de Jong's "hit it hard and early" paper

Over the weekend Menno de Jong and his many collaborators published a detailed clinical study of 18 H5N1 patients diagnosed and treated in Vietnam in 2004 - 2005 (Nature Medicine, subscription only). Thirteen of the cases died and five survived. Like many papers that have received media attention, it has important and interesting findings but has also been over interpreted, or at least its findings have been generalized too much. In this case, it is the fault of the authors.

de Jong et al. did virological and immunological studies in 18 bird flu cases and 8 other cases with the more usual human influenza subtype infections (5 were H3N2 and 3 were H1N1 cases). The two groups differed both in severity of disease (none of the non-H5N1 flu cases died) and in the timing of their presentation to the hospital. The authors speculate the non-H5N1 cases presented later because they were from more outlying areas. This is plausible as a less severe flu case is more likely to be hospitalized closer to home than a very severe one. But the difference is more than just average time of presentation. Examination of Supplementary Figure 1 in the online supporting material shows that all of the non-H5N1 cases presented before all of the H5N1 cases (with the exception of the two earliest H5N1 cases who presented on day four of illness, that same day as the single latest presenting non-H5N1 case).

One interesting finding was a non-finding: genetic differences did not show any signature changes characteristic of increased virulence in either the human cases of avian influenza or in the fatal cases, in particular. Mutations associated with PB2 (specificaly E627K) did occur, but in only half of the viruses isolated, and equally in the fatal and non-fatal cases. Some other mutations in the polymerase complex associated with mammalian adaption were seen, reinforcing the probable importance of viral genetic replication elements in determining host response. The same general finding was reported recently in an analysis by Chen et al.

Previously there has been much discussion of mutations in the HA protein, which has sites on it that bind to cellular docking sites ("receptors") thought to be specific to birds versus humans. The last set of papers to receive media attention even stated that the reason the virus was not easily transmitted from birds to humans and between humans was that the cells with avian virus receptors in humans were so deep in the lung the virus had a hard time getting there and getting out again. We have previously pointed out that there is good evidence that receptors for bird-associated HA (α2,3SA) exist in the upper respiratory tract of humans and perhaps other tissues and the de Jong paper seems to bear this out, with high viral loads in the nose, throat and trachea, all parts of the upper tract.

In the non-H5N1 infections, viral loads in the nose and throat were about equal, but in the H5N1 cases viral loads in both nose and throat were higher than in non-H5N1 cases and higher in the throat than the nose of H5N1 cases. Remember, however, that the H5N1 cases were all later in the course of their illness than the non-H5N1 cases. The point here is that with these data it isn't possible to know if the higher viral loads are due to increased efficiency of replication or because a host factor like decreased immunity allowed the infection to go on longer, or (less likely), that if the non-H5N1 viral loads had been measured at the same duration of illness, they too would have been higher. The difference in clinical course argues against this last, possibility, in our view.

Viral genetic material was also found in rectal swabs and in the blood of H5N1 cases. There were no rectal swabs available for the non-H5N1 cases, nor was viral isolation attempted for blood in these instances. No viral genetic material was found in the blood of non-H5N1 cases. These data are one more indication that influenza virus may infect tissues outside the respiratory tract and that H5N1 may be more likely to do so. We know very little about the tissues that H5N1 can or does infect and the question of infection of the intestinal tract, particularly, remains open and of vital consequence.

This paper also presents more data on the levels of cytokines in H5N1 and non-H5N1 cases. Cytokines are local chemical signals the immune system uses to synchronize the delicate choreography of its many moving parts. Some signals call for inflammatory cells to come to an area of infection to fight microbial invaders while other signals tell the system to stop sending reinforcements. If the signalling system gets out of kilter, there is danger of a "cytokine storm," a dysregulated signalling system that can have fatal consequences. Scientists believe cytokine dysregulation is one of the possible consequences of H5N1 infection and may contribute to a rapidly fatal outcome in some (but not all) cases. The de Jong paper found significantly higher levels of cytokines in the blood (likely reflective of increased local levels in the lung), especially those cytokines that call for more inflammatory cells. The signal to come help was there in abundance but the signal to stop sending inflammatory cells didn't happen. At the same time the balance of infection fighting white blood cells in the blood was altered, with a marked decrease in white blood cells in the peripheral blood. (This same effect was seen recently in a drug-induced cytokine storm syndrome we posted on here,) Cytokines were increased in both H5N1 and non-H5N1 cases, but more so in H5N1 and still more in the fatal cases versus the non-fatal H5N1 cases. The question remains what causes the cytokine dysregulation, in particular if it is a specific effect of the virus, and if so, how it works. The authors of this study state that viral load is the trigger for increased cytokines and they present data that show good correlations between cytokines and viral load in the throat. However both cytokines and viral load could be caused by a third factor. If the virus is able to disable the cytokine system and with it the cell mediated immune reaction, then this prior effect would produce both higher cytokine levels and higher viral loads.

This affects the last paragraph of the paper, which forms the punchline of much of the media coverage:

Our observations point to a central role for high viral burden in the pathogenesis of human H5N1 disease and suggest that timely suppression of viral replication soul remain the mainstay for treatment of influenza H5N1. This is supported by our previous data demonstrating that successful control of viral replication by antivirals was associated with a good clinical outcome.

The previous data come from his New England Journal article in 2005 on oseltamivir resistance, cited here by de Jong as support for hitting the disease hard and early with antivirals. It does not demonstrate that oseltamivir made a difference, only that in oseltamivir treated cases those whose viral load decreased to below detection survived and those whose viral load didn't, succumbed. It is neither a showing that oseltamivir was responsible for the decrease in viral load nor that the sustained viral load was the cause rather than a sign of a failing patient. Neither paper demonstrates that vigorous treatment with antivirals affects cytokine dysregulation, whose cause remains unknown.

In the last analysis de Jong may be right about all of this, and with some luck and hard work we will be able to find out. However we believe this study, like the earlier ones about deep lung involvement being the explanation for lack of transmissibility, has been over interpreted, not just by the press but by the authors.


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Revere: I'm with you on this one. All of my research and thinking points to very early cytokine dysregulation by H5N1 (within a few hours of infection). The argument about higher viral load (higher replication efficiency) is also a function of virulence that is hard to examine separately. Higher replication efficiency will just make any cytokine dysfunction worse. Besides the famed PB2 E627K point, we should also be taking a hard look at NS1 mutations. NS1 in H5N1 has a number of interesting changes.

Is it okay to ask questions here? Most posts seem to be informed comments...I'll ask, but I understand if this isn't the right forum.

What do the Reveres think of using substances like curcumin or skullcap or reservatol in the absence of any other treatment? I've read articles like this one:
at fluwiki and wondered a) if these are valid possibilites for trying to deal with an inflammatory response, b) whether to choose one or use them all if the need should arise, c) whether to take them throughout a flu pandemic or only if sick and d) how much to take (a range is suggested for curcumin; I guess I'd tend to go for the maximum amount.)


Karen: I have no knowledge or expertise with these agents, I'm afraid. Maybe others can help.

There is a theory that Spirulina, which is a blue/green algae that can be found in the world's oceans; will slow down a cytokine storm. You can buy 500 mg tabs in health food stores.
And Lomatium is one of the only plants that can attack a virus, according to the theory. So Lomatium might kill H5N1.
It comes in a small bottle in liquid form you can buy in a health food stroe. It is about the only plant known that can kill a virus.
And if you get bird flu, or I get bird flu, and we die after taking all of these plants, please do not get mad at me.
It is only a theory.
Have a good day, and don't worry too much about bird flu, since the worse it can do is kill you. That is why I do not worrry about it.

On the intestinal infection issue in humans, it is worth noting that the natural transmission cycle of avian influenzas in their waterfowl reservior hosts is through the oral-fecal route with water as the medium of transfer (i.e., ducks contract virus while feeding in -- or drinking -- water contaminated with AI virus).

Most non-human mammalian cases outside of laboratory settings, and most early confirmed human cases, appear to have come through eating uncooked blood or tissues from infected birds (zoo tigers and leopards, feral cats, dogs, etc).

Natural route of infection for AI viruses -- evolutionarily speaking -- is through ingestion and intestines rather than respiration and respiratory tract (although this may be changing for the Asian H5N1 at issue here). This points out importance of identifying and tracking atypical fatal H5N1 cases involving gastroenteritis in absence of respiratory symptoms as reported by de Jong in an earlier paper.

Revere, good stuff. So with this in mind, what happened to those two girls that were sent home from De Jongs hospital in Vietnam supposedly "clear" of H5N1. Was it an in body mutation perhaps post infection and recovery, or was it just more likely hanging around in their respiratory tracts? They got Tamiflu as I recall too.

It sure killed them like getting their heads smacked on a flat rock. 24 hours and gone.


By M. Randolph Kruger (not verified) on 12 Sep 2006 #permalink

OK... Correct me if I am wrong. The patients who are suspected of having H5N1 are being treated with tamiflu, now, right? The mortality rate for those patients is still 50% (or greater). Granted, a lot of them are not receiving tamiflu until they present at the hospital, which may be day 2 or 3 (or even 4), But, many are still dying....

Don't get me wrong, I am all in favor of doing everything possible to treat a patient, especially since our arsenal is so small. But, shouldn't there be a higer rate of survival for patients treated at any time with tamiflu versus patients that are not treated with tamiflu at all? Or is tamiflu rendered worthless after the viral load is detectable or reaches a certain level?

By floridagirl (not verified) on 12 Sep 2006 #permalink

Zo Kun: Yes, intestinal route is "normal" for birds. One question has always been where the receptors for &alpha:2,3 are in humans. We don't know their distribution, or whether intestinal cells can be infected in humans. I would guess "yes" but the data arent' in yet.

Randy: No useful comments. Just don't know.

Floridagirl: Tamiflu doesn't work that well for treatment and almost not at all after the first 48 hours, so we shouldn't expect a survival difference in late treated cases. Too late to stop whatever disease process is going on.

Revere and William, thanks for your responses--and for the chuckle, William. I'll look into your suggestions.

The high levels of H5N1 in the upper respiratory tract of patients in Vietnam, coupled with the high levels of Indonesian H1N1 in the noses of ferrets in the CDC reassortment experiments, as well as high levels reported in the nose and throats of fataly infected members of the Karo cluster, all suggest that efficient H2H transmission of a very lethal H5N1 is just one sneeze away.


I am convinced that these strains of H5N1 with the residue 92 polymorph on the NS1 gene segment are rapid replicants due to the Interferon pathway interference. The in vivo interference, likely a stultification of NF-kappaB, allows the early viral particles a headstart on the immune system, a temporary invisibility.

I surmise that the Cytokinic Dysregulation is closely mediated by the late start of the immune response due to this early stage failure to detect and respond. By the time the H5N1 infection is fully detected, numerous polymerase activities are already completed. The CD is a fail-safe, even a limited self-destruct, because the virus evaded the early sentinals.

I've additionally conjectured early this year that this particular glutamic acid polymorphism on the NS1 gene segment may also mediate higher mortality rates in pregnant women and the young due to the involvement again with NF-kappaB, which is also required in a number of phases of cell differentiation.

I can't seem to get to the bottom of the actual mechanism as yet.

Perhaps the bulk of a particular cell's NF-kappaB population is consistently engaged for cell differentiation in the pregnant and the young leaving the H5N1-infected cell short of resources when NF-kappaB is needed to unbind and translocate to spur the Interferon pathways?

Perhaps we are all short of supply from the start of an important mediative factor?

Some discussion touching this topic:

Floridagirl and Revere

Don't forget with tamiflu, it was found in the Vietnam cases that some strains of h5n1 were resistant to Tamiflu. Which is why the US health department is recommending Relenza, for the following reasons. They state:

''Theoretically, there is less risk of resistance with Relenza compared with Tamiflu. Also, the most common Tamiflu resistant mutant is susceptible to Relenza.''

Thought some of you would find this article useful. It is Bloomberg news article on the study that was completed recently on Relenza / Tamiflu resistance.…

This following paragraph from the head of the microbiology department at the University of Hong Kong.

'' If an intravenous or oral preparation of zanamivir is finally available, it will replace oseltamivir whose only superiority is good systemic blood level,'' said Yuen.

NS1: Your conjectures are plausible, but conjectures in this area are a dime a dozen. It will have to be examined empirically.

cpg: The Tamiflu resistance in the Vietnam papers is not very solidly established. I pointed out (in a post on the old site) that the data in the paper are not correctly interpreted, statistically. The question about resistant strains remains whether they are also genetically fit. Resistant strains exist but they replicate less well and therefore we don't know their significance.

Karen, most of these treatments have been proposed in the medical literature, but none have been tested. Cinnamaldehyde, the active ingredient of Guizhi-Tang, was tested in cell culture, and found to be effective, though this is only a first step (Guo JY, Huo HR, Zhao BS, et al (2006). Cinnamaldehyde reduces Il-1 β-induced cycloxygenase activity in rat cerebral microvascular endothelial cells. Eur J Pharmacol 537:174-180)

NS1: I agree with you. Check out the following ref if you haven't seen it: Bornholdt ZA, Prasad BV. X-ray structure of influenza virus NS1 effector domain. Nat Struct Mol Biol 2006;13:559-60.

Revere: the interesting question is whether H274Y has popped up in other places; unless you tested the HA subunit, you would be blissfully unaware. I would say that the strain discussed in the Vietnam papers is probably quite fit enough to cause fatality, though I agree with you there are flaws with de Jong's study. No study is ever perfect!

Marissa: I'm not concerned with flaws in the study. It is an observational study which by its nature will have uncontrolle variables. As an epidemiologist, that's the world I live in. My concern was over interpretation of the results. In this case, the authors' interpretation has some pretty strong policy implications and I was urging caution about that.

My view (guess) about the polymorphism question is that the RNP complex is important in host range, while NS1 may be the important (or one of the important) virulence determinants. I have no opinion about transmissibility. HA and NA probably fit in here somewhere, but maybe not as centrally as we have assumed.

Thank you, Marissa.

Revere wrote:
"NS1: Your conjectures are plausible, but conjectures in this area are a dime a dozen. It will have to be examined empirically."

That's why I called it conjecture.

With all the available research, we should be chasing each individual immune-mediative, transcription factor until we know if it is implicated in late response to H5N1, ergo, allowing the cytokinic dysregulation, ARDS, et al.

Empirical Examination

Isn't that what you guys called scientists purport to do?