of Medicine and Dentistry of New Jersey-New Jersey Medical School

Robert A Schwartz, MD, MPH is a member of the following medical societies: Alpha Omega Alpha, American Academy of Dermatology, American College of Physicians, and Sigma Xi

Disclosure: Nothing to disclose.

Richard H Sinert, DO Professor of Emergency Medicine, Clinical Assistant Professor of Medicine, Research Director, State University of New York College of Medicine; Consulting Staff, Department of Emergency Medicine, Kings County Hospital Center

Richard H Sinert, DO is a member of the following medical societies: American College of Physicians and Society for Academic Emergency Medicine

Disclosure: Nothing to disclose.

Francisco Talavera, PharmD, PhD Adjunct Assistant Professor, University of Nebraska Medical Center College of Pharmacy; Editor-in-Chief, Medscape Drug Reference

Disclosure: Medscape Salary Employment

Florian P Thomas, MD, MA, PhD, Drmed Director, Spinal Cord Injury Unit, St Louis Veterans Affairs Medical Center; Director, National MS Society Multiple Sclerosis Center; Director, Neuropathy Association Center of Excellence, Professor, Department of Neurology and Psychiatry, Associate Professor, Institute for Molecular Virology, and Department of Molecular Microbiology and Immunology, St Louis University School of Medicine

Florian P Thomas, MD, MA, PhD, Drmed is a member of the following medical societies: American Academy of Neurology, American Neurological Association, American Paraplegia Society, Consortium of Multiple Sclerosis Centers, and National Multiple Sclerosis Society

Disclosure: Nothing to disclose.

Richard P Vinson, MD Assistant Clinical Professor, Department of Dermatology, Texas Tech University Health Sciences Center, Paul L Foster School of Medicine; Consulting Staff, Mountain View Dermatology, PA

Richard P Vinson, MD is a member of the following medical societies: American Academy of Dermatology, Association of Military Dermatologists, Texas Dermatological Society, and Texas Medical Association

Disclosure: Nothing to disclose.

R Christopher Walton, MD Professor, Director of Uveitis and Ocular Inflammatory Disease Service, Department of Ophthalmology, University of Tennessee College of Medicine

R Christopher Walton, MD is a member of the following medical societies: American Academy of Ophthalmology, American College of Healthcare Executives, American Uveitis Society, Association for Research in Vision and Ophthalmology, and Retina Society

Disclosure: Nothing to disclose.

Mary L Windle, PharmD Adjunct Associate Professor, University of Nebraska Medical Center College of Pharmacy; Editor-in-Chief, Medscape Drug Reference

Disclosure: Nothing to disclose.

Sarah L Wingerter, MD Attending Physician, Department of Emergency Medicine, St Christopher’s Hospital for Children; Clinical Assistant Professor of Pediatrics (Adjunct), Temple University School of Medicine

Sarah L Wingerter, MD is a member of the following medical societies: American Academy of Pediatrics

Disclosure: Nothing to disclose.

Suyung Wu, MD Consulting Staff, Neuroscience Department, Elkhart Clinic

Suyung Wu, MD is a member of the following medical societies: American Academy of Neurology and American Academy of Sleep Medicine

Disclosure: Nothing to disclose.

The standard coronavirus test, if available, works well—but can new diagnostics help in this pandemic? | Science

A doctor in Germany prepares a swab to test for coronavirus infection.

Daniel Reinhardt/picture alliance via Getty Images

By Robert F. ServiceMar. 22, 2020 , 12:15 PM

As the United States races to ramp up testing for the pandemic coronavirus using technology based on the tried-and-true polymerase chain reaction (PCR), alternative approaches are beginning to roll out that could make it easier and quicker for people to learn whether they have been infected. Some methods modify the standard PCR test, which amplifies tiny bits of genetic material to enable detection, whereas others sequence the virus directly or use the genome editor CRISPR.

Faster and cheaper tests are coming, says Evan Jones, CEO of OpGen, a rapid diagnostics company. However, he adds, developing new kinds of tests is “going to take time.” Some of the new tests are coming online now, but others will likely take months to validate and ready for widespread distribution.

“Testing, testing, testing” has been the mantra repeated again and again by World Health Organization Director-General Tedros Adhanom Ghebreyesus. Diagnostic assays that identify active infections in people are vitally important for public health efforts, not just for individuals’ health concerns. Widespread diagnostic testing, along with isolation of the infected, contact tracing, and quarantining of those contacts, seems to have been key in South Korea’s work to suppress virus spread.

Related

In the United States, the slow rollout of coronavirus PCR tests has been widely attributed to a combination of stringent rules aimed at ensuring their reliability and a complex web of companies and health care systems responsible for developing, carrying out, and paying for tests. The Trump administration says testing is accelerating. On 16 March, at a White House press conference, U.S. Health and Human Services Assistant Secretary for Health Brett Giroir said the country would be able to process 1 million tests by the end of the week, and 2 million the following week. But the actual numbers aren’t close to that yet. According to data compiled by the COVID Tracking Project, a nonprofit collaboration of public health officials and journalists counting tests given in the United States, 191,541 PCR diagnostics have been performed as of 22 March, with 24,345 of them positive for the virus.

On 29 February, the U.S. Food and Drug Administration (FDA) posted new rules to allow for emergency use authorizations of coronavirus tests beyond the ones being made and distributed by the U.S. Centers for Disease Control and Prevention. Academic virology labs, public health departments, and companies sprang to work creating their own PCR tests. Today, some four dozen organizations have received FDA approval for their tests. Among the largest are diagnostic companies, such as Roche Molecular Systems, which received FDA’s green light for its test this week. It will initially supply some 400,000 tests per week in the United States and 3 million globally, according to Alexandra Valsamakis, the company’s chief medical officer. Other large companies have recently gained approval for their tests as well, including Thermo Fisher Scientific and Abbott Laboratories.

University virology labs have also leaped into the breach to help diagnose cases in their vicinity. This past week, for example, doctors at the University of Pittsburgh Medical Center (UPMC) began to use a homemade PCR test to check for infection in Allegheny county. For now, its number of tests remains small, about 100 per week. “We definitely wished we had started it sooner,” says Alan Wells, who heads UPMC’s clinical laboratories.

PCR is the most commonly used test for diagnosing coronavirus because it’s highly accurate. (See How does the most common coronavirus test work?) But other problems limit it. “It’s not getting the turnaround we need,” says Steven Wolinsky, an infectious diseases physician at Northwestern University. Each test takes about 4 hours once a sample reaches a centralized testing lab, with the time split between sample preparation and the actual PCR test. With transport and queues, getting a result can take 2 to 4 days. In that time, infected people may spread the virus to many others.

Self-swabbing

Another new dimension is now being added to the coronavirus diagnostic landscape: “home” tests, which involve mailing a sample taken at home to a lab. Tomorrow, for example, Everlywell expects to begin to ship kits to homes and retail pharmacies. These tests will start with screening questions, either online or at a retailer, to determine whether a person is likely to have been exposed to the virus. If they are, they can receive a nasopharyngeal sampling kit by mail or can buy one from a local retailer. A person will be given detailed instructions to administer their own swab, insert it into a protective vial, and overnight mail it to one of dozens of diagnostic labs (which partnered with Everlywell and already have FDA approval) for PCR analysis.

Frank Ong, Everlywell’s chief medical and scientific officer, says the company expects to quickly ramp up from offering thousands of such tests per day to tens of thousands. Although each test will still likely require a 4-day wait for results, Ong says, this home sampling strategy carries major benefits: It will protect health care workers from exposure to potential infection and free up their time. “We need to make sure we give them the bandwidth to take care of patients,” Ong says. Other companies, including Nurx and Carbon Health, say they’re now shipping limited supplies of their own home sampling kits.

Most PCR tests for the new virus are being done with big, expensive automated machines that do many tests at once. Major hospitals or diagnostic facilities have them, but another option beginning to roll out now is smaller, less expensive devices that also do nucleic acid amplification. These could be used by smaller hospitals and even individual doctor’s offices.

On Friday, for example, Cepheid, which sells small PCR systems for rapidly detecting influenza viruses, tuberculosis bacteria, and other microbes, received FDA emergency use approval for a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) test cartridge that slots into the company’s GenXpert system, a device the size of espresso machine that can produce a diagnostic result in as little as 45 minutes. Cepheid officials say that 23,000 such systems are in place worldwide, with 5000 in the United States. On 19 March, GenMark received FDA emergency use approval for its own coronavirus tests, which run on similar-size machines that use a proprietary electrochemical approach to detect target genetic material in less than 2 hours. Other companies rushing to deliver point-of-care diagnostic machines include Mesa Biotech, HiberGene, Mobidiag, and QuantuMDx.

So, too, are companies such as Oxford Nanopore and Fulgent Genetics, which instead of using PCR directly sequence any genetic material in a sample and then look for matches to, say, the new coronavirus. This high-speed gene sequencing approach could help characterize the coronavirus’ genome to better understand how the virus is evolving, but it could also be a diagnostic in certain situations, such as remote sites without access to PCR. Oxford Nanopore’s handheld devices have been used in Ebola outbreaks, and the company sent many to China early in the pandemic.

CRISPR for testing

The fastest way to test for the coronavirus may ultimately be offered by companies using the CRISPR genome editor, better known for adding or deleting DNA in cells. Two U.S. companies, Mammoth Biosciences and Sherlock Biosciences, say they have created CRISPR-based tests and are in the process of validating them with patient samples before seeking emergency use approval by FDA. The technique starts with a patient sample, extracts viral RNA, and uses a fast nucleic acid amplification test called loop-mediated amplification to make just enough RNA for the test to detect. Researchers then add two components of a CRISPR genome editor, a protein called CAS12 that cuts DNA or RNA and a “guide” RNA that slots into CAS12 and helps it search out a sequence corresponding to a piece of the coronavirus genome. If CAS12 and its guide find a match in the RNA, CAS12 binds to that matched RNA, which activates CAS12 to cut it and go on to cut any other short RNA or DNA strands in the vicinity, including copies of a strand designed to liberate color-changing molecules when CAS12 cuts them free. The upshot can be a simple color change on a test strip.

The technique excels at hunting for small snippets of genetic material, says Jennifer Doudna, a biochemist at the University of California (UC), Berkeley, a CRISPR pioneer, who is chair of Mammoth Biosciences’s science advisory board.

In a preprint posted 10 March on medRxiv, researchers at Mammoth Biosciences and UC San Francisco report that tests on clinical samples produced results with accuracy rates comparable to PCR in just 30 minutes. It uses a simple paperlike strip with a colored line that appears with a positive result. The company is discussing with partners manufacturing test kits that would allow rapid and cheap diagnosis of SARS-CoV-2 infection at home without requiring medical know-how, says Trevor Martin, Mammoth’s CEO.

It may take months to finalize the test and get regulatory approval for it, so it likely won’t be ready in the crucial weeks ahead. But it could be ready if the spread of the coronavirus continues. Some predict the virus will also recede but then have a resurgence of infections in the fall. Getting results wouldn’t require PCR machines operated by trained technicians, Martin says. “It would be a game changer for our response” to emerging diseases, Martin says.

Supply problems

Even as companies and academic labs are scaling up their PCR-based diagnostic efforts, hospitals and testing sites around the country report that they are facing a more immediate crunch: Many are running out of chemicals and other materials that enable the tests, such as the swabs to collect samples from patients and the reagents needed by PCR. Benjamin Pinsky, a Stanford University pathologist who developed a PCR-based diagnostic test in use in Northern California, says his lab is facing rolling shortages of different supplies, most notably the kits used to extract RNA from viral samples, before it can be loaded into PCR machines.

“This has been a big challenge,” Pinsky says. “We’ve had to be very nimble in dealing with this,” constantly switching suppliers or even chemical procedures, which must be validated before they can be used on patient samples. His team has even sent pleas over Twitter to the Stanford community and regional biotech companies calling for donations of reagent assemblies, such as kits from Zymo and Qiagen. And even though donations have been pouring in, supplies are still running short, Pinsky says.

Reagent companies are trying to respond. For example, Qiagen, a major supplier of RNA extraction kits, announced Tuesday that its employees are working around the clock to increase production from 1.5 million kits per month to 6.5 million per month by the end of April and further increases later.

Pinsky, for one, says he’s ready for companies to take over coronavirus testing entirely from academics such as himself. “I’m hopeful these companies will be able to provide the testing they have promised,” Pinsky says. “That remains to be seen.”

Polymerase Chain Reaction (PCR) | Services

Cancer/Leukemia

Polymerase Chain Reaction (PCR)

What is Polymerase Chain Reaction (PCR)?

The Polymerase Chain Reaction (PCR) is a molecular biology technique that enables cytogenetic technologists to detect and analyze specific gene sequences in a patient’s sample. The PCR method allows for the amplification of small sections of DNA. For this reason, it is sometimes referred to as “molecular photocopying“.

The purpose of Polymerase Chain Reaction testing is to diagnose and monitor hematologic malignancies and solid tumors. Peripheral blood, bone marrow, and paraffin embedded tissue are acceptable specimen types for Polymerase Chain Reaction (PCR) testing. We use Polymerase Chain Reaction at Genetics Associates in the diagnosis, monitoring, and treatment of patients suffering from Leukemia. This is done in the form of targeted cancer therapy.

How does Polmerase Chain Reaction (PCR) work?

Four things are required in the Polymerase Chain Reaction (PCR) cycle. The first is a target sample.  The target sample the patient whose DNA is going to be amplified by the PCR process.

The second requirement is a primer. The primer acts as an adhesive to the target sample and provides a starting point for the amplification process. Basically, it selects the portion of DNA that will be copied.

The third component of the PCR cycle is the Taq polymerase.  This is the enzyme that replicates the DNA.

Lastly, the fourth step in the PCR process is that Nucleotides must be introduced to act as “building blocks” for the new DNA strands.

The Three Steps of Polymerase Chain Reaction (PCR)

Step one is to heat the target sample in order to denature the DNA. Denaturing is the process of unwinding the two strands of DNA into a single strand.

Step two is to reduce the temperature so that the primer can be added. This allows the added molecules to bind, or anneal, to pieces of the single strand DNA. This part of the process “labels” the starting points of segments of DNA that are to be amplified and replicated.

Step three is to create new segments of the single strand DNA. This  is accomplished by adding the Taq polymerase. It moves along the primed segments of DNA in order to create a template for replication as it moves along. Doing this over and over creates a “chain reaction“, hence the name Polymerase Chain Reaction (PCR).

Polymerase Chain Reaction Testing for Leukemia

The results of this testing is determined by Nashville based laboratory, Genetics Associates. All results are interpreted by board certified and Tennessee licensed cytogenetic directors at our laboratory, who are available to discuss results and offer clinical advice. Contact us to learn more about our PCR testing.

The PCR test | RIVM

Q&As: What does the Ct value on the PCR test mean?

To explain the Cycle Threshold (Ct), we first need to explain what happens during a PCR test. After a swab is used to collect a specimen (the testing sample), this sample is sent to the lab. In the laboratory, lab technicians use equipment to check whether genetic material from SARS-CoV-2 is present in the sample.

To ensure that the test will be as sensitive as possible, a specific fragment of the genetic material is amplified (copied) using the polymerase chain reaction method, known as PCR. This fragment of genetic material is specific to the coronavirus SARS-CoV-2. That means that the test cannot give a positive result for other viruses, such as a flu or cold virus. 

The genetic material is copied in ‘cycles’. During each cycle, the genetic material doubles in volume; this continues until it is detected by the PCR device. The Cycle threshold or Ct value is the number of amplification cycles required to detect the first signal that the virus is present.

What does a high or low Ct value mean?

A low Ct value means that it did not take many cycles to detect the presence of the virus. In that case, there were a lot of virus particles in the sample. This does not necessarily mean that the patient is very ill, or very contagious. Not every patient becomes equally ill as a result of the virus, and not every patient spreads the virus easily. If the Ct value is high, then the patient had a lower viral load (fewer virus particles in the sample), so many amplification cycles were needed to detect the presence of the virus.

A PCR test is very sensitive. Relatively little genetic material from the virus is needed for a positive test result.

At the beginning of the COVID-19 epidemic, RIVMNational Institute for Public Health and the Environment
advised taking a closer look at the PCR results of samples with a Ct value greater than 30. Based on the added experience gained with the PCR test for SARS-CoV-2 since then, it can be stated that the results below a Ct value of 35 are completely reliable. Taking an extra look to see if there is also a good S-curve is therefore only useful at Ct values of 35 or higher. Shifting the checkpoint from Ct 30 to Ct 35 does not increase the number of positive results, but it does save lab technicians a lot of time.

More information about PCR tests and Ct values (in Dutch). 

90,000 Covid-19

COVID-19 became a reality in early 2020 and continues to spread. We try to do our best to keep you and your loved ones safe.

We ask you not to forget about preventive measures and apply for a diagnosis of coronavirus infection in time. Diagnosis of COVID-19 implies the detection of a virus, confirmation of a previous infection, an assessment of the severity of inflammation, and possible complications.

In our medical center “Mederika” you can get all the most necessary services to check your body.

The first thing that can help in the diagnosis of coronavirus is tests that will give a quick and accurate result:

We are also taking additional measures to ensure the health and safety of our patients:

• All local government safety protocols followed
• Hand antiseptic
• We monitor the health of our guests
• First aid kit available
• Access to health care
• There are masks for guests
• Regular disinfection of the room
• Pay no cash
• Physical distancing rules followed
• Shields provided

We wish you and your loved ones health!

diagnostics – prices for PCR tests in Moscow

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Where to take a coronavirus test in Kazan, get tested

The COVID-19 coronavirus was first detected in Wuhan, China at the end of 2019, but already at the beginning of 2020, a pandemic of this virus captured the whole world, including Russia.

People have not been sick with this virus before, and therefore we do not have immunity to it yet.

In severe cases, many patients require a long stay in the intensive care unit and artificial ventilation of the lungs. The virus infects the lungs, and people die from bilateral pneumonia if they do not receive medical attention on time.

The new coronavirus has a set of properties that allows it to spread quickly: about 80% of carriers of the virus get sick easily or do not have any symptoms at all, and the symptoms, if any, are similar to those of a common cold or SARS.

Thus, a person, not knowing about his illness, becomes a carrier of a dangerous infection, continues to go to shops, use public transport, and go to work.

In addition, the disease caused by COVID-19 is severe in older people over 65 years of age.

Therefore, if you suspect that you may have come into contact with a carrier (in transport, a store, at work, etc.), it is important to get tested for coronavirus.

What is this virus?

Coronaviruses are a family of viruses that primarily infect animals.But some of them, such as COVID-19, are beginning to affect people, causing them to become seriously ill. These RNA viruses contain glycoproteins that resemble thorns and give the virus a corona-like appearance. This is where the name of this viral group comes from.

Who needs to get tested for coronavirus?

If you suspect that you may have come into contact with a carrier (in transport, a store, at work, etc.), it is important to get tested for coronavirus.

The analysis is intended for those who do not notice their symptoms, but have reason to worry: for example, they often use public transport (especially during rush hours).

The sooner doctors detect a virus in a patient, the more effective the treatment of complications caused by it will be.

How is the test done?

Two types of tests are used to detect COVID-19:

• PCR smear;
• Serological blood test.

The main type of testing is PCR (polymerase chain reaction). A coronavirus test can only be done in a laboratory. In its process, molecular genetic research is performed to detect the genetic material of COVID-19.

If antibodies to this virus have not yet appeared in the blood or the necessary biological material was not obtained during the collection of a smear, the results of the analysis for coronavirus may turn out to be false negative. Therefore, if patients have a high risk of infection, it is recommended that testing be carried out twice with an interval of several days.

Where can I take a coronavirus test in Kazan?

Residents of Tatarstan can get tested for coronavirus at the Medexpert medical center.The sampling of material for analysis is carried out by appointment at the address: Kazan, Polyclinic Sibgata Khakim, 52.

Get tested for coronavirus (PCR) in Kazan

To pass the test analysis, you must first make an appointment by calling the clinic 590-33-33.

All safety measures taken:

• There is a separate entrance for people who come to take the test;
• only one patient can enter;
• temperature is measured at each inlet.If it exceeds 37.1 ° C, the material is not taken.
• reception time is clearly scheduled, for each one is allocated exactly 15 minutes.

In case of a positive or doubtful result, the data are sent within 2 hours to the Rospotrebnadzor laboratory.

Cost of a coronavirus test in Kazan

90,000 PCR analysis in Saratov in the TANMED clinic to hand over at an affordable price, decoding, norm

No one is immune from sexually transmitted infections.The best way to fight is timely diagnosis. PCR analysis or polymerase chain reaction is performed to detect Trichomonas by searching for similar DNA and RNA. The method allows to confirm or deny the presence in the human body of gonococci, gardnerella, microplasmas, chlamydia.

You can undergo a comprehensive examination to identify sexually transmitted diseases at the TANMED clinic.

Who should be examined?

We offer a survey on modern, high-tech equipment, with the definition of accurate results.

It is recommended to consult a doctor if:

• itching appeared
• burning sensation in the genital area
• unpleasant odor
• discomfort when urinating
• the nature of the discharge has changed: amount, consistency, smell, color
• there was unsecured contact with new partner
• pregnancy planning

Principle of operation and advantages of PCR analysis

Research by the PCR method is based on the creation of a reference detector of the genetic material DNA and RNA, inherent in a particular causative agent of the disease.Thanks to the rule of complementarity, the standard is able to detect a related fragment of genetic material in the analyzed sample and start copying. It takes very little time to create billions of copies. Thus, it is easy to detect the presence of infection in any biological organism.

The main advantages of polymerase reaction assisted treatment are:

• versatility – often used when other ways of establishing are not effective;
• accuracy of identification of the type of pathogen;
• high sensitivity – contributes to the detection of the disease at an early stage, when there are no clinical or laboratory manifestations.

At TANMED LLC, only biomaterial is taken, then it is sent to a specialized laboratory. The analysis time is on average 2-3 working days.

Entrust your health care to professionals

In case of any ailment, consult a doctor. Timely diagnosis will allow you to identify the problem at an early stage and quickly prescribe the appropriate treatment. The medical staff of the TANMED clinic provides qualified medical care at affordable prices.

How do coronavirus tests work and can they be wrong? We analyze the pros and cons of the most popular

05/19/2020, TV Channel 360

Russian companies are developing and registering tests to detect SARS-CoV-2. Some are based on reliable PCR techniques. Others release express systems. “360” learned from manufacturers and scientists what are the advantages and disadvantages of those and other tests, and most importantly, why they are sometimes unreliable.

Test types

Rapid tests are less diagnostic than simple tests. They are excellent for determining the boundaries of the focus, identifying the carrier of the infection in the population. But, explained “360” the deputy director of the FBUN MNIIEM named after Gabrichevsky Evgeny Selkova, together with them you need to do an enzyme-linked immunosorbent assay (ELISA) or PCR diagnostics.

PCR – polymerase chain reaction, the same swab from the nasopharynx. RNA is extracted from the sputum. DNA is modeled on its basis and duplicated many times using a polymerase enzyme.This process is called amplification. In this case, only the part that meets the specified conditions is copied – it coincides with a particle of the virus, and only if it is in the sputum. Due to this, the concentration of pieces of RNA / DNA in the sample increases and indicates whether a person has a disease.

The ELISA method with high accuracy detects antibodies to coronavirus in the blood serum. Moreover, even in those cases when the pathogen did not lead to an obvious illness and the person tolerated it asymptomatically.On May 15, free ELISA testing of the population will begin in Moscow.

“You can determine whether a person was sick or is now in an acute phase by those proteins that are detected – immunoglobulin G-class or immunoglobulin M-class. M is an acute phase, G means that a person has recently been ill and his immunity has begun to form, ”Selkova said.

The ELISA examination, the head of the department of microbiology of latent infections of the Gamaleya Institute, Viktor Zuev, told 360, will show the extent to which the residents of the capital have developed herd immunity.And based on this information, they will make a forecast about the end of the pandemic. In order for the disease to stop spreading in Moscow, 60–70% of the population must have immunity to it.

Why tests lie

Some of the test developers interviewed by “360” explained that the result can be affected by the violation of the storage or transportation conditions of the analyzes, as well as errors during the testing itself.

In every diagnostic procedure, there is such a concept as sensitivity.Some tests reveal the presence of the virus at a lower concentration of its particles in sputum, others at a higher concentration, Zuev said. The latter can give a negative result if this concentration is small. The sensitivity of tests from different manufacturers is different, so not everyone shows the presence of a virus the first time. Another important characteristic is the specificity of the method.

“Viruses have a number of closely related components. If a test for two measles “viruses” detects measles, but gives a positive result for a thousand “viruses” of influenza, this is a non-specific test.In addition to sensitivity, the test should be specific, ”concluded Zuev.

Test systems EMG

Test systems for the detection of the SARS-CoV-2 strain of the Russian-Japanese company EMG were created with the participation of the Russian Direct Investment Fund (RDIF). They are based on advanced Smart Amp technology. Thanks to it, the accuracy reaches 99.9%, and for most analogs it is below 95%. Sensitivity – 50-100 copies of the virus in the sample, and not 1000-10,000 copies, as in others.

“This is an innovative test because it allows analysis in 30 minutes, rather than the hours that an existing test takes with the same accuracy.And it allows testing in mobile labs. That is, it is not necessary to have a large-scale laboratory, ”said Kirill Dmitriev, RDIF CEO, to 360.

The mobile laboratory is two compact suitcases that allow you to test people anywhere. One is intended for the isolation of RNA in sterile boxes, the second is an amplifier that copies its pieces. The productivity of one portable laboratory for coronavirus is 24 tests per hour.

Now the tests are used in the metropolitan KDL laboratories and at some large Russian enterprises.The manufacturer also launched a joint project with Yandex for testing at home.

“Polivir SARS-CoV-2”

The specificity of the PCR kit “Polivir SARS-CoV-2” developed by the Scientific and Production Company “Litekh” is 100%. The sensitivity of ELISA diagnostics is 97.5%, and the specificity is 97.9%. PCR kits are designed for 100 tests. In comparison with analogs, they can be classified as express tests.

“Our kits, among other PCR kits, can definitely be called express kits due to the unique RNA-express isolation, which is included in the kit and greatly speeds up the testing process by one and a half to two hours.To determine antibodies to coronavirus, our company supplies standard ELISA kits. The analysis time is about one and a half hours, “- said” 360 “Deputy Director of the company Lyudmila Petrova.

PCR kits are delivered to the capital’s health department, then they are distributed to hospitals and clinics. According to doctors, Petrova noted, the tests are distinguished by high specificity, speed of research, ease of use and low cost.

Tests “Medsi”

Sistema-Biotech LLC has developed PCR tests for the Medsi network of clinics and institutions subordinate to the Moscow Department of Health.An express system is currently undergoing testing, which will allow identifying cases in a mobile mode, the director general of the organization Dmitry Mordvintsev told 360.

“In the case of PCR, the advantage is that it is a very accurate, sensitive and highly reproducible test. But it requires specific and expensive equipment and personnel who know how to use it. And it’s long, ”added Mordvintsev.

Express tests do not need expensive equipment, and they are done in just 30 minutes.True, their accuracy is somewhat lower. According to Mordvintsev, the company has never encountered false positive or false negative results of its products. They estimate the accuracy of their PCR tests at 97-100%, and the express method at 80-85%. But they plan to raise the figure to 90–95%. The sensitivity is higher than most analogs.

A distinctive feature of the tests is that all reagents are in one reaction mixture. And all processes take place in one test tube. Other technologies simplify the process and allow you to control the release of RNA in each tube, and not do it in parallel.That is, no matter how SARS-CoV-2 mutates, the test will always determine it.

“Isotherm SARS-CoV-2 RNA-screen”

This is an express test developed by Generium JSC in collaboration with the Novosibirsk group of companies Medico-Biological Union. The sensitivity of the kit exceeds 1000 copies of the virus RNA in a milliliter of sputum.

“When developing the test, we proceeded from such shortcomings of classical PCR tests as the low speed of obtaining the test result and, as a result, the high workload of the current equipment.The main task was to create a test that allows using standard equipment to detect the presence of a virus with the highest accuracy and speed, “Generium CEO Dmitry Kudlai told 360.

Thanks to the extremely simplified procedure for preparing a sample for analysis and the use of standard equipment, the duration of the analysis is reduced to 40 minutes, which allows up to 1500 analyzes per day. With the help of such kits of reagents for the detection of coronavirus, residents of 65 regions of Russia are tested.

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