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Testing Procedures (Human) H5N1

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  • Testing Procedures (Human) H5N1

    Hi All,

    I am new to this forum and I apologize if I am asking some questions that others have covered. I am a scientist with training in medical microbiology, molecular biology and immunology, but do not have any expertise in influenza or bird flu. I am however genuinely alarmed by the possibility of a H5N1 pandemic, and the probably very real possibility that the virus will eventually mutate to a strain that can be easily transmitted from human to human.

    I have some specific questions that I would like to throw out there, not only for my own personal education, but perhaps as food for thought. Given the premise as a scientist that under uncontrolled conditions that what can go wrong will go wrong and probably all ready has, here are my questions.

    1) Who is watching? Is it just the WHO keeping records and responding to possible outbreaks? If say, there is a reported cluster of cases tomorrow in Bangladesh, Indonesia, Thailand, Vietnam, or god forbid North Korea, does anyone from WHO even bother to go anymore to collect data and specimens. What are the qualifications of the people who respond? Are we relying on each governments own ability to collect data, specimens, quarantine the area, etc.... Is there a standard process for this that governments strictly adhere to?

    2) Who does the testing to determine whether or not it is "Bird Flu." What kind of test is it? Is it an antibody based test that relies on say hemmaglutination? What company makes this test? Is there adequate quality control or testing to ensure adequate results? Or are we just relying on untrained veterinary personeel from third world countries to perform this test. Are specimens from each outbreak required by WHO to be sent out to a central collection center for verification/genotyping?

    3) I have seen an influenza gene section on the GenBank website. Who is actively sequencing the outbreak isolates? Is there central funding for this, or is this like the rest of science with having to fight and scramble for funding to perform the basic research necessary? Are they required to deposit their sequences in a public databank such as GenBank?

    4) Is anyone routinely given the "job" of constantly performing recombination and phylogenetic analyses on these sequences to determine how fast the virus in different areas may be mutating, recombining? Has it been shown using some of these DNA recombination programs (which admittedly I don't understand the math behind) that influenza does recombine, or does it simply mutate? If it does recombine with host sequences or other viruses, does it have segments that are more "recombigenic" than others?

    Sorry for so many questions, and if they have been adequately covered elsewhere.

    Curious and Concerned

  • #2
    Re: Who is Watching?

    Welcome Curious - Excellent questions. We will have them answered this morning for you.

    The general situation is that the various countries are in control of the access, testing, and results of all H5N1 issues. Sometimes they encourage and allow international help. Sometimes they do not.

    I have a meeting now. Please check back at 12h00 EST for specific answers.



    • #3
      Re: Who is Watching?

      As a scientists, I am sure you will be appalled by the answers. The only one paying close attention is H5N1.

      There are mandatory requirements for reporting H5N1 in domestic animals. However, there are many examples of countries generating false negatives or just not testing. Indonesia is in the news because of a Nature news report showing no sequences from birds have been generated in the past year. Similarly. there has only been one human sequence from Indonesia made public.

      WHO has labs than confirm samples. but they don't get negative or untested samples. NIAID will do complete sequencing for all 8 genes as long as the sequences are made public. I am working on getting samples sent from west Africa, but that is the only example that I know of that takes advantage of this free service.

      WHO largely just looks for reassortment. A paper will come out next week blowing reassortment in H5N1 out of the water.

      The WHO scientists really have no interest in recombination and regularly publish papers with obvious recombination that is never mentioned.

      I could go on and on, but I'm sure the monitoring falls well shy of your expectations.
      Last edited by HenryN; July 29, 2006, 11:46 AM.


      • #4
        Re: Who is Watching?

        As Dr. Niman says above, the process of quantifying the H5N1 stuation is flawed.

        1) WHO needs to be invited by each country to participate. This does not always happen. The situation is managed by each individual country.

        2) Data on the test process:

        Detection of antibody to avian influenza A (H5N1) virus in human serum by using a combination of serologic assays.

        Rowe T, Abernathy RA, Hu-Primmer J, Thompson WW, Lu X, Lim W, Fukuda K, Cox NJ, Katz JM.

        Influenza Branch, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.

        From May to December 1997, 18 cases of mild to severe respiratory illness caused by avian influenza A (H5N1) viruses were identified in Hong Kong. The emergence of an avian virus in the human population prompted an epidemiological investigation to determine the extent of human-to-human transmission of the virus and risk factors associated with infection. The hemagglutination inhibition (HI) assay, the standard method for serologic detection of influenza virus infection in humans, has been shown to be less sensitive for the detection of antibodies induced by avian influenza viruses. Therefore, we developed a more sensitive microneutralization assay to detect antibodies to avian influenza in humans. Direct comparison of an HI assay and the microneutralization assay demonstrated that the latter was substantially more sensitive in detecting human antibodies to H5N1 virus in infected individuals. An H5-specific indirect enzyme-linked immunosorbent assay (ELISA) was also established to test children's sera. The sensitivity and specificity of the microneutralization assay were compared with those of an H5-specific indirect ELISA. When combined with a confirmatory H5-specific Western blot test, the specificities of both assays were improved. Maximum sensitivity (80%) and specificity (96%) for the detection of anti-H5 antibody in adults aged 18 to 59 years were achieved by using the microneutralization assay combined with Western blotting. Maximum sensitivity (100%) and specificity (100%) in detecting anti-H5 antibody in sera obtained from children less than 15 years of age were achieved by using ELISA combined with Western blotting. This new test algorithm is being used for the seroepidemiologic investigations of the avian H5N1 influenza outbreak.

        PMID: 10074505 [PubMed - indexed for MEDLINE]


        Details on testing for H5N1 in the US.
        The test was approved in Feb 2006. It is called a real time virus PCR test. Oropharyngeal swab specimens and lower respiratory tract specimens (e.g., bronchoalveolar lavage or tracheal aspirates) are obtained from a patient on a Dacron swab tip with aluminum shaft. The specimen is placed in nucleic acid extraction lysis buffer (for virus inactivation and RNA stabilization). The specimen is then stored and shipped at 4°C to a CDC Laboratory Response Network (LRN) lab. There are 140 LRN labs in the US with at least one lab in every one of the 50 states. The test is performed in a BSL level 2 facility. Results are available within 4 hours of receipt of the specimen.

        Indications for testing were disseminated in a CDC Health Alert Bulletin on June 7, 2006.

        One real life exercise with this procedure has been recently reported from Texas.

        WHO has provided guidelines for the rapid detection of H5N1.
        Direct testing is done on specimens from pulmonary secretions and is preferred for the management of an acute outbreak.

        Other types of tests are possible, see -
        Recommended laboratory Tests (WHO) July 2005
        Serological (blood) testing is possible. The microneutraliization assay for detection of H5N1 antibody is recommended. It requires live virus (as the reagent) and must be done in a BSL level 3 facility.

        Serological testing is done to confirm an infection. It requires paired serum samples. The first sample is taken at the first signs of infection and the second approximately two weeks after onset of symptoms. Serological testing is not helpful in the management of an acute outbreak.
        - CR -<!-- / message -->

        3) Sequencing is done at several places. The results are sometimes made public.

        4) Once the sequences are released they are analyzed by various experts. Dr Niman is one.
        Last edited by sharon sanders; July 29, 2006, 11:37 AM. Reason: added data from CR


        • #5
          Re: Who is Watching?

          Curious - more info:

          Development of vaccines against influenza H5

          A veterinarian's experience of the spring 2004 avian influenza outbreak in Laos

          Avian influenza and sialic acid receptors: more than meets the eye?

          Influenza Report 2006


          • #6
            Re: Who is Watching?

            Thanks everyone who responded to my question. I have read the abstracts , and looked at many of the links. It really is disheartening that there isn't a more coordinated international effort that responds to each and every outbreak. It seems that some of the countries such as Thailand are genuinely trying to quarantine cases and act responsibly by destroying all birds in the area that could carry the virus, and other countries are either responding poorly or trying to cover up or both. It just makes common sense that if there was an international team that could be dispatched each and every time to ensure that proper precautions are taken, antivirals are given to close contacts, vaccine (soon to be available from Glaxo) is administered to those in the surrounding community, quarantines are enforced, samples are collected and sent off to a central repository for analysis and sequencing that our odds of heading off a potential pandemic would be much greater. It may be the only way to do it. Another suggestion would be the setting up of a fund to pay individual farmers for their poultry should they become infected. That way, poor villagers would have an economic incentive to report the death of birds.

            Also, thanks for the heads up on the testing and sequencing. About time that a quantitative PCR test became available. The news that it is in plasma though is somewhat of a double edged sword. Makes more easily accessible a source of genetic material early in the course of the disease for testing and sequencing, but also increases the risk to medical and laboratory personnel not suspecting this as a source of possible contagion.

            Curious and Saddened


            • #7
              Re: Who is Watching?

              If you have time to browse these threads you will quickly come to realise nobody is very happy with the sampling, sample release, sequence release, clinical data release or epidemiological data release.

              In theory the WHO is in overall charge and the terms of its relationship with the nations states, on whose territory events occur, are codified in the International Health Regulations (2005). On paper this should lead to immediate notification of the WHO of any event that may have implications beyond that state?s boundaries. The theoretical sequence of events after that would be local preliminary testing with conformation by one of a handful of WHO reference labs, only after conformation by one of these labs will the event ?count? in the WHO?s H5N1 statistics. Animal outbreaks are covered by other similar legislation with reporting to the FAO.

              The reality is rather different. The WHO?s position vis-?-vis the states is weak in that they can only gain access to anything by invitation and when states are less than transparent the WHO will tends not to push the point for fear of getting even less access and information.

              The reasons for a lack of transparency are all the usual ones ?

              Economic ? damage to domestic poultry industry through reduced domestic consumption and export bans, costs of compensation in the event of culling.

              Pride ? not wishing to admit they do not have the expertise/labs to manage the problem in house or not whishing to expose failures in surveillance.

              Secretiveness ? just not wanting anyone else wondering around in their territory asking questions.

              Once sequencing has been performed they are placed in a secure database and are theoretically available for release if the state wishes. In practice it is less clear why they are not released, some seem to miraculously appear in the public domain after peer review publications from the sequencers and we have the absurd situation where Indonesia has said they would consider release if they were asked by the WHO while the WHO will not ask despite saying they would prefer release if it were up to them.

              In a letter to the Parliamentary minister in charge of the UK?s WHO reference lab sent in mid March I have just received a reply from DEFRA stating

              ?The VLA does ensure that preliminary key sequence information is placed, within hours, on the University of California Los Almos influenza database (http:/ To which any influenza researchers, in both the public and commercial fields, may apply for access.?

              Valuable information is sometimes gleaned from leaks or phylogenic trees based on the sequestered data. It seems unclear as to exactly how much scientists with access to the data can getaway with in this kind of partial release.

              There are major problems with false negatives possibly due to initial infection occurring in the LRT not URT and therefore failing to appear in swab tests. Retesting post-mortem or through blood tests in late stage disease gives more reliable results but is not always performed, particularly if the local authorities are ?happy? with the initial diagnosis. A high level of scepticism has been generated by some of the initial diagnosis and denials that have later been reversed once an outbreak has become too widespread to hide.

              What type of sample is taken is up to the local authority and if found positive they should be forwarded to the WHO reference lab (this does not always occur) how they culture them (Eggs or MDCK) seems to vary between labs.

              I hope this is of some help and I hope you will continue to post here wherever you feel your expertise may be able to shed some light. We need all the help we can get!


              • #8
                Re: Who is Watching?

                Originally posted by curious
                It really is disheartening that there isn't a more coordinated international effort that responds to each and every outbreak.
                It really is, Curious. Over the last year, I've been appalled by how political this issue has become. We are dealing with a biological entity out to get us, and it is brushed under the carpet, denied, minimized, and it goes on and on, with quarter-measures and half measures, in country after country, with pandemic preparations that look more like picnic parties than anything else, with drug and vaccine development that looks more like a first attempt at preparing sushi. The scientific data is never obtained, and then never released, and then never analyzed, except by a handful, who think it too frightening to share with anyone else. And the world's public has come to the conclusion that it's better not to tell anyone if their farms are infected, because then their neighbors will attack them for it or their livelihoods will be destroyed. If they themselves get infected, they don't go to the hospital because of the stigma involved. H5N1 doesn't care about all of this though--it just wants warm bodies, and it's growing tired of birds.


                • #9
                  Re: Who is Watching?

                  And the very few of us that are aware of all of this.....are almost intimidated to tell our friends/associates, for fear that they will think we are CRAZY. It's the most screwed up situation, I have ever, ever, witnessed.


                  • #10
                    Re: Who is Watching?

                    Print these and give to your associates.

                    Pandemic Planning Update 2 - Report from Secretary Leavitt dated June 29, 2006


                    "While pandemics have happened several times in the past, never before have we had all of the tools of today. Never before have we possessed the wealth of knowledge on the problem and the ability to prepare. The challenge is immense, but so is our will to protect and preserve."

                    "Last March, I wrote that we are in a race, a race against a fast-moving, highly pathogenic avian H5N1 flu virus; a race to prepare in every possible way against a potential human flu pandemic. Three months later, the pace has not slackened.
                    The deadly avian H5N1 virus has now been confirmed in 53 countries, an increase of 16 nations since March. Three additional nations have reported human cases. The number of reported human cases now stands at 228, an increase of thirty percent, and more than half of the people infected have died."....

                    Pandemic Preparedness

                    Five Priorities

                    • Monitoring disease spread to support rapid response
                    • Developing vaccines and vaccine production capacity
                    • Stockpiling antivirals and other countermeasures
                    • Coordinating federal, state and local preparation
                    • Enhancing outreach and communications planning

                    Ask your associates why do they think the U.S. government is doing this?

                    and Osterholm on July 14, 2006:

                    ...According to Osterholm, it’s time the world prepares for the worldwide spread of the avian flu. He said, “This is the one topic that keeps me up at night.” ....

                    So far the H5N1 bird flu hasn’t spread widely among humans. That is expected to change. According to Osterholm, there are several ways the H5N1 can mutate into a virus that does spread and is foreign enough that people's immune defenses can’t fight it. When that happens, he says the virus will spread across the world (causing a so-called pandemic) and even strong, healthy people will become sick and die.



                    • #11
                      Re: Who is Watching?

                      Just thought you'd like to see this, WHO does issue regional alerts. Thanks to Rick at P4P

                      BUSINESS CONTINUITY
                      PLANNING GUIDE

                      October 2005

                      " WHO has not yet issued a world pandemic alert, although it has issued a regional alert for South East Asia and Sealand."



                      • #12
                        Re: Who is Watching?

                        Thanks Siam, interesting document. In contrast to Indonesia, Singapore appears to have a relatively well thought out plan.


                        • #13
                          Re: Who is Watching?

                          Although I think this comment from DA Henderson is relevant re 'border health control measures'

                          Once-common border controls and inspections have proved to be of no value in the prevention of disease, as was clearly shown during the 2003 SARS epidemic. More than 35 million passengers were screened with the intent of quarantining those with fever. No cases were found. If travelers had been infected, they were most likely in the silent, incubation phase of illness and could not have been identified, whatever screening measures had been employed. We are now experiencing population movement of a magnitude and speed such as has never before been witnessed.


                          • #14
                            Re: Who is Watching?

                            Another good plan from Vancouver

                            Good section on vaccines and antivirals (a lot of assumptions but at least they're thinking about it)

                            and I imagine there wil be a lot of pressure to at least tie sequence data to antiviral therapy


                            • #15
                              Re: Who is Watching?

                              Originally posted by kent nickell
                              We are now experiencing population movement of a magnitude and speed such as has never before been witnessed.
                              All the rules change. Think of it like this. There are two businessmen, who have each been given the right to set up exciting franchises in two different countries. The first businessman is given 2006 China or India, and the second is a sparsely populated island far from any other land. In China, you have an extensive road network, rivers, canals, railroads, factories, cars, motorcycles, planes, natural resources, people to trade with, banks to receive loans from, people who understand the concept of franchising, potential partners, telephones, internet, email to communicate, television, radio to advertise. On the island, you have a much smaller number of people, with little to no exposure to franchises, little transportation or trade, no financing, perhaps not even a normal currency to trade with. Both of you work really hard over the next five years. Which of you will end up doing the best? That is how different our present world is from 1918 or before. This virus can enter any country it wants, in thousands of ways, and if or when it becomes a pandemic virus, no Earthly force could slow it down. Just look at the 25 biggest cities of the world--they are the heart of our world civilization. There are 413 million people in them. If you add the next 75 cities, that number about doubles. 50-100 million people died in 1918. The world is so small and densely populated now that you could find that number of people in 3-5 cities today!!!