Comment on H5N1 lethality in humans

In a brief letter to Biosecurity and Bioterrorism, Alan Zelicoff notes a problem with serosurveys for influenza H5N1 infection:

…peak titers after H5N1 infection occur at about 4 to 6 weeks postinfection and may drop by as much as 32-fold over the course of a year, probably decreasing the sensitivity of serologic testing for past asymptomatic infections. Micro-neutralization testing may be more sensitive.

He cites a serological survey carried out on poultry workers in South Korea, in which 9 of 2,500 subjects were found to have antibodies to H5N1 virus, in the absence of illness. These seropositive individuals carried antibodies that neutralize H5N1 virus infectivity. Assays for antibodies that block infection may be more specific for infection than hemagglutination-based assays. His conclusion:

One can anticipate additional serological surveys that will better inform public health practitioners of the threat to humans from circulating H5N1 clades….morbidity from novel influenza strains does not equate with an impending pandemic, let alone one with high mortality. It would appear likely that a systematic, prospective cohort study is in order to adequately capture the frequency of asymptomatic infection.

Human infections with influenza H5N1 virus: How many?

The lethality of avian influenza H5N1 infections in humans has been a matter of extensive debate. The >50% case fatality rate established by WHO is high, but the lethality of the virus might be lower if there are many infections accompanied by mild or no disease. One way to answer this question is to determine how many individuals carry antibodies to the virus in populations that are at risk for infection. A number of such studies have been done, and some have concluded that the results imply a low but substantial level of infection (even less than one percent of millions of people is a lot of infections). The conclusion of a new meta-analysis of H5N1 serosurveys is that most of the studies are flawed, and that the frequency of H5 infections appears to be low.

Twenty-nine different H5N1 serological studies were included in this meta-analysis. None of these are particularly satisfactory according to the authors:

None of the 29 serostudies included what we would consider to be optimal, blinded unexposed controls in their published methodologies, i.e., including in the serology runs blinded samples from individuals with essentially no chance of H5N1 infection. Serological assays can easily produce misleading results, especially when paired sera are not available.

Some of the problems identified in the serological surveys include the possibility that many H5N1 positive sera are the result of false positives, that is, cross reaction with antigens from other influenza virus strains. In addition, many studies utilized H5N1 strains that are no longer circulating.

It is clear that most of the H5N1 serosurveys have not been done as well as they should have been. The authors conclude that “it is essential that future serological studies adhere to WHO criteria and include unexposed control groups in their laboratory assays to limit the likelihood of misinterpreting false positive results.”

Let’s not forget that a completely different way of assessing H5N1 infection – by looking for virus-specific T cells – has been reported. The results provide further evidence for subclinical H5N1 infection and are not subject to the caveats noted here for antibody surveys.

I come away from this meta-analysis with an uneasy sense that the authors are not being sufficiently objective, and that they firmly believe that there are no mild or asymptomatic H5N1 infections. One reason is the authors’ use of ‘only’ to describe their findings. For example: “Of studies that used WHO criteria, only [italics mine] 4 found any seropositive results to clades/genotypes of H5N1 that are currently circulating”. The use of ‘only’ in this context implies a judgement, rather than an objective statement of fact. Furthermore, despite the authors stated problems with all H5N1 serosurveys, they nonetheless conclude that there is little evidence for asymptomatic H5N1 infection. If the studies are flawed, how can this conclusion be drawn?

My concern about the authors’ objectivity is further heightened by the fact that they are members of the Center for Biosecurity at the University of Pittsburgh. These are individuals whose job it is to find dangerous viruses that could be used as weapons. On the front page of the website for the Center for Biosecurity is a summary of the meta-analyis article which concludes that “In the article, Assessment of Serosurveys for H5N1, Eric Toner and colleagues discuss their extensive review of past studies and conclude that there is little evidence to suggest that the 60% rate is too high.”

I would argue that if the H5N1 serosurveys are flawed, then do them properly; it is incorrect to simply assume that the H5N1 virus is as lethal as WHO suggests. The World Health Organization should call for and coordinate a study that satisfies criteria established by virologists and epidemiologists for a robust analysis of human H5N1 exposure.

Evidence for influenza H5N1 infections in humans

The fatality rate for human infections with avian influenza H5N1 is widely quoted at >50%, based on the number of deaths among the fewer than 600 cases confirmed by the World Health Organization. Wang, Parides, and Palese suggest that this number is an overestimate:

…the stringent criteria for confirmation of a human case of H5N1 by WHO does not account for a majority of infections, but rather, the select few hospitalized cases that are more likely to be severe and result  in poor clinical outcome.

To address this problem, the authors summarized the results of serological surveys in which human sera were examined for the presence of antibodies to influenza H5N1 virus. Because antibodies are part of our immune defenses, they are a good indicator of a previous infection.

The authors searched the scientific literature and identified 20 studies in which human sera were examined for the presence of H5N1 antibodies according to WHO guidelines (a 4-fold or greater increase in neutralizing antibody titer in paired acute and convalescent sera, with the convalescent serum having a titer of ≥1:80, or an antibody titer of ≥1:80 in a single serum collected at day 14 or later after onset of symptoms and a positive result using a different serological assay).

Studies that used the WHO criteria included 7,304 study participants. Rates of seropositivity were from 0 – 5.3%, with one study reporting 11.7% positivity. The meta-analysis yielded a seropositivity rate of 1.2% (95% confidence interval 0.6% – 2.1%). When only poultry workers were considered, the seropositivity rate was 1.4%.

Other studies were separately analyzed that did not utilize WHO guidelines; these included 6,774 participants and yielded a seropositivity rate of 1.9% (95% confidence interval 0.5 – 3.4%).

A total of 12,677 study participants from 20 studies were included in this meta-analysis, of which 1-2% had evidence for prior H5N1 infection. The authors conclude:

…avian H5N1 viruses can cause a rate of mild or subclinical infections in humans that is not currently accounted for and thus, the true fatality rate for H5N1 influenza viruses is likely to be less than the frequently reported rate of more than 50%.

It seems very clear that standardized, large scale studies are needed to determine the real number of human H5N1 infections. This information is critical for assessing the actual threat of H5N1 influenza for humans.