Scientific Method —

Making HIV tests visible to the naked eye

Pregnancy-test-style diagnosis gives the blue stripe a whole new meaning.

One of the recurring themes in healthcare is that a lot of what works in the developed world doesn't work in the developing world. There may be limited access to basics like power or communications facilities, never mind the medical technologies that make a hospital one of the modern wonders of the world. Beyond simple access to expensive technologies and medicines, even something as basic as a diagnostic test might be too expensive or require skilled technicians to use.

Though cures might remain expensive—drug companies like their profits—every dime saved on diagnosis is a dime more for prevention and cure. That makes cheap, accurate, and simple diagnostic tests very, very desirable.

I was thinking about this when I came across an older paper that somehow didn't attract any attention when it came out. Last year, a group of researchers showed that they could detect HIV at extremely low concentrations. That by itself is nothing special: people are always improving diagnostic tests. What is special is that the test is very much like a pregnancy test, in that a simple visible color change indicates a positive result. Even better, it seems to work in real-life tests.

The similarity to a pregnancy test goes a bit deeper than how the test results appear. In a pregnancy test, the blue color comes from a reaction that causes gold to come out of a solution and form small irregular clumps. Similarly, the blue color in the researcher's HIV test comes from slowing the formation of gold particles.

Let's take the case of a sample from a patient that is HIV negative. In that case, the blood will not have any of the viral particles or viral proteins (called antigens). The researchers rely on a targeted reaction between antibodies and the antigens they recognize to bind a chain of antibodies together on a substrate. The last antibody in the chain is attached to an enzyme that catalytically destroys one of the reactants required to form the gold particles. If no antigen is present, the enzyme is free to diffuse and its concentration remains low. As a result, the gold can quickly precipitate out and form individual spherical gold particles that turn the solution red.

If the patient is HIV positive, the enzyme's concentration becomes high at the sites where the antibodies attach to HIV. Once again, it removes one of the reactants, slowing the precipitation of gold. But, because it's no longer diffuse, there are regions where the slow reaction produces agglomerates of lumpy gold crystals that turn the solution blue.

Obviously, the degree of color change depends on the amount of HIV antigen in the sample, so the solution goes from red through green to blue over a narrow range of HIV antigen concentration.

The most interesting part of this study, however, is the way it was tested. Papers claiming extraordinary sensitivity to some cancer-indicating or infection-indicating antigens are regularly published, but the tests never get marketed. Why is that? The test is all on artificial samples: take the antigen, and dilute it with salty water. If you are feeling especially adventurous, add a single confounding antigen. Publish results proclaiming miraculous sensitivity and dirt cheap tests.

Unfortunately, those results probably depend on the highly purified samples that were tested. In reality the effectiveness (and cost) of diagnosis has been shifted from the test to purifying the samples the tests were run with. Not very useful.

In this work, the researchers started with whole serum. For HIV, the test was, ultimately, performed on people who were HIV positive with detectable viral loads, HIV positive people with undetectable viral loads, and HIV negative people. The test actually performed better than standard HIV tests, because it could pick out the patients whose viral load was below the limits of detection via the standard method.

That is all pretty cool, but I am not sure that these tests are going to make it to Zimbabwe or Uganda any time soon. There are at least three issues that I (a non-expert) spotted. First is that the HIV test is only sensitive to HIV-1. That's the most significant threat to human health, but HIV-2 is also a hazard. I am sure that it would be very simple to make a test that is also able to detect to other strains of HIV, but that may well make it more expensive and less sensitive.

The second issue is the use of antibodies and enzymes. If you look in a chemistry catalog for antibodies and enzymes, they are often sold by the microliter. That indicates a certain amount of expense, which is exactly the opposite of the stated goals of the project.

The last issue isn't directly related to the HIV test, but rather comes from a second test the researchers performed. They tested for the recurrence of a cancer using a different antigen. Antigen tests for different cancers have been "under development" for years. But, the big issue is that they often produce false positives: you have the antigen, but not the cancer. This is not something that the researchers can do anything about in terms of their test, but it's a reminder that any diagnosis of that nature needs to be very carefully tested before it is released upon unsuspecting general practitioners.

Chris Lee
Chris writes for Ars Technica's science section. A physicist by day and science writer by night, he specializes in quantum physics and optics. He lives and works in Eindhoven, the Netherlands. Emailchris.lee@arstechnica.com//Twitter@exMamaku

Let's assume we get a test with a 25% false positive rate, which is pretty high, but a very low false negative rate. Someone thinks they have HIV. They get the test.It says negative, so they can go on their merry way.It says positive, they go in for further testing.

However, the false negative rate needs to be effectively zero. Not 0.01% but rather zero. If someone takes the test, and they get a negative (but are actually positive) they are endangering the lives of other people. Pregnancy test error rates aren't a huge deal because worst case scenario only affects the mother. Imagine the uproar if those false tests led to additional pregnancies.

Let's assume we get a test with a 25% false positive rate, which is pretty high, but a very low false negative rate. Someone thinks they have HIV. They get the test.It says negative, so they can go on their merry way.It says positive, they go in for further testing.

However, the false negative rate needs to be effectively zero. Not 0.01% but rather zero. If someone takes the test, and they get a negative (but are actually positive) they are endangering the lives of other people. Pregnancy test error rates aren't a huge deal because worst case scenario only affects the mother. Imagine the uproar if those false tests led to additional pregnancies.

Only affects the mother, and usually they find out on their own in relatively short order.

One more problem that I ( also as a non expert) see with this is that HIV can take a number of months to seroconvert. This could cause someone to take the test and get a negative result while truly being infected. Don't get me wrong, it is a good step forward for testing, but there will need to be education with this type of product. Perhaps they should include two in a pack so that you can do a baseline test and then another 6 months down the road to ensure the first test was accurate.

you can buy HIV tests at your local pharmacy that work by taking swabs from the inside of your mouth. $40.

has to be 3 months after the probable infection, when the body builds up a certain number of antibodies to HIV that the test can detect.

"OraQuick In-Home HIV Test"

I agree that OraQuick is good (it gives an easy to read diagnosis and also tests for HIV 2), but the first paragraph of the article talked about bringing costs down to make the tests viable in the developing world. $40 for one test just isn't going to work in many places. Even 40 cents might be too high in some places.

One more problem that I ( also as a non expert) see with this is that HIV can take a number of months to seroconvert. This could cause someone to take the test and get a negative result while truly being infected. Don't get me wrong, it is a good step forward for testing, but there will need to be education with this type of product. Perhaps they should include two in a pack so that you can do a baseline test and then another 6 months down the road to ensure the first test was accurate.

Presumably the antigen they target is a viral protein that is much less likely to mutate than other proteins, making this much less of an issue. Well, that's what *I* would do, however it's entirely possible that a) we don't know what proteins are less likely to mutate and/or b) all of the proteins are likely to mutate in a few generations

I'm not very impressed by this article, however. The technology described is a simple sandwich ELISA, the same technology used in many (or most) home pregnancy tests. And yes, they too use antibodies, so this in itself is not necessarily a cost burden, given that ELISAs are exquisitely sensitive (and therefore require extremely small amounts of antibody) and mass production of the antibodies would drastically lower the price.

Furthermore, this article tells me nothing about what these researchers did that was unique. What is described in the article is completely unremarkable because, again, it's simply a sandwich ELISA with a special enzyme conjugated to the secondary antibody. I'd be shocked if this is the only group to use this approach to detecting HIV, so what did they do that was so special?

EDIT: I just skimmed the paper and I also could not find what they did that was so damn special. It appears that they used entirely existing technologies and simply applied them to the problem of detecting HIV. That's great and useful, but I am surprised this was published in Nature Nanotechnology given the seeming lack of novelty behind the technology.

I've had family members who have worked in hospital labs and many of the tests done cost just pennies between the chemicals needed and the time of the lab tech. However, the hospitals charge 40-50-even 100 dollars for those tests. Looking at what a hospital charges for a test is not a viable way to determine how much the testing really costs.

In the casse of the $40 In-home HIV test, we can't just look at the retail cost. To see if it is viable for a developing country, we need to look at the costs of raw materials, manufacturing, and shipping. This could be something that costs pennies to make and ship, but then the compnay making the test charges far more than that to the distributor, then the distributor charges even more to the pharmacy, then pharmacy charges more to the customer.

With a disease that is as serious and carrying as big a stigma such as HIV, you need a simple preliminary test that actually will give quite a few false-positive results, but the results of the test are only used to see if a person should get a full HIV test. While having false-positives isn't a good thing, if you want a fast, cheap test, it should be considered as a preliminary test, because it would cut the number of people who will take the more expensive, but more conclusive test. You would rather have the initial test to give false-positives than false-negatives when if comes to an untreatable, commutative disease like HIV.

One super important thing to note is that the test for HIV is only a small part of any HIV test. The more important part is the human interaction available if a person does test positive. Because of the (incorrect) stigmas associated with HIV, even by those that are educated on the topic, it can cause severe mental/emotional problems. Going to a clinic and getting tested will always be the best way and should be done regularly if you are sexually active.

Home tests like the already available OraQuick do provide phone counseling if you tested positive, but that still takes a step by the person. A local clinic will have much better resources available to help people out.

One more problem that I ( also as a non expert) see with this is that HIV can take a number of months to seroconvert. This could cause someone to take the test and get a negative result while truly being infected. Don't get me wrong, it is a good step forward for testing, but there will need to be education with this type of product. Perhaps they should include two in a pack so that you can do a baseline test and then another 6 months down the road to ensure the first test was accurate.

Presumably the antigen they target is a viral protein that is much less likely to mutate than other proteins, making this much less of an issue. Well, that's what *I* would do, however it's entirely possible that a) we don't know what proteins are less likely to mutate and/or b) all of the proteins are likely to mutate in a few generations

I'm not very impressed by this article, however. The technology described is a simple sandwich ELISA, the same technology used in many (or most) home pregnancy tests. And yes, they too use antibodies, so this in itself is not necessarily a cost burden, given that ELISAs are exquisitely sensitive (and therefore require extremely small amounts of antibody) and mass production of the antibodies would drastically lower the price.

Furthermore, this article tells me nothing about what these researchers did that was unique. What is described in the article is completely unremarkable because, again, it's simply a sandwich ELISA with a special enzyme conjugated to the secondary antibody. I'd be shocked if this is the only group to use this approach to detecting HIV, so what did they do that was so special?

EDIT: I just skimmed the paper and I also could not find what they did that was so damn special. It appears that they used entirely existing technologies and simply applied them to the problem of detecting HIV. That's great and useful, but I am surprised this was published in Nature Nanotechnology given the seeming lack of novelty behind the technology.

Not everything that goes in nature-type publications contains earth shattering new results. In this case, the general interest is what sells it. As someone above said, in the western world, a 40 buck test is fine. In the developing world that is far too expensive. Yet, there is bugger all profit to be made in making a really really cheap test. That is what sells the paper.

One more problem that I ( also as a non expert) see with this is that HIV can take a number of months to seroconvert. This could cause someone to take the test and get a negative result while truly being infected.

It all depends what you're actually testing for. Seroconversion is the point at which your immune system produces antibodies to an infectious organism. If the test is looking for the presence of such antibodies, then yes, these may take months to appear.

However, in the paper under discussion, the investigators made a test that was sensitive to a protein in the HIV-1 capsid (shell). So the test should produce results as soon as the number of viral particles in serum is measurable.

Obviously, there is still going to be a slight delay before the viral load is significant, so the test can still give a negative result immediately after initial infection. But testing for viral capsid proteins doesn't depend on an immune system response, and is probably as good as practically possible.

I've got to say that I was pretty impressed by the headline sensitivity of 1 × 10−18 g ml−1.

One more problem that I ( also as a non expert) see with this is that HIV can take a number of months to seroconvert. This could cause someone to take the test and get a negative result while truly being infected.

It all depends what you're actually testing for. Seroconversion is the point at which your immune system produces antibodies to an infectious organism. If the test is looking for the presence of such antibodies, then yes, these may take months to appear.

However, in the paper under discussion, the investigators made a test that was sensitive to a protein in the HIV-1 capsid (shell). So the test should produce results as soon as the number of viral particles in serum is measurable.

Obviously, there is still going to be a slight delay before the viral load is significant, so the test can still give a negative result immediately after initial infection. But testing for viral capsid proteins doesn't depend on an immune system response, and is probably as good as practically possible.

I've got to say that I was pretty impressed by the headline sensitivity of 1 × 10−18 g ml−1.

With a disease that is as serious and carrying as big a stigma such as HIV, you need a simple preliminary test that actually will give quite a few false-positive results, but the results of the test are only used to see if a person should get a full HIV test. While having false-positives isn't a good thing, if you want a fast, cheap test, it should be considered as a preliminary test, because it would cut the number of people who will take the more expensive, but more conclusive test. You would rather have the initial test to give false-positives than false-negatives when if comes to an untreatable, commutative disease like HIV.

That depends on a lot of factors. There are two purposes of the test: (1) to identify people who need further testing and need treatment (2) to inform people already infected that they are infectious so they can modify their behavior and thereby minimize the risk to others. With respect to (1), false positives are the risk -- they result in wasted health care spending. Also, an infected person will delay treatment and may get sicker than otherwise. With respect to (2), other peoples' health and lives are put at risk.

How this affects the population is a complex problem. If the escape rate is low and the population's probability of infection is high, false negatives probably don't matter all that much because the person's belief that he is disease-free is not likely to persist for many sexual encounters after the failed test. But if the population rate of infection is low, he will believe himself noninfectious potentially for a long time and many people could be exposed to the risk of infection from that individual before he thinks he ought to get tested again.

In either case, of course, the spouse of the person who gets a false negative can be put at great risk by the false negative but not by a false positive.

there is more to it than that. I focused on HIV testing, but the false positives in something like prostate cancer can lead to an unnecessary seriously invasive procedure to simply confirm the positive result.

So, yeah, false negatives are worse than false positives. But that does not make false positives trivial.

Let's assume we get a test with a 25% false positive rate, which is pretty high, but a very low false negative rate. Someone thinks they have HIV. They get the test.It says negative, so they can go on their merry way.It says positive, they go in for further testing.

However, the false negative rate needs to be effectively zero. Not 0.01% but rather zero. If someone takes the test, and they get a negative (but are actually positive) they are endangering the lives of other people. Pregnancy test error rates aren't a huge deal because worst case scenario only affects the mother. Imagine the uproar if those false tests led to additional pregnancies.

As I understand this disease, and frankly many diseases, they are a lot trickier than a simple binary on/off. Most diseases are very hard to find in the early stages as low concentrations of anything is hard to detect. On the bright side, it probably also makes you less contagious. So probably a fairly accurate test is useful, though we would all wish for a “perfect” one.

Over all, people need to be more careful and act in a way that assumes the worst. In truth, we have all the tools we need to stop this disease and have for a while, in theory. Much of this story is really about the difficulty in modifying human behavior.

With a disease that is as serious and carrying as big a stigma such as HIV, you need a simple preliminary test that actually will give quite a few false-positive results,

No, finding out that you may have an incurable disease is not something you want false positives on. That would be more than a little mentally traumatic for a lot of people.

Most of the people you would be giving the quick, cheap test wouldn't be mentally traumatized by a false positive. For one, if it's just being use as a preliminary test, people would be fully informed that a positive on this test wouldn't be conclusive if its allowed to have a high chance of false-positives so to avoid the possibility of false-negatives. A quick test would be to test people who are at high risk of contracting HIV and would likely end up spreading it (i.g. prostitutes, intravenous drug users). The test would be aimed towards people who need to be tested on a regular basis, but you don't want to spend the time, money, or resource on because they are putting themselves at risk. It wouldn't be used with people who were inadvertently exposed like those from Dr. Wayne Harrington dental office; you would use the more precise test because you know that they were exposed to the virus. Those people you wouldn't do the quick test unless they ask for it. Those would be the ones who would be traumatized if they received a false-positive, but some may opt for it if given the option.

With a disease that is as serious and carrying as big a stigma such as HIV, you need a simple preliminary test that actually will give quite a few false-positive results, but the results of the test are only used to see if a person should get a full HIV test. While having false-positives isn't a good thing, if you want a fast, cheap test, it should be considered as a preliminary test, because it would cut the number of people who will take the more expensive, but more conclusive test. You would rather have the initial test to give false-positives than false-negatives when if comes to an untreatable, commutative disease like HIV.

That depends on a lot of factors. There are two purposes of the test: (1) to identify people who need further testing and need treatment (2) to inform people already infected that they are infectious so they can modify their behavior and thereby minimize the risk to others. With respect to (1), false positives are the risk -- they result in wasted health care spending. Also, an infected person will delay treatment and may get sicker than otherwise. With respect to (2), other peoples' health and lives are put at risk.

How this affects the population is a complex problem. If the escape rate is low and the population's probability of infection is high, false negatives probably don't matter all that much because the person's belief that he is disease-free is not likely to persist for many sexual encounters after the failed test. But if the population rate of infection is low, he will believe himself noninfectious potentially for a long time and many people could be exposed to the risk of infection from that individual before he thinks he ought to get tested again.

In either case, of course, the spouse of the person who gets a false negative can be put at great risk by the false negative but not by a false positive.

For scenario 1, the person's lifestyle is the cause of wasted spending not the results of a false-positive. If you are believed to have been exposed to HIV, you are put on antivirals whether there is a false-positive or not, because you don't know if the person has the virus until the results can be verified. And if a person does have HIV, it's in the public's interest to have a person's status known as early as possible, even if the test results are less reliable than desired. There is no harm telling a person that they cannot be premiscuous because they would spread the disease. The only harm would come IF the person received a false-positive AND the person decided that he didn't need to worry about partaking in activities with other with the disease because he believed he already had the disease. But if he abstained from such activities until it was verified he had the disease, no harm is done.