A little over a
year ago, the first known human patient got sick with avian influenza virus(H7N9).
The number of H7N9 cases rose and fell in the spring of 2013, and a total of
134 people were known to have contracted H7N9 before June, 2013. Since then,
sporadic cases appeared in the summer and fall, and by the end of December,
2013, new cases started to pick up again. We have now seen a second wave rise
and fall, although several new cases still being reported each week. As of the
time of this writing, just over 400 people are known to have been infected with
H7N9. The case fatality rate (CFR) – roughly speaking, the percentage of people
infected with H7N9 who die from it – for these known cases is almost 40%.
One question
that is on a lot of people's minds is, how many other H7N9 cases are out there
that we don't know about? How many mild cases are there that never get tested? How
many asymptomatic cases are there that are missed? If there were a lot of
undetected cases out there, that would mean that H7N9 is a lot less fatal than
the known cases would make us think. On the other hand, if we were somehow
miraculously seeing every single actual case, then the CFR would be as bad as
all of these cases make it out to be. (And imagine what the CFR would be like
without hospitals, ventilators and oseltamivir!)
How do we find
out if there are cases that we're missing? One way is to do what is called a
seroprevalence study. This means collecting blood samples from as wide a swath
of a population as possible and testing to see how many of these samples have
antibodies to H7N9. Antibodies are molecules that are made by cells of the
immune system and that stick to specific pathogens to help our bodies to rid
themselves of these pathogens. If someone gets sick with H7N9 influenza, his or
her body would most likely continue to produce a significant amount of antibodies
specifically against that strain for at least a good number of months after
infection and possibly much longer. In general, people who are infected with
influenza but who do not develop symptoms will also produce such antibodies,
but their bodies will make fewer of them, and, on average, they won't make as many
of them for as long. We don't know exactly what the pattern of antibody
production is for people who are infected with H7N9 but don't develop symptoms,
though, because researchers haven't identified enough of these individuals to
study.
It is very
important that we get these seroprevalence studies right. If they're done wrong
and we miss a lot of cases, then we will simultaneously underestimate how
common H7N9 cases are and overestimate how deadly the strain is. On the other
hand, if seroprevalence studies are done wrong and we think a lot of people
were infected with H7N9 when they weren't, then we will overestimate how common
H7N9 cases are but underestimate how deadly the strain is. Facts can help us to
respond to H7N9, and if we get the facts wrong, then we can't respond properly.
For example, if we come to think mild H7N9 cases are far more numerous than the
severe ones that actually get diagnosed, then we might not worry as much about H7N9
as we should.
What I'd like to
talk about here are some of the important ways that seroprevalence studies can go
wrong. To answer my title question, no, we cannot always believe the
conclusions of every seroprevalence study we see. Scientists make mistakes,
just like everyone else, and sometimes things just go wrong, too. I'd like for
you to understand just how some of these mistakes can arise, so that you can
better judge for yourself whether a study is likely to be reliable or not, or
so that you can at least know that there are things out there that can go
wrong.
How are seroprevalence studies done?
There are 2
types of laboratory assays (tests) that are usually used in seroprevalence
studies (although there are others): hemagglutination inhibition (HI) assays1
and microneutralization (MN) assays.2 (For more information about
the HI assay in general, see a nice description by Dr. Racaniello.3)
MN assays are considered better (more sensitive and specific) than HI assays,
but they are harder to do. MN assays require a significant amount of extra work
at the end that HI assays don't. But, more importantly for H7N9 studies, HI
assays can be done with either "killed," modified or "live"
virus, whereas MN assays require "live" H7N9 virus. In other words,
HI assays can be done in almost any lab, but MN assays require a BSL-3 lab. A
neutralization assay4 has been developed that uses a
"pseudovirus" instead of live H7N9 and is therefore far less hazardous
to work with, but formal WHO diagnostic criteria still require standard HI
and/or MN assays.
First, blood samples are collected. Each blood sample is drawn into a tube, and after 15-30 minutes, the tube is centrifuged to separate clotted red blood cells from the rest of the blood. The red blood cells are discarded; what's left is called serum, and that's what's studied. The serum samples should then be put in a refrigerator if they'll be studied within a few days; if they'll be studied later, they should be frozen. Once a researcher is ready to study the serum samples, the serum samples are thawed. Virus is also used for the assay, so one or more tubes of virus are thawed, too. Different types of mammalian or bird cells are prepared: typically horse, turkey or chicken red blood cells for HI assays, or a special type of dog kidney cells ("MDCK" cells) for MN assays. Various solutions are prepared. Serum samples, virus preparations and cells are diluted as needed, and everything is transferred into little wells in a plastic "plate" in just the right way. In the HI assay, the plate then sits at room temperature for 1 hour, after which it is "read" by eye. In the MN assay, the plate then sits at 37°C (body temperature) for 19-21 hours, after which it is read by a machine (an "ELISA reader"). The assay is done. The results of the assay are then written down and analyzed, and voilà , a paper appears in the scientific literature.
What could possibly go wrong with these blood tests?
Let's start with
some things that can go wrong with the lab work:
- If blood samples are left
sitting around for a long time without being centrifuged, the red blood cells
will start to break apart, and enzymes released from the red blood cells will
start to destroy antibodies (and everything else) in the blood samples. This
happens even faster if blood samples are not refrigerated.
- If serum samples are left in
the fridge too long, things can start to deteriorate, just like food in your
fridge would. The antibodies that you would like to measure start to be broken
down. (Sometimes, for many different kinds of studies, people study serum
samples left over after patients' blood tests at hospitals. Those samples
sometimes sit around in a fridge for quite a while. Some of them can even be green from stuff growing in them while
they're sitting around. Yuck.)
- If plasma (what's left in blood
after unclotted red blood cells are removed) is used instead of serum
(what's left in blood after clotted red blood cells are removed),
then the assay can read artificially high. Serum should always be used, not
plasma.
- Every time serum is frozen and
thawed, some of the antibodies are effectively destroyed. This should not be
done over and over. Serum samples should be put into the right size tubes that
the researcher will want to use, so that the samples are put through only 1-2
"freeze-thaw cycles" before they are tested. And all serum samples
should go through the same number of freeze-thaw cycles.
- The same thing is true for
virus samples used in MN assays. A single freeze-thaw cycle can reduce virus
infectivity by a factor of 10. Virus samples also need to be kept on ice when they're being worked with.
- The plate can be read wrong. It's
hard to imagine reading an HI assay plate wrong, but a special procedure
(ELISA) and special equipment (ELISA plate reader) are used in the MN assay,
and ELISA assays can go wrong.
But, hopefully
all of that was done right. Not all researchers, students and technicians are
created equal, but hopefully the lab "PI" (Principal Investigator;
the person running the lab) is competent and ensures that everyone is doing
things correctly.
What could go wrong with the data analysis?
What else could
go wrong? The data analysis might not be done correctly. And it's here where perfectly
good data can be ruined and where you have to look at seroprevalence studies
most closely.
Suppose you've
measured your antibody amounts ("titers") in your serum samples. How
do you decide which titers mean the sample came from someone who was infected
with H7N9, and which titers mean they didn't? Do you just pick a number out of
thin air? If you don't have data to tell you which titers mean
what, then all you are doing is measuring antibody levels in a population, and
you can make no interpretation about what those levels mean. You can't say that
they mean any people have or have not been infected with H7N9 at all.
Instead, you
need actual measurements using serum samples from people who are known to have
been infected with H7N9 to tell you what your titers mean. Someone has to study
a number of patients to see what their actual H7N9 antibody titers are, and
then a mathematical analysis of that data is done to come up with a threshold
titer value, above which serum samples can be said to have come from people
infected with H7N9 with some large degree of certainty, and below which they
are thought to have come from people who were not infected. We've seen almost
no asymptomatic cases (cases with no symptoms), so we really can't say much
about them. So we have to go with data from H7N9 patients who have had symptoms.
Here's a great graph showing antibody titers, as measured using the HI assay, in
serum samples from H7N9 patients:5
As you can see
in the graph above (Figure 1), by around 3 weeks after infection onset, all samples from patients
whose HI titer was measured had titers ≥40.
The graph below (Figure 2),
from a different study,4 shows that the HI titer for all H7N9 samples
studied by this set of authors was also ≥40. In addition, this graph shows titers from "control"
samples (i.e., samples from people who did not have H7N9 infections); all
control samples had titers that were <40.
Finally, below (Figure 3) is
another nice graph, from a third study,6 showing anti-H7N9 antibody
levels ("IgG"), "HI" assay results and MN assay ("NAb")
results for several H7N9 patients, again showing that all samples from the H7N9
patients studied had HI titers ≥40. This graph
also shows that all H7N9 patient serum samples had an MN titer of ≥20, if samples were taken after enough time had elapsed since their
infections had started.
In other words,
if an individual's anti-H7N9 antibody titer is ≥40 by the HI assay or ≥20 by the MN
assay, these data suggest that we could pretty safely say that he or she has
had a symptomatic H7N9 infection within the past few months, and if the
HI or MN titers are below those cutoffs, then the individual probably hasn't
had a symptomatic H7N9 infection. We don't know to what extent asymptomatic
H7N9 infections will be captured by these cutoffs, but it is likely that some
asymptomatic cases would be missed using these cutoffs. It is also possible
that some mild infections could be missed using these cutoffs. However, it
would be a great step forward just to get estimates of what percentages of any
regional population or occupational group of people have had any kind of H7N9
infection. A comparison of antibody titers for asymptomatically infected and
symptomatically infected H5N1 cases may be instructive when thinking about
H7N9.7
WHO guidelines are
even stricter than the cutoffs discussed in the paragraph above. WHO guidelines
say that, using the HI assay, only single samples with titers of ≥160 can be considered "seropositive": "Paired sera
(acute and convalescent sera) with a 4-fold rise in HI titer or single sera
collected in convalescent phase with HI titer of ≥160 could be considered as
H7N9 HI antibody positive. Sera with HI titer of 20-80 should be confirmed by
MN or WB assay."1 For the MN assay, however, the WHO does not
give specific cutoffs: "With single-serum samples, care must be taken in
interpreting low titers such as 20 and 40. Generally, knowledge of the antibody
titers in an age-matched control population is needed to determine the minimum
titer that is indicative of a specific antibody response to the virus used in
the assay."2
Now, it should
be noted that WHO assay instructions recommend the use of horse red blood cells
for the HI assay, and not everyone uses horse red blood cells. Some people use
chicken, turkey, guinea pig or other kinds of red blood cells. That starts
making comparisons between different groups' assays difficult. Horse red blood
cells are better to use than turkey red blood cells for H7N9 because they have
more a2,3-linked ("bird") sialic acids (influenza receptors); HI
results are more sensitive with horse red blood cells. In other words, it may
take less antibody in the assays to get the same result using horse red blood
cells than it would using turkey red blood cells. This would translate into a
higher number, when discussing H7N9 patient titers, for HI assays using horse
red blood cells, compared to assays using turkey red blood cells. I have not
seen direct comparisons of titers obtained using different types of red blood
cells in HI assays specifically for H7N9, but the situation is probably similar
to that for H5N1.8
Figures 1 and 3
above were made with HI data obtained using horse red blood cells. Figure 2
used guinea pig red blood cells. Are they completely comparable? No. Are they
pretty comparable? Yes.
Are you getting
a feeling for how complicated it is to interpret a seroprevalence paper? And
for how difficult it is to compare results across studies?
Why does all of this matter?
It matters because
some seroprevalence studies don't use appropriate cutoffs. And because it can
be hard to determine even what an appropriate cutoff is when red blood cells
from different species are used in an HI assay. This is where the reader has to be really careful. Cutoffs for
seropositivity have been a big issue9 with H5N1 seroprevalence
studies; some researchers have used cutoffs that were too low, and hence they have
almost certainly overestimated how common H5N1-specific antibodies were in the
populations studied.
So far, only one
H7N9 serology paper published to date has reported probable seropositive samples,
and this paper simply reported HI titers without using any specific threshold
for seropositivity. Only one used study horse red blood cells in HI assays. The
one paper that used an MN assay did use appropriate cutoffs. It should be noted that the new WHO HI guidelines were only
published in December, 2013, after a couple of these papers were already published.
Here are the
studies that have been published so far (I hope I haven't left any out):
- Bai et al.10 looked
at serum samples collected before November, 2012 from poultry workers in
eastern China and found no H7N9-positive samples. The study used HI and MN
assays. Turkey red blood cells were used in the HI assay. Appropriate cutoffs were
used for the MN assay.
- Hsieh et al.11 studied
14 close contacts of the first H7N9 case in Taiwan. The authors took blood
samples within 18-28 days after the contacts' earliest exposures. The authors
used an HI assay but not an MN assay. They used turkey red blood cells for the HI
assay. They found all contacts to have an HI titer £10, and
declared all to be seronegative. The HI titer for the H7N9 patient in their
study was 1:80. These conclusions seem very sound.
- Yang et al.12 looked
at serum samples from 1129 people from regions of China in which H7N9 cases had
been seen, and from 396 poultry workers from 10 districts in which H7N9 cases
had been seen. None of the samples from the general population was found to be
seropositive, whereas >6% of the poultry workers were found to be seropositive. The authors
also examined serum samples from several H7N9 patients. The study used an HI
assay but not an MN assay. The authors used a cutoff of ≥80, along with turkey red blood cells, for the HI assay. Because the
authors examined serum samples from H7N9 patients using their methods and got
results that are reasonably similar to other results, their cutoffs are most
likely reasonable, and their conclusions are probably quite sound. The authors
report:
- "Of the 1129 serum samples collected from individuals (age range, 1–88 years) in the general population, 9 (0.8%) had an HI titer of≥40 to influenza A(H7N9), but no serum samples with an HI titer of≥80 were found (Table 1). In contrast, among poultry workers, 13.9% (55/396) and 6.3% (25/396) had influenza A(H7N9) antibody titers of ≥40 and ≥80 (20 had an HI titer of 80, and 5 had an HI titer of 160), respectively."
It is hard to imagine that an HI titer of 160 can be a spurious finding ("non-specific," to the initiated). Thus, these data strongly suggest that at least some H7N9 cases have been going undetected among poultry workers. Suppose we consider only the poultry workers with HI titer ≥80, or 6.3% of the poultry workers. If we then consider how many poultry workers there are, total, in districts from which H7N9 cases have emerged, then this study suggests that it's possible that quite a large number of poultry workers have been exposed to H7N9. Still, this study examined only a very small number of people, and we should be cautious about reading too much into these results.
- Qiu et al.13 looked
at 3 H7N9 patients and 3 close household contacts of the patients who were
exposed before infection control practices were put in place. The authors
looked for viral RNA using a sensitive test (PCR) and examined serum samples
drawn 15-26 days post-exposure using both an HI assay and a pseudovirus-based
neutralization assay. They found no contacts to be seropositive. The H7N9
patients had HI titers that reached 160-640 during this time, and the patient
contacts all had titers <10. The authors used horse red blood cells for the HI assay. These
findings also seem sound.
To summarize,
the conclusions from all of these papers do seem sound. But, it would be wise
to keep all of these issues in mind as subsequent studies appear over time.
An additional study14
looked at antibody titers in 1723 serum samples collected in Vietnam using a
very different kind of assay (a protein microarray). Because seropositivity
cutoff levels had not been determined with authors' assay methods using actual
H7N9 patient samples, these authors were appropriately very careful not to
attempt to draw any conclusions about H7N9 seroprevalence from their data:
"Because titers calculated from our assay are not directly
comparable to HI or microneutralization tests, no cutoff is chosen to represent
positivity or clinical protection. It is not possible to associate these titers
with past exposure or past infection, as serological assays have not yet been
validated for H7N9."
For the future
So, as new H7N9 serology studies gradually come out, you be the judge. Figure out whether they're believable or not. Ask yourself the following:
- What assay(s) were used? Did the authors use an MN assay? They get bonus points if they did.
- If only an HI assay was used, then the conclusions are slightly less certain than if an MN assay was used.
- If the authors used an HI assay, what species were the red blood cells from?
- If horse red blood cells weren't used, then HI titer cutoffs lower than 160 are probably appropriate, but there is also more uncertainty about what an appropriate cutoff would be.
- What cutoff(s) did they use for seropositivity in their assay(s)? Do these cutoffs mesh with WHO guidelines? Do they mesh with what we know about H7N9 patient HI and MN antibody titers?
References
- http://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_hai_a_h7n9_20131220.pdf
- http://www.who.int/influenza/gisrs_laboratory/cnic_serological_diagnosis_microneutralization_a_h7n9.pdf
- http://www.virology.ws/2009/05/27/influenza-hemagglutination-inhibition-assay/
- Qiu C, Huang Y, Zhang A, Tian D, Wan Y, Zhang X, Zhang W, Zhang Z, Yuan Z, Hu Y, Zhang X, Xu J. Safe pseudovirus-based assay for neutralization antibodies against influenza A(H7N9) virus. Emerg Infect Dis. 2013 Oct;19(10):1685-7
- Zhang A, Huang Y, Tian D, Lau EH, Wan Y, Liu X, Dong Y, Song Z, Zhang X, Zhang J, Bao M, Zhou M, Yuan S, Sun J, Zhu Z, Hu Y, Chen L, Leung CY, Wu JT, Zhang Z, Zhang X, Peiris JS, Xu J. Kinetics of serological responses in influenza A(H7N9)-infected patients correlate with clinical outcome in China, 2013. Euro Surveill. 2013 Dec 12;18(50):20657
- Guo L, Zhang X, Ren L, Yu X, Chen L, Zhou H, Gao X, Teng Z, Li J, Hu J, Wu C, Xiao X, Zhu Y, Wang Q, Pang X, Jin Q, Wu F, Wang J. Human antibody responses to avian influenza A(H7N9) virus, 2013. Emerg Infect Dis. 2014 Feb;20(2):192-200
- Buchy P et al., PLoS One. 2010 May
27;5(5):e10864
- See, e.g., Table
4 in Pawar SD et al., Virol J. 2012 Oct 30;9:251
- Osterholm MT and
Kelley NS, MBio. 2012 Feb
24;3(2):e00045-12
- Bai T et al., N Engl J Med. 2013 Jun
13;368(24):2339-40
- Hsieh SM et al., J Infect. 2013
Nov;67(5):494-5
- Yang S et al., J Infect Dis. 2014 Jan
15;209(2):265-9
- Qiu C et al., J Clin Virol. 2014
Feb;59(2):129-31
- Boni MF et al., J Infect Dis. 2013 Aug
15;208(4):554-8
NOTE: I did not have a hand in writing this post and thus take no credit for it. This was entirely the work of the Guest Writer.
