NUCLEIC-ACID AMPLIFICATION TESTS

NUCLEIC-ACID AMPLIFICATION TESTS

Nucleic-acid amplification tests, also known as NATs or NAATs, are used to identify small amounts of DNA or RNA in test samples. When it comes to STD testing, there are NATs available that can detect a variety of different STDs. In fact, most urine tests for STDs are performed using nucleic-acid amplification tests.

MECHANISM

A NAT uses a series of repeated chemical reactions to make numerous copies of the DNA or RNA that doctors are trying to detect. These reactions amplify the signal of the nucleic acids in the test sample so that they are easier to identify. It's much simpler to find 10,000 copies of a gene than 10. 

once the amount of DNA or RNA has been increased in the sample using PCR or LCR, more conventional tests are used to detect it. These tests usually involve some form of nucleic acid hybridization. In those tests, the sample is probed with an artificially produced complementary strand of DNA or RNA that has been labelled in some way that makes it easy to detect. It may help to picture it as a glow in the dark tag that only sticks to one very specific piece of identifying information.

STD TESTING

Nucleic-acid amplification tests are incredibly useful for STD testing. They allow doctors to detect an STD pathogen even when only a very small number of organisms are present. It is this sort of technology that has made it possible to do urine testing for STDs that were previously only detectable by swab.

Furthermore, since nucleic-acid amplification tests are incredibly sensitive to even small amounts of viral DNA, they are very important for screening the blood supply. These tests make it possible to detect tiny amounts of HIV and other blood-borne pathogens that might otherwise be missed.

There are also non-amplified nucleic acid tests available for certain STDs, such as gonorrhea and chlamydia. Non-amplified nucleic acid hybridization tests are more likely to be used when large amounts of bacterial or viral DNA (or RNA) would be expected to be present, such as in a urethral swab or in a bacterial culture sample. In such circumstances, no amplification is necessary. In these samples, if DNA or RNA is present, it should be present in detectable amounts.

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No more ‘sledgehammer’: As gonorrhea grows resistant to antibiotics, researchers look to bespoke treatments

With antibiotic resistance on the rise, the days when doctors and clinics could rely on one treatment to cure all gonorrhea cases may be waning.

In fact, clinicians may find that some of their patients respond best to drugs of the past. But how will they know which patients?

To answer that question, a handful of researchers and companies are trying to develop rapid, point-of-care diagnostics that would signal which drugs work for a given patient and permit clinicians to tailor treatment to the bacterial strain. A future, in other words, of bespoke treatment, in which a greater variety of drugs are used to treat a very common sexually transmitted disease — potentially prolonging the utility of several antibiotics.

“It would allow for smarter medicine,” Dr. Jeffrey D. Klausner, a professor of infectious diseases medicine at the University of California, Los Angeles, told STAT. “Right now, we’re treating gonorrhea with a sledgehammer, we’re treating everything with the same exact regime. And it’s not a surprise that the organism will become resistant to what we’re currently using.”

Klausner has collaborated with several companies working in this area but has not received payment from the companies. The companies have made contributions to UCLA, he said.

Gonorrhea is spread by sexual contact, and infection can occur in the genitals, the rectum, and the throat. The World Health Organization estimates that about 78 million people a year are infected with gonorrhea; roughly 820,000 of those infections occur in the United States.

Left untreated, gonorrhea can cause pelvic inflammatory disease and infertility in women and sterility in men. Infection increases a person’s risk of contracting HIV. Babies born to an infected woman can develop blindness.

From a treatment point of view, the ideal regimen for curing gonorrhea is an oral antibiotic — and was long the standard of care. But gonorrhea is a wily bacterium, adept at learning tricks that allow it to evade the power of antibiotics. One after another, the available drugs have started to fail.

For several years, the recommended regimen for gonorrhea has been a dual-therapy approach, the thinking being that it is harder for resistance to develop to a drug combination than to a single drug. The current recommended therapy is one injection of the antibiotic ceftriaxone followed by a multiday course of azithromycin. But even that approach is starting to show signs of weakness.

At the same time, there has been a belief that, even as medicine has moved on, there is life left in the drugs that have been abandoned — if you could figure out in whom they would work.

“We’ve been treating empirically and a lot of times ignoring the fact that the majority of organisms, the majority of infections we’re seeing, are actually fully susceptible to the old drugs,” said Fred Tenover, vice president for scientific affairs at Cepheid Inc., a California-based maker of molecular diagnostics.

“So now we have to have a change in philosophy and say: You know what? Now it’s time to go and do what we do for every other infection, which is actually define what the best therapeutic is up front.”

Cepheid is exploring the idea of developing such a test, Tenover said. “We’re trying to figure out what it would look like and what it would cost,” he said. “This is not in our pipeline officially. It’s something we’re looking at, something we haven’t committed to, but we have figured out how to make the assay.”

Other smaller companies are in the hunt. SpeeDx, an Australian-American firm, has a test in development that it hopes will receive licensing approval in Europe this autumn. The company is currently conducting a clinical trial in the United States in hopes of with the goal of filing for approval with the Food and Drug Administration.

The test both diagnoses gonorrhea and indicates whether the infecting strain can be treated with Cipro, said CEO Colin Denver. It currently would need to be processed by a central lab, but the plan is to develop the assay as a point-of-care test that could be used in a clinic or doctor’s office.

Shield Diagnostics, of Santa Clara, Calif., has a similar idea in mind. The company has a proprietary testing platform; CEO Fred Turner said it will roll out its first test this fall. The test can diagnose gonorrhea and ascertain if the strain is susceptible to Cipro.

For now, it is not a point-of-care test; that may be three or four years down the road, Turner said. In the meantime, Shield will process the tests in its own laboratories. Turner said the testing will likely take two to three days. Ideally, over time, both companies hope their assays will be expanded to give a broader resistance profile, one that indicates whether other drugs would be effective.

“Our ultimate goal is to have clinicians utilize a test-and-treat algorithm so that you don’t have to use these empiric therapies,” Denver said.

A patient would come into a clinic and specimens to test would be taken. While he or she is seeing the doctor, the test would be run. Within an hour or so, the doctor would know whether a single dose of Cipro or cefixime (also an oral drug) would do, or whether the ceftriaxone-azithromycin combination would be the better option.

“It’s like personalized medicine — your individual infection treated with the antibiotic you know is going to work,” Denver said.

But in order for these types of tests to be truly useful, they’ll need to be affordable and produce a rapid answer, said Dr. Lindley Barbee, an assistant professor of infectious diseases at the University of Washington and medical director of the Public Health – Seattle & King County STD Clinic. Not a day or two later, but while the person was in the clinic.

Many clinics that treat sexually transmitted infections are government funded, Barbee noted: “You have to get the cost low enough to make it worthwhile.”

That’s clearly a factor that has slowed development of these tests. “If you say to somebody who’s paying $10 for this test now, ‘Well, I can better define your therapy but it’s going to cost $17 a test, not $10 a test,’ that may be beyond the reach of a lot of public health clinics or STI clinics. And that’s probably what it would take,” said Cepheid’s Tenover.

“To date the return on investment has not been positive enough to make these commercially available,” he said. “That may be changing. And I think as we talk more and more about the spread of superbug gonorrhea, people may be more and more interested in doing this. But in the past there hasn’t been a market for these types of tests.”

Source: https://www.statnews.com/2018/07/17/gonorrhea-grows-resistant-to-antibiotics-researchers-look-to-bespoke-treatments/

Montreal scientists score possible breakthrough for rapid diagnostic medical tests

A simple and fast chemical process developed by scientists in Montreal could allow family doctors to one day use equipment as straightforward and cheap as a diabetic's blood sugar tests to diagnose a range of diseases in minutes from their offices, instead of waiting days for results to come back from a lab.

In the biosensor developed by Montreal researchers, if an an antibody or other protein (green or red) is present, it binds to a DNA strand (green and red spirals), affecting how it then pairs up with its complementary strand on the surface of a gold electrode. A decrease in electrical current results. (Ryan & Peter Allen/University of Montreal)

In a paper in yesterday's issue of the Journal of the American Chemical Society, researchers from the University of Montreal describe a novel way to detect large molecules like antibodies in blood using a quick, one-step process involving electricity and DNA.

The technique could give rise to a proliferation of new so-called point-of-care medical tests — blood tests that can be performed in the field, possibly even by patients themselves, and that yield results in minutes.

"Nowadays if you go see a physician to have a blood test, they'll send you to a clinic. A couple vials of blood will have to be analyzed by a technician with specialized techniques, and the results will only come back a week later," said paper co-author Alexis Vallée-Belisle, a University of Montreal chemistry professor and the Canada Research Chair in bioengineering and bio-nanotechnology.

"What these tests are looking for is antibodies — proteins that are generated by the body in response to viruses or bacteria. The highlight of the technique we developed is you put a drop of blood on our electrodes, and in five or 10 minutes you can know if you have the antibody. And in principle, you can detect any antibody you want."

Electrical current

Vallée-Belisle and his team, working with a chemist from the University of Rome, came up with a way to detect antibodies by having them bind to loose, single strands of DNA. Those single strands then attempt to pair up with nearby complementary strands to form the characteristic DNA double helix.

The researchers designed the single DNA strands so that if no antibodies are present when they bind with their complementary pairs, detectable electrical current flows. But if antibodies are present and attached to the loose strands, the current drops. The drop is so precisely measurable that the test can even be used to determine how much antibody is in a sample, instead of simply indicating its presence or absence.

They call the process "electrochemical steric-hindrance hybridization assay," or eSHHA, because it takes advantage of how the DNA molecules react in the presence of other larger particles, an effect known as "steric hindrance."

Biochemistry professor Kevin Plaxco of the University of California at Santa Barbara, who supervised Vallée-Belisle's previous postdoctoral work and who is himself an expert in electrochemical methods to detect antibodies, called the results "clever" and "very simple and elegant."

"They're detecting the molecules in a way that has some rather interesting advantages," he said Thursday.

There are currently point-of-care antibody tests on the market for diagnosing HIV within minutes, Plaxco pointed out, but they give qualitative yes-or-no results and not nuanced, quantifiable data on how much antibody was found.

That might not be needed for HIV, but it could be useful for doctors treating autoimmune diseases, or for using the eSHHA technique to detect things like how much of a chemotherapy drug a patient is metabolizing so that their dose can be fine-tuned.

STD screening in minutes

The Montreal researchers' method would also allow for simultaneous testing for the presence of more than a dozen different antibodies, meaning one small drop of blood and one quick test would be enough to screen for almost every sexually transmitted disease within minutes, at a doctor's office.

The doctor could then immediately notify the patient and prescribe medication right away if any of the results came back positive.

"Currently a large percentage of people don't come back to see the results, because you need to take another appointment, so you waste a lot of time and a lot of money," Vallée-Bellisle said.

He said so far, his team has shown their method works to detect five different typical "model" proteins and will soon publish research showing they can also detect three of the various HIV antibodies. After that, they're also aiming to detect antibodies for syphilis and herpes.

One of the big advantages of their method is its cost: The electrodes used to detect the electrical current can be had for five to 10 cents each and creating the necessary DNA sequences can be as cheap as $10 for a large enough strand to run "thousands of tests," Vallée-Bellisle said.

"The basic infrastructure for all this is dirt cheap," Plaxco confirmed.

Vallée-Bellisle said the next step in commercializing their findings will be signing up a business partner to work on developing the right niche for their work.

Source: https://www.cbc.ca/news/technology/montreal-scientists-score-possible-breakthrough-for-rapid-diagnostic-medical-tests-1.3243022

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