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Pandemic Flu False Negatives in Vietnam and Korea
May 8, 2005
>>Genetic sequences from more recent versions of H5N1 are needed to produce more up-to-date test components, called primers.
Primers are tiny strands of synthetic nucleic acid used in PCR or polymerase chain reaction testing. If they are a perfect match for the influenza strain, primers used in flu tests should bind to the RNA of the virus.
Kobasa and lab technician Laura Hart spent several weeks at the National Institute for Hygiene and Epidemiology in Hanoi, sharing diagnostic expertise and helping Vietnamese scientists assess their testing proficiency.
That is when the problem with the primers came to light.
"There've been enough changes in the viruses between last year and this year that we found some of our PCR primers did not work that well," says Kobasa, a researcher in the division of respiratory viruses.
The comments above offer some explanation for the false negatives in southern Vietnam. Earlier this year, tests from the National Institute for Infectious Diseases (NIID) in Tokyo found that several samples that tested negative at the Pasteur Institute in Ho Chi Minh City tested positive in Tokyo. The number of false negatives was initially reported as 7 of 30. The number was raised to 11 of 30 in follow-up NY Times report.
These numbers indicated that approximately 80% of the H5N1 positive cases in southern Vietnam were being missed. Recent reports indicated a missing amino acid has been detected in isolates in northern Vietnam, suggesting those isolates were recombinants between isolates from Vietnam and isolates from China. It seems likely that there have been primer problems in northern Vietnam also, which may explain why northern Vietnam sent 1000 samples to CDC for testing.
The lowered sensitivity for tests in the south may be do in part to an evolving H5N1, which continues to recombine with the many versions of the virus in Vietnam and new versions brought into the area by migratory birds. This primer related sensitivity problem may also explain why Thailand failed to identify any human cases this season when H5N1 was detected throughout the country in a wide variety of domestic and wild birds. Human Influenza A cases were testing negative for H5N1. The testing was almost certainly also based on the 2004 sequences, which had changed in 2005.
For Vietnam and Thailand, new probes relied in part on new sequences. However, primer issues may have also played a role in the WHO's failure to find WSN/33 sequences in swine in Korea. However, for the Korean sequences, the 2004 sequences were known in 2004 and had been deposited at GenBank in 2004. However animal quarantine in Korea as well as consultants to the WHO failed to confirm the data. In 2005 they did find H1N2 sequences in Korean swine, but these sequences had been reported previously in swine from the United States and Korea. They were quite different from the human WSN/33 sequences, or the original WSN/33 sequences or the closely related sequences found in swine.
The WHO had written up a release that described these 27 isolates from 2 farms in Korea. Although a draft was distributed to media, it remains unclear if the release ever went out to a larger audience. A small number of these isolates were sequenced and no WSN/33 was found in the limited number of sequences generated. However, it remains unclear if the primers used to generate the H1N2 sequences would have detected WSN/33 sequences.
Thus, the lack of primer specificity may have generated false negatives in Vietnam and Korea. The inability of the WHO to monitor H5N1 and WSN/33 seriously impacts any intervention strategy. The results from the 1000 samples from northern Vietnam have not been released. It remains unclear as to how much sample collection has been done from patients with flu-like symptoms. In northern Vietnam the case fatality rate has fallen to 20%. Many of the recovered patients are H5N1 positive, but have a relatively short hospital stay, which suggests a large number of milder cases may not be seen by physicians or hospitals.
Similarly, it is not clear where the WSN/33 sequences are in Korea, or how the 1933 human lab virus made its way into swine in Korea. Since Korea imports pigs from the United States and H1N2 was detected in the United States prior to being detected in Korea, the H1N2 and WSN/33 infections could date back to 2001 or earlier.
Strategies based on control of H5N1 bird flu or WSN/33 pandemic flu dependent on early detection have limited chance of success if the presence of the virus is largely unknown.