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False Negatives in H5 Positive Montana Pintail Ducks
Recombinomics Commentary
October 7, 2006

The USDA National Veterinary Services Laboratories (NVSL) confirmed the presence of low pathogenic H5N3 avian influenza through virus isolation in two of the 16 samples collected from wild pintails in Cascade County, Montana. Initial screening results announced on Sept. 21 indicated that H5 and N1 subtypes might be present in the collected samples

Because these rapid screening tests are highly sensitive, it is not uncommon to have positive results for a specific subtype on the initial screen test and yet not be able to isolate a virus of that subtype. This was the case for the N1 subtype in this sample which tested as a weak positive in the initial screen test. During confirmatory testing, an N1 subtype was not isolated but instead an N3 was found.

As previously announced, genetic testing ruled out the possibility that the samples carried the highly pathogenic strain of H5N1 avian influenza that is circulating overseas.

The above comments on the failure to isolate H5N1 from the N1 positive duck samples highlight the limitations of the testing procedures.  The most likely explanation of the above data is a dual infection by H5N3 and H5N1.  The H5N3 was likely present at a higher concentration, and the H5N3 serotype was isolated.  Isolating a serotype not detected in the original sample is not uncommon

Dual infections in wild birds are common.

However, the above comments leave the status of the H5N1 unknown.  Genetic testing merely shows that the H5N1 was below the testing limits.  The results do not rule out the presence of HPAI H5N1.  Since the highly sensitive rapid screening gave a weak positive, the failure to detect the H5N1 sequence was not unexpected, and did not rule out the presence of low levels of H5N1.

In this case, the lack of an H5N1 isolate raises clear concerns.  However, the presence of low path H5N1 in other samples raises similar concerns because the low path may be present at higher levels and obscure the HPAI H5N1.  However, unlike the above case, there would not be clear evidence of a second serotype.

Detection of H5N1 HPAI in live wild birds is rare.  It has been detected in Russia, but most countries that detect HPAI find the virus in dead wild or domestic birds.  It can also be found in sick poultry, but that detection is usually after other birds begin to die.

H5 was detected in a dead goose from a farm on Prince Edward Island.  The H5 confirmatory PCR test was also said to be weak and the size of teh insert was withheld.  However, the deaths of four geese after showing symptoms that matched those of Qinghai H5N1 infections strongly suggests HPAI H5N1 is in North America.

The above data raises serious questions about false negatives.  H5N1 frequently recombines in dually infected hosts, and the presence of the second virus can be seen in the sequence of the first virus.  Recent sequences of H5N1 from wild and domestic birds in Northern China have extensive evidence of recombination.  Those sequences have been made public in initial and confirming deposits.

Sequences from the H5N1 isolates should be made public immediately, so appropriate sequence analysis can be conducted.

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