Hello, my name is H1N1.

April 29, 2009 10:11 AM

How is the swine infuenza virus identified?

I'm asking because I was surprised to learn today that my country doesn't have the equipment necessary to identify the virus. Even if I assume that there are no affordable sequencing facilities in Bulgaria, and that producing/importing the antibodies necessary for serological tests of the capsid is too difficult, shouldn't identification still be possible by growing the virus, PCR, restriction digest and running it through a gel? The virus has been fully sequenced, after all.

Maybe there isn't a company in the country that can synthesize oligos/primers for PCR in the first place (business idea, here I come)? Other than that, any standard lab should be able to complete the procedures above, unless I am wrong in my assumptions and it doesn't even work that way.

posted by halogen to science & nature (11 comments total)

How can human infections with swine influenza be diagnosed?

To diagnose swine influenza A infection, a respiratory specimen would generally need to be collected within the first 4 to 5 days of illness (when an infected person is most likely to be shedding virus). However, some persons, especially children, may shed virus for 10 days or longer. Identification as a swine flu influenza A virus requires sending the specimen to CDC for laboratory testing.
posted by Pollomacho at 10:48 AM on April 29

Case Definitions for Infection with Swine-origin Influenza A (H1N1) Virus (S-OIV)

A confirmed case of S-OIV infection is defined as a person with an acute febrile respiratory illness with laboratory confirmed S-OIV infection at CDC by one or more of the following tests:

1. real-time RT-PCR
2. viral culture
posted by benzenedream at 10:59 AM on April 29

Right, it's an RNA virus so it'd have to be RT-PCR. So, presumably, the hybridization probes just aren't readily available in Bulgaria?
posted by halogen at 11:04 AM on April 29

I think it's a matter of accuracy and ease. You'd have to isolate, possibly replicate the virus and then extract the RNA, reverse transcribe it, PCR up a region of interest, find an appropriate restriction site (there's no guarantee that even an esoteric restriction enzyme will cut into a useful site). If there are no usable sites then you've got to clone restriction sites into the sequence at known points or onto the ends of the sequence, and transfer it to a plasmid, grow it up again and then cut it along side a control.

I just ran a quick comparison of a Hemagglutinin gene from H5N1 and H3N2 genes that are ~1800 bp (available on PubMed), and there were very few long stretches of similarity. Which means that in the best case you'd likely be looking for small variations in small final products after digestion. I'm thinking that you're going to be doing more work than it would take to sequence it and then try and make a final determination by comparing restriction gels that might come down to a few base pairs worth of difference in size. The whole thing would need to be repeated a couple of times.Then you should probably go back and resequence all your intermediate steps to make sure you didn't mess up the DNA something awful.

So do all that work and still not be sure, or toss a vial of blood on a plane and let another lab do it in a day.

I don't think it's a lack of skill or probes (my friend works in a lab in the Midwest and routinely sends off to South Korea for hundreds of primers at a time, and gets them damn fast). Any synthesis company around the world could get probes around the world in a few days for enough cash.

Perhaps Bulgaria simply doesn't have the diagnostic labs because it's been cheaper to send stuff off for a while, or they aren't regularly using sequencing for diagnosis. It would certainly be cheaper and faster to keep sending samples out of the country now than to try and start up a lab.
posted by Science! at 11:12 AM on April 29

The other thing to remember is, this is not just a doing an experiment to see what results you get kind of thing. These are patients you're talking about. Once you have somethign that really works (not just kind of works) you really need do some validation work and show that it's really accurate, specific, precise, etc. That's not a trivial task that you're going to leap on just in case.

So bottom line, what Science! says. It's easier to send them off to someone with a proven method and transfer that method only if the sample load increases.
posted by Kid Charlemagne at 11:58 AM on April 29

I'm asking because I was surprised to learn today that my country doesn't have the equipment necessary to identify the virus.

I doubt this. I think I could do this with very little equipment. RT-PCR, as mentioned, would be one way to do this. Western blotting would be another way but antibodies won't be available at this stage I guess.

The only thing that would concern me is the lab security. You may need a lab with the highest security to work with dangerous or possibly dangerous organisms. Such a lab may not be available.
posted by yoyo_nyc at 12:53 PM on April 29

According to this article the CDC authorizes a Taqman probe-based assay for swine flu diagnosis. The article also mentions other (real time PCR) systems that are not yet approved. I suspect that a molecular beacon type system would work as well. All of these could probably be performed on any thermocycler capable of real time (fluorescence) detection.

I don't believe that biosafety would be an issue since the conditions aren't too stringent according to the CDC guidelines. Enhanced BSL2 facilities are common.

My guess is that the specific labeled probes aren't available in Bulgaria but all of the facilities and equipment are.

Restriction digests? Do people still do those?
posted by euphorb at 3:46 PM on April 29

No, no! Do a Northern Blot with P32 labelled probes for old school cred.

Biosafety shouldn't be a problem since you will usually be dropping the swab directly into an RNAse inactivating reagent, which should be sufficient to kill influenza.
posted by benzenedream at 6:21 PM on April 29

Kid Charlemagne has it. But more wordily:

You misquoted the source in your question; the actual statement was: "Bulgaria's health minister says his country has no facility to identify swine flu virus ". I'm sure the required equipment to run a test exists in Bulgaria; what doesn't exist is a facility equipped and staffed to do what would be experimental testing for an emerging infectious disease, without risk of misidentification.

The likes of the US CDC are still scrambling to make a standardized, approved test for swine flu. As of today they say this:

Recommended Tests
Real-time RT-PCR for influenza A, B, H1, H3 at a State Health Department Laboratory is recommended. Currently, swine-origin influenza A (H1N1) virus will test positive for influenza A and negative for H1 and H3 by real-time RT-PCR. If reactivity of real-time RT-PCR for influenza A is strong (e.g. Ct <3>
Other influenza tests
Rapid influenza antigen test: Also, these tests have unknown sensitivity and specificity to detect human infection with swine-origin influenza A (H1N1) virus in clinical specimens, and have suboptimal sensitivity to detect seasonal influenza viruses. Therefore, a negative rapid test could be a false negative and should not be assumed a final diagnostic test for swine-origin influenza infection.

Immunofluorescence (DFA or IFA): These tests can distinguish between influenza A and B viruses. A patient with a positive for influenza A by immunofluorescence may meet criteria for a suspected case. However, it is not possible to differentiate from seasonal influenza A viruses. Immunofluorescence depends upon the quality of a clinical specimen, operator skills, and has unknown sensitivity and specificity to detect human infection with swine-origin influenza A (H1N1) virus in clinical specimens. Therefore, a negative immunofluorescence could be a false negative and should not be assumed a final diagnostic test for swine-origin influenza infection.

Viral culture: Isolation of swine-origin influenza A (H1N1) virus is diagnostic of infection, but may not yield timely results for clinical management. A negative viral culture does not exclude infection with swine-origin influenza A (H1N1) virus.

The point is that none of the standard, off-the-shelf infuenza tests will definitively identify this strain; until tests are developed that are nearly 100% reliable tests need to be sent off to genuine experts in the field. The lab techs who routinely do flu testing in most places in the world are not equipped for this. I'm sure within a week or two there will be a set of RT-PCR tests that unambiguously identifies this strain with proven reliability and no false positives.

When there is a standardized, bulletproof test developed somewhere like the CDC, local testing will be possible worldwide. It will probably be a set of RT-PCR tests as suggested in the question.
I don't know what regulatory hurdles a newly-invented test might have to clear to be cleared for use in Bulgaria. The Taqman assay euphorb links to may well not be available in Bulgaria for red tape reasons, or because that particular machine isn't present in any hospital labs. They're very expensive and the article mentioned that Mexico just had to buy some to do the testing.

Also: Northern blotting? Restriction analysis? You're not helping.
posted by nowonmai at 9:05 PM on April 29

Yes, yes, my question assumed that however poor Bulgaria is, most labs will have basic equipment such as thermal cyclers. I'm sorry I didn't make that clear, but that's why I was wondering whether it is consumables such as oligos, antibodies, etc. they can't easily get.
posted by halogen at 9:15 PM on April 29

The WHO has now published a reliable test for swine flu; it's a simple RT-PCR test requiring only commercially available reagents and standard PCR and sequencing technology, not the super-expensive real-time RT-PCR machinery we talked about upthread. So now there is certainly no material obstacle to doing the tests in Bulgaria, although it might still be more convenient to use labs in other countries for non-technical reasons.
posted by nowonmai at 3:56 PM on May 4