1
DEPARTMENT OF HEALTH AND HUMAN
SERVICES
FOOD AND DRUG
ADMINISTRATION
CENTER FOR BIOLOGICS EVALUATION
AND RESEARCH
BLOOD PRODUCTS ADVISORY COMMITTEE
This
transcript has not been edited or corrected, but appears as received from the
commercial transcribing service:
Accordingly the Food and Drug Administration makes no representation as
to its accuracy.
Friday, July 23, 2004
8:00 a.m.
Gaithersburg Holiday
Inn
2 Montgomery Village
Avenue
Gaithersburg, Maryland
20877
2
PARTICIPANTS
Kenrad E. Nelson, M.D., Chair
Linda A. Smallwood, Ph.D., Executive
Secretary
Pearline K. Muckelvene, Scientific
Advisors
& Consultants Staff
MEMBERS:
James R. Allen, M.D., M.P.H.
Kenneth Davis, Jr., M.D.
Donna M. DiMichele, M.D.
Samuel H. Doppelt, M.D.
Jonathan C. Goldsmith, M.D.
Harvey G. Klein, M.D.
Suman Laal, Ph.D.
Katherine E. Knowles,
Acting Consumer Representative
D. Michael Strong,
Non-Voting Industry Representative
TEMPORARY VOTING MEMBERS:
Liana Harvath, Ph.D.
F. Blaine Hollinger, M.D.
Katharine E. Knowles
Matthew J. Kuehnert, M.D.
Susan F. Leitman, M.D.
Keith C. Quirolo, M.D.
George B. Schreiber, Sc.D.
Donna S. Whittaker, Ph.D.
3
C O N T E N T S
PAGE
Update on West Nile Virus, Hira Nakhasi,
Ph.D. 6
IV. Hepatitis B Virus Nucleic Acid
Testing (NAT)
for Donors of Whole
Blood:
A. Introduction and
Background,
Gerardo Kaplan, Ph.D.,
Laboratory
of Hepatitis and Related
Emerging
Viruses, DETTD, OBRR, FDA 28
B. Serological Course of
Hepatitis B,
F. Blaine Hollinger, M.D.,
Baylor College of
Medicine 32
C. Preclinical and Clinical
Data for
HBV MP NAT, Steven Herman, Ph.D.,
Roche Molecular
Systems 51
Allan Frank M.D., M.S.,
Roche Molecular
Systems 65
Open Public Hearing:
Michael Busch, Blood Centers
of the Pacific 103
William Andrew Heaton,
Chiron 121
Sherrol McDonough,
Gen-Probe 129
Richard Smith, NGI 136
Harvey Alter, AABB 144
IV. Hepatitis B Virus Nucleic Acid
Testing (NAT)
for Donors of Whole
Blood:
E. Committee Discussion and
Recommendations 154
V. Current Trends in Plasma Product
Manufacturing
A. Introduction and
Background,
Mark Weinstein, Ph.D.,
Associate
Deputy Director, OBRR, FDA 223
B. Presentation, Jan M.
Bult, CEO,
Plasma Protein
Therapeutics
Association 225
Open Public Hearing:
Patrick Schmidt, CEO, FFF
Enterprises 258
4
1 P R O C E E D I N G S
2 DR. SMALLWOOD:
May I ask all advisory
3
committee members to, please, take your seats?
4
Welcome to the second day of the Blood Products
5
Advisory Committee meeting.
Yesterday I read the
6
conflict of interest statement that applies to this
7
meeting, however, we have a new process now and we
8
will read a conflict of interest statement for each
9
day.
10 So, if you will indulge me, I will read
11
that at this point. This brief
announcement is in
12
addition to the conflict of interest statement read
13
at the beginning of the meeting yesterday, and is
14
part of the public record for the Blood Products
15
Advisory Committee meeting on July 23, 2004. This
16
announcement addresses conflicts of interest for
17
topic V.
18 Drs. Liana Harvath, Blaine Hollinger,
19
Matthew Kuehnert, Susan Leitman, Keith Quirolo,
20
George Schreiber, Donna Whittaker and Ms. Katherine
21
Knowles have been appointed as temporary voting
22
members for this meeting
5
1
Dr. Michael Strong is participating in this meeting
2
as the non-voting industry representative, acting
3
on behalf of regulated industry.
The Food and Drug
4
Administration has prepared general matters waivers
5
for the special government employees participating
6
in this meeting who required a waiver under Title
7
XVIII, United States Code 208.
8 In addition, there are regulated industry
9
and other outside organization speakers making
10
presentations. These speakers
have financial
11
interests associated with their employers and with
12
other regulated firms. They were
not screened for
13
these conflicts of interest. I
would just like to
14
remind everyone participating to, please, make
15
known, if you have not already done so, any
16
affiliation you may have and your status with that
17
affiliation prior to speaking.
18 Our committee chairman, Dr. Kenrad Nelson
19
has joined us this morning, and we also have Dr.
20
Blaine Hollinger who will also be part of the
21
committee this morning.
22 I just wanted to announce to those who
6
1
were not here yesterday that the next date, which
2
is tentative however pretty much firm, for the next
3
Blood Products Advisory Committee meeting will be
4
October 21st and 22nd, 2004.
5 At this time I will turn over the
6
proceedings of the meeting to the chairman, Dr.
7
Kenrad Nelson.
8 Update on West Nile Virus
9 DR. NELSON:
Thank you, Dr. Smallwood. I
10
will try to keep awake after the 24-hour airplane
11
ride. I came in last night but I
feel really
12
pretty good and I am very interested in the topic
13
today so I think that will help.
14 The first topic is an update on West Nile
15
virus by Hira Nakhasi.
16 DR. NAKHASI:
Good morning. I just want
17
to give you an update, as Dr. Kenrad Nelson
18
mentioned, on the West Nile epidemic and donor
19
testing which is happening now, in 2004. First I
20
will try to wrap up last year's things and then
21
come up to 2004.
22 Next slide, please.
The topics which I
7
1
will update you on are, as I said, last year's
2
epidemiology and the investigational West Nile
3
testing outcome of that, and some of the
4
transfusion-transmitted cases, and then the trigger
5
for the ID-NAT testing. Then I
will update you on
6
the West Nile donor and product management
7
recommendations with the recent revelations we have
8
got. Then I will update you on
the 2004 epidemic
9
and investigational West Nile testing, and also our
10
efforts in-house on the panel development and other
11
scientific issues--you know, the variation among
12
the strains of viruses infectivity of these
13
studies.
14 Next slide, please.
If you summarize in
15
one slide the last year's epidemic, it really
16
basically sums up that we had approximately 1000
17
[sic] cases or, to be precise, 9862 cases, human
18
cases, and 264 deaths. And, the
proportion of the
19
West Nile meningitis/encephalitis was 29 percent,
20
whereas, the fever was 69 percent in the human
21
cases.
22 Forty-six states, including
Washington,
8
1
D.C., were endemic, and donor testing started, as
2
all of you know, in July of 2003, using two
3
investigational NAT testing. In
some cases, a
4
small proportion started in the middle of June.
5
Despite this testing, I think these two
6
investigational NAT testing--these are minipool and
7
the two tests were the Gen-Probe test and the Roche
8
test, and Roche tested, as you know, in pools of 6
9
and the Gen-Probe test involves a pool of 16.
10 Despite testing, there were some
11
transfusion-transmitted cases and CDC had
12
investigated a total of 23 cases.
They were
13 confirmed by NAT and IgM reactivity and also by
14
follow-up of both the donor and the recipient. Out
15
of the 23, 6 were confirmed cases.
Only 4/6, you
16
may recall, had very low viremia, around 0.1
17
pfu/ml. Eleven cases did not confirm;
3 were
18
inconclusive because of the follow-up situation;
19
and 3 were open investigations.
20 Next slide, please.
As I said, since it
21
started on July 1 of last year, screening using
22
minipool NAT and IND, all geographic regions of the
9
1
U.S. were screening at that time.
With that, what
2
happened 1000 units of West Nile infected blood
3
donors were interdicted after screening
4
approximately 8 million donations.
So, I think it
5
was a very, very vast improvement over the year
6
before when there was no testing.
The last
7
positive donation was reported in the middle of
8
December in 2003.
9
Despite this testing, as you
see, the
10
majority of cases were interdicted, more than 75
11
percent, but there was a small percentage which
12
went through because, as you know, this was done in
13
minipool NAT.
14 Next slide, please.
This slide is Mike
15
Busch's slide where he showed why we were missing
16
some of these cases, and we knew that minipool NAT
17
sensitivity was such. The areas,
you know, where
18
the wrap-up takes place when--you know, he calls it
19
stage I, II, III, IV and V, and in stage I and II
20
they are ID-NAT positive but minipool NAT negative,
21
IgM negative. So, it could be
plus/minus. So,
22
during that stage they become IgM positive but they
10
1
become minipool negative and they are still ID-NAT
2
positive. So, this region and
this region were the
3
ones where they went through.
But, you know, these
4
were IgM negative and these were IgM positive so
5
the question is what is the infection of these
6
types of samples.
7 Next slide, please.
So there was a
8
potential for transmission of West Nile through
9
minipool NAT negative blood of low viremia in some
10
patients. Therefore, what
happened at that time is
11
that limited prospective ID-NAT testing started in
12
high incidence areas. If you
remember last year,
13
Colorado, Kansas and certain other areas, and
14
Nebraska were hot spots and ID-NAT was triggered at
15
that time, and the trigger was based on if the
16
preceding the rate of 1/200 minipool NAT positive
17
rate of 1/250, then they would start testing with
18
ID-NAT testing. Also, what happened
at that time
19
is that there was voluntary withdrawal of the
20
frozen transfusables in the high incidence areas
21
before the ID-NAT was initiated by some blood
22
establishments.
11
1 Next slide, please.
There was also
2
another initiative started at that time. The
3
initiative was to go back to do the retrospective
4
study on the minipool NAT negative samples and test
5
them by ID-NAT to find out how many we missed. It
6
would also let us know what was the low level of
7
viremic high incidence samples in high incidence
8
areas where minipool NAT did not pick them up.
9 The other purpose of the study was also to
10
identify samples which are like minipool NAT low
11
titer, minipool NAT negative but ID-NAT positive
12
for infectivity studies. I told
you that we do not
13
know whether those samples are still infectious at
14
low levels, and what is the level of infectivity.
15
So, these samples would be tested in various animal
16
models including non-human primates.
Also, the
17
purpose of these samples is to really find out the
18
relative clinical sensitivity of various West Nile
19
investigational testing. I will
report in a minute
20
what is happening with the infectivity state.
21 Next slide, please.
Based on the
22
observation that we had minipool testing and we
12
1
missed some of the samples because the viremia was
2
low, and also in the ID-NAT testing in the high
3
incidence areas--based on those studies and based
4
on the logistics issues, the question was what
5
should be the trigger for ID-NAT, and also logistic
6
issues such the availability of adequate resources,
7
recruitment, reagents and trained technologists.
8 So, the discussion about the trigger for
9
ID-NAT was held in collaboration with the AABB task
10
force. By the way, we are very
indebted to the
11
AABB task force for the biweekly meetings almost
12
throughout the year, and weekly meetings with the
13
task force during the epidemic to update us and
14
jointly discuss the strategies for how to go
15
forward with the testing performance, as well as
16
the epidemic.
17 So, based on that discussion, which was
18
held in February, the recommendations were the
19
following for the ID-NAT trigger:
It was discussed
20
that we should monitor reactive rates by zones
21
daily, enrolled 7 days when the epidemic was
22
starting, which was usually, you know, around the
13
1
beginning of July and early June even and this year
2
even May some cases were found.
The trigger was
3
that if you have 2-4 cases in any geographic
4
area--that is the blood collection, and the
5
frequency of 1/1000. This was
based on the fact
6
that every 1/4 would be missed by minipool NAT and
7
require ID-NAT. This was the
study done by ARC and
8
BSL and they found out that that would be the
9
trigger. And, you go back to
minipool NAT only
10
when you see ID-NAT reactivity and you don't find
11
zero cases in a consecutive 3-4 day period or the
12
rate is less than 1/1000. So,
that was the trigger
13
because, you know, we wanted to be prepared this
14
year because last year it was on an ad hoc basis to
15
start ID-NAT testing in those hot areas. So, we
16
wanted to be prepared this year if these areas
17
become hot so that we get the logistics present
18
there so we can start without interruption of the
19
ID-NAT testing.
20 Next slide, please.
Now we come to 2004,
21
where are we now? As of July 20,
which is a couple
22
of days back--as you see, every week the numbers
14
1
keep changing. Last week there
were 108. This
2
week it is 182 human cases out of which there were
3 4
deaths. There are 2 from Arizona, 1
from Texas
4
and 1 from Iowa. Out of total
infections, 74
5
percent of cases are neuroinvasive West Nile
6
illness and 26 percent cases are West Nile fever.
7
At the moment there are 35 states endemic for West
8
Nile. This slide has been kindly
provided by Jen
9
Brown, from CDC, and other slides which I will
10
mention later.
11 The total number of presumptive West Nile
12
viremic donors reported to the CDC ArboNet--that is
13
why I highlighted this, is 23.
There are more
14
cases than that but, as you know, there is a delay
15
in reporting to the ArboNet from the health
16
departments. So, using minipool
NAT as well as
17
ID-NAT in select areas, starting on May 4. Out of
18
these 23 presumptive West Nile viremic donors, 21
19
are from Arizona. The majority
are from the
20
Maricopa county near Phoenix, in Arizona; 1 from
21
New Mexico and 1 from Iowa. But
this is the tip of
22
the iceberg.
15
1
Next slide, please. This slide, again, is
2
provided by Jen. You can see the
distribution of
3
the West Nile, both the animal, avian and mosquito
4
infection, which is in this color, and the blue
5
color shows you the human cases.
You can see it is
6
very high in Arizona and California.
I am telling
7
Mike Strong that it is creeping up in Washington
8
soon. So, he has been telling me
we don't see
9
anything and I said, well, wait and watch! As you
10
remember, in 1999, how this started and how it is
11
spreading and, you know, it just keeps on going. I
12
hope it will end up in the ocean sometime.
13 Next slide, please.
This is just to give
14
you how early the human cases can be detected. As
15
you see from the slide, the earliest one was in
16
April. So, you know, there is an
expansion of this
17
epidemic, it looks like. We were
told in the
18
textbooks it is mostly in August and September or
19
late July but you can see it as early as April now,
20
and last year we saw it as late as December, in the
21
middle of December. So, you
know, it is almost a
22
year-round activity now.
16
1 Next slide, please.
Thanks to all the
2
blood establishments and testing establishments, I
3
got these data from several folks and I will
4
acknowledge them as I speak. The
total number,
5
according to my calculations but this may not be
6
right, is 61 presumptive viremic donors reported,
7
starting in May, 2004. As I
said, some of them are
8
reported to ArboNet and some of them are not. So,
9
it is not in addition to that; it is inclusive of
10
the ArboNet reports. ARC has
told me--Sue Stramer
11
gave the data from June 16 to July 20, 7 hard
12
cases. Again, this is also in
the Arizona area.
13
But she says no region has their ID-NAT trigger.
14 Mike Strong gave me this data from Roche.
15
There are 2 positive confirmed by ID-NAT--around
16
300,000 donations screened.
17 BSL, Sally Caglioti and Mike Busch told me
18
that there are 23 confirmed, out of which 16 came
19
from minipool NAT and 7 came from ID-NAT, confirmed
20
positives. There are 14 pending
and he was saying
21
that some of them are ID-NAT and would have been
22
missed by minipool NAT. Also,
some of them are low
17
1
viremic and also there are some which are IgM
2
positive. The denominator is
around 400,000.
3 Gen-Probe, Leanne Kiviharju, gave the
4
data. These are non-ARC data but
I am not sure--I
5
sent an email to Leanne--whether this is also
6
non-BSL but I am not sure; maybe we can find out
7
from here, but 21 confirmed positive and 7 are
8
pending. I am glad that you guys
sent me several
9
slides. I was basically trying
to summarize what
10
the presumptive donors are and, you know, I really
11
appreciate your sending extra slides.
12 The Department of Defense, Ron Hagey sent
13
me the data which has 8 confirmed out of 62,774
14
since January of 2004.
15 So, you know, this is the majority of the
16
screening going on at this time and there may be a
17
few cases which have not been reported yet, but
18
this is where we stand as of today.
19 Next slide, please.
I just wanted to sort
20
of briefly remind you that FDA is still continuing
21
to work closely with the test kit manufacturers and
22
we would like to facilitate implementation of these
18
1
tests and expediter test licensure.
I just want to
2
remind you that we issued two guidances in October,
3
2002 and May, 2003. There are 3
INDs for West Nile
4
minipool-NAT. One is from Roche,
one from
5
Gen-Probe and one from ARC. This
is public
6
information. FDA is continuing
to work with the
7
AABB task force. I think that
has been a
8
wonderful, wonderful collaboration with the AABB
9
task force and the people on the task force are
10
really helpful in doing this project together, and
11
with the CDC, NIH help, and to monitor the epidemic
12
and monitor the testing.
13 Next slide, please.
Both ARC and BSL did
14 a
study, which is unpublished observation.
We had
15 a
small discussion at the task force on what they
16
found out in some of the viremic donors when they
17
followed up. They wanted to find
out what is the
18
rate of the disappearance of RNA when they convert
19
IgM and IgG. As you remember, in
the last years
20
before the testing started the literature was that
21
it can go as long as 28 days of viremia. But from
22
their studies, and I don't want to go into detail
19
1
here because these are unpublished and, you know, I
2
don't want to divulge information--the gist of that
3
was that what they found out in both cases is that
4
the viremia may last up to 49 days in one case and
5 39
days in the ARC study, and in the BSL 49 days,
6
and West Nile RNA may go coexist with IgM.
7
Therefore, this sort of started us thinking. In
8
the guidance document we put 28-day donor deferral
9
and so we may have to rethink the deferral for
10
that.
11 Next slide, please.
We have not discussed
12
it with the AABB task force but we will be
13
discussing with the task force that, you know, the
14
integration of West Nile testing information. We
15 are thinking about maybe 56-day deferral for
West
16
Nile diagnosis of symptoms, including headache and
17
fever, or 14 days after symptom resolution if it is
18
more than 56 days. Potential
reinstatement of
19
donor deferral for West Nile symptoms only
20
following 30 days without symptoms, and negative by
21
West Nile IgM or ID-NAT. Again,
this is current
22
thinking. We have nothing in the
works yet but we
20
1
have internal discussions, and we will discuss it
2
at our next regular AABB task force before we come
3
up with a recommendation. Dr.
Alan Williams is
4
spearheading this initiative.
5 Next slide, please.
With regard to our
6
activities in-house, as I mentioned last year also,
7
we are still working on the panel development. The
8
purpose is to monitor sensitivity of assays to
9
detect viral nucleic acid antibodies, and also
10
trying to isolate and characterize West Nile
11
strains from human samples during 2003 and 2002
12
epidemics. The purpose of this study, which is done
13
by Dr. Maria Rios in our group--and all these
14
studies actually really are done by Dr. Maria Rios'
15
group--is the genetic variation of viral strains;
16
detection by currently available West Nile assays.
17
The purpose is to really see if there is any
18
genetic variation and also infectivity studies
19
using animal models. Currently,
the samples have
20
been identified which could be used for infectivity
21
studies. However, there are
logistic issues about
22
the animals, baboons, which are being worked out
21
1
with the Southeast Medical Center.
I guess the
2
task force is working on that.
Hopefully, we will
3
get some information by fall and we will be set to
4
do those studies.
5 Next slide, please.
Briefly, they have
6
two isolates, NY99 in 2002, which have been
7
characterized by genetic sequencing which I can
8
show you in a minute. The viral
infectivity is
9
determined by in vitro studies using cell lines and
10
primary human blood cell cultures.
Final panel
11
specifications are being established through the
12
collaborative studies, and the range of
13
concentration ranges between 1000-5 copies/ml.
14 Next slide, please.
Just a piece of
15
information here that Maria was kind enough to
16
provide to me. You know, she did
the comparison of
17
the human 2002 strain and the NY99 flamingo isolate
18
and then passed through the Vero cells.
She found
19
there were 20 nucleotide mutations and one
20
insertion. The mutations are
distributed all
21
across the region which result in 5 amino acid
22
substitutions. She is
characterizing more isolates
22
1
and she already has 6 from 2002, 11 from 2003 and 6
2
from 2004. So, the purpose is to
really compare
3
and to see what the differences are and how those
4
differences impact on our tests.
5 Next slide please.
The outcome of the
6
panel testing--six laboratories participated in
7
that. She tells me there were no
false-positive
8
results reported. More
variability in detection
9
was found towards the lower end of the viral
10
concentration, i.e., 80 percent of the time
11
detected 100 copies/ml member but all laboratories
12
detected 100 percent of the time the panel members
13
of 500-1000 copies/ml. Further
testing is going to
14
define the consensus copy number.
15 Next slide, please.
This is the important
16
slide. I would like to thank all
the people who
17
really helped to make this talk possible. Jennifer
18
Brown, whom I have always been bugging to provide
19
the slides. Thank you,
Jennifer. Dr. Sue Stramer,
20
Dr. Mike Busch and Sally Caglioti, Dr. Mike Strong,
21
Leanne Kiviharju, Roland, Maria and all these
22
people--whoever I send an email they are kind
23
1
enough to respond quickly. Also
my colleagues at
2
the FDA, Maria Rios, Alan Williams, Dr. Epstein,
3
Martin Ruta, Indira Hewlett--always helping in this
4
whole project and, last but not the least, the AABB
5
task force. I am really, really
grateful to them
6
for providing all the information and helpful
7
discussion. Thank you very much.
8 DR. NELSON:
Thank you. Any questions or
9
comments? Yes?
10 DR. GOLDSMITH:
Do you have additional
11 data on the level of viremia in these samples that
12
you have been studying? What is
the maximum level
13
of viremia?
14 DR. NAKHASI:
Which samples are you
15
talking about?
16 DR. GOLDSMITH:
The ones that you
17
recovered from the viremic donors.
18 DR. NAKHASI:
From the viremic donors, I
19
don't know. Maria, do you know
what the levels
20
are?
21 DR. RIOS:
Between 10
5 and 106 is the high
22
level of viremia that we have found.
Are you
24
1
asking for the range of viremia or the high level
2
of viremia?
3 DR. GOLDSMITH:
I was just curious about
4
the high but it is fine to give the range.
5 DR. RIOS: It
varies. It varies. The
6
assays, in general, that use lower volumes do not
7
detect them. Assays that have higher
volume and
8
high throughput detect, but do not give accurate
9
quantitation, to 10
6 copies/ml.
10 DR. NELSON:
One of your slides had 23
11
positives with 16 by minipool and 7 by ID. Were
12
those 7 not detectable by minipool or was it just
13
that ID screening was triggered and they weren't
14
tested by minipool?
15 DR. NAKHASI: I
think they came for the
16
ID-NAT testing. Is that
true? Yes. You know, in
17
BSL they had already started ID-NAT testing in
18
Maricopa County. The trigger had
started earlier.
19 DR. NELSON:
So, they were negative by
20
minipool?
21 DR. STRONG:
No, the trigger was activated
22
and they started doing ID screening so they haven't
25
1
gone back yet, I think, to see if those would have
2
been picked up by minipool.
3
DR. BUSCH: Actually, 7/12 that were
4
picked up in the region that had been converted to
5
ID-NAT, 7 of them had been fully worked up and 5 of
6
those 7 are negative at 1:16 dilutions so they
7
would have been missed by minipool.
Of those 5, 1
8
of them is antibody negative and 4 have IgM and
9
IgG.
10 DR. NELSON:
Has anybody looked at the
11
characteristics of the donors that have low levels?
12
Are there host factors that might influence whether
13
somebody has high level or low level?
I know one
14
feature may be antibody but in those that are
15
antibody negative, I wonder if there are any donor
16
characteristics that influence the level of
17
viremia.
18 DR. BUSCH: Sue
has looked at that I think
19
more formally and there wasn't any correlation.
20
These are representative donors of the donor pool
21
in terms of the viremics, non-viremics and low
22
viremics. I think it is just by
chance. This
26
1
phase of early viremia is completely asymptomatic.
2 DR. RIOS: It
may have some inherited
3
characteristics that limit the viral replication.
4
The reason why we think that is because we have
5
performed some in vitro studies with human primary
6
macrophages and there is a great variability not
7
only in the day of the viral peak, but some
8
individuals can have a very steady and low titer
9
that doesn't progress to peak.
So, that indicates
10
that some inheritance variability may interfere
11
with replication.
12 DR. NELSON:
That is interesting. Other
13
questions?
14 DR. LAAL:
Unless I misunderstood, I
15
noticed that in 2003 we had a majority of your
16
isolates from people who had fever, and about
17
one-quarter were from neuroinvasive cases. In 2004
18
it is reversed.
19 DR. NAKHASI:
Yes, that is an important
20
point. I discussed it with the
CDC folks and they
21
said, you know, don't pay attention to that because
22
the fever cases were--you know, this year they are
27
1
paying more attention so some of the fever cases
2
were not real fever cases. You
are right, you saw
3
the switch.
4 DR. LAAL: But
then in the isolates that
5
you are picking up now for the genetic studies, are
6
you carefully making sure that you look at both
7
types?
8 DR. NAKHASI:
Maybe Maria can say; I don't
9
know.
10 DR. RIOS: The
isolates that have been
11
studied so far don't come from patients. Actually,
12
that is the effort we are going to move towards
13
now. They are identified through
the blood
14
screening. So, in order to
evaluate if there is
15
any isolate that may not be picked up by the blood
16
screening we need to acquire samples from cases
17
that are non-blood donors to investigate this
18
possibility.
19 DR. NELSON:
Yes, Mike?
20 DR. STRONG:
Just a quick comment on the
21
donors. In the studies that were
done last year,
22
many of the donors that were interviewed, in fact,
28
1
were symptomatic either shortly before or shortly
2
after their donations but the screening questions
3
just didn't pick them up.
4
IV. Hepatitis B Virus Nucleic Acid Testing (NAT)
5 for Donors of Whole Blood
6 DR. NELSON:
Thanks. The next topic is
7
hepatitis B virus nucleic acid testing for donors
8
of whole blood. Dr. Gerardo
Kaplan will introduce
9
this and give us background.
10 A. Introduction and Background
11 DR. KAPLAN:
Good morning.
12 [Slide]
13 I am Gerardo Kaplan, Chief of the Lab of
14
Hepatitis and Related Virus Emerging Agents. I am
15
with the Office of Blood, and I will introduce for
16
you the hepatitis B virus n nucleic acid testing
17
for donors of whole blood.
18 [Slide]
19 The general agenda for this meeting is
20
that after the introduction and background, Dr.
21
Blaine Hollinger will give us an update on the
22
serology of hepatitis G. This
will be followed by
29
1
two presentations from the Roche Molecular Systems
2
and their preclinical and clinical data in support
3
of their application. Finally, I
will come back to
4
give you the FDA perspective on hepatitis B MP-NAT
5
and present the questions for the committee. I
6
understand that there will be a break and then a
7
public hearing.
8 [Slide]
9 So, the issue is that the FDA is seeking
10
the opinion of the committee on the performance of
11
the Roche COBAS AmpliScreen HBV test in minipools
12
of 24 samples to screen blood for transfusion by
13
nucleic acid testing, and its proposed intended use
14
as an alternative to hepatitis B surface antigen
15
testing in conjunction with testing for antibodies
16
to hepatitis B core antigen.
17 [Slide]
18 In support of their claims, Roche
19
Molecular Systems performed a clinical trial. The
20
study objectives of the clinical trial were to
21
determine whether the COBAS AmpliScreen test, which
22
is a minipool of 24 samples of plasma from
30
1
volunteer blood donors, can detect hepatitis by DNA
2
in hepatitis B surface antigen-anti-core negative
3
window period cases. This is the
primary objective
4
of the study. In hepatitis B
surface
5
antigen-positive donors, who are acutely infected
6
or chronic carries, and in persons previously
7 exposed
to hepatitis B as the secondary objective.
8 [Slide]
9 During the clinical trial, Roche Molecular
10
Systems identified two window period cases in about
11
600,000 volunteer whole because donations screened
12
by hepatitis B NAT using their minipools of 24
13
samples.
14 RMS, Roche Molecular Systems, claims that
15
the use of the COBAS AmpliScreen HBV test in
16
conjunction with the anti-core test would reduced
17
the residual risk of transfusion-transmitted
18
hepatitis B, and also that this test, the COBAS
19
AmpliScreen, could be used as an alternative to the
20
commonly used hepatitis B surface antigen donor
21
screening test. I would like to
point out that
22
blood in the U.S. is currently being tested by
31
1
surface antigen and core antibodies.
2 [Slide]
3 So, we would like to request comment of
4 the
committee on three questions.
Basically, the
5
firs would be do the sensitivity and specificity of
6
the Roche COBAS AmpliScreen hepatitis B test in
7
minipools of 24 samples support licensing of the
8
assay as a donor screen?
9 [Slide]
10 If so, assuming continued use of screening
11
tests for anti-hepatitis B-core, do the data
12
support use of the Roche COBAS AmpliScreen
13
hepatitis B test in minipools of 24 samples to
14
screen blood for transfusion as an equivalent
15
alternative to the surface test?
If not, which
16
studies would be required to validated such a new
17
test?
18 The third question is do the data support
19
use of the Roche COBAS AmpliScreen hepatitis B test
20
on minipools of 24 samples to screen blood for
21
transfusion as an added test in conjunction with
22
licensed donor screening tests for hepatitis B
32
1
surface and anti-core?
2 Now I would like to introduce Dr. Blaine
3
Hollinger. He will give us an
update on hepatitis
4 B
serology.
5 B. Serological Course of Hepatitis B
6 DR. HOLLINGER:
Thank you, Gerardo. It is
7
always a pleasure to come to these meetings and I
8
always tend to learn more than I think anyone here
9
because there is always so much information.
10 [Slide]
11 The talk today is about serology, and
12
serology can be confusing. It is
confusing to our
13
GI residents and fellows in trying to determine
14
what happens after hepatitis B infection. The
15
clinical and serologic changes that occur following
16
infection represent a complex interaction between
17
the host, the virus and specific antigens. If you
18
have an understanding of these items, I think it
19
becomes very easy to understand what happens.
20 There are certain changes that occur after
21
infection with all viruses that seem to be very
22
similar. Viruses are either
enveloped or
33
1
non-enveloped. Hepatitis B virus
happens outcome
2
have a lipoprotein envelope that contains hepatitis
3 B
surface antigen as the envelope protein.
There
4
are also some other particles in this serum which
5
are seen in excess of the infectious virion, and
6
these are the small particles here, and these
7
tubular forms which actually come off, in many
8
cases, from the hepatitis B virion.
9 The inside, or the nucleocapsid of the
10
virion is comprised of the hepatitis B core
11
antigen, and this encloses a relaxed, circular,
12
partially double-stranded DNA molecule.
Another
13
antigen which is seen also is the hepatitis B
14
antigen, but this is not part of the virion in any
15
way. It is a secretory protein,
about 16-17 kd in
16
size, that is secreted during active infection, and
17
is representative usually of high concentrations of
18
virus in the bloodstream. We
don't really know
19
what is the reason for this particular antigen.
20 [Slide]
21 This is the HBe genome. It has four open
22
reading frames. One of them is
responsible for the
34
1
hepatitis B-surface antigen and is comprised of a
2
small, a medium and a large protein.
A second one
3
is the core gene which expresses the core-antigen,
4
the hepatitis B core-antigen and also the e-antigen
5
as well. There is a DNA
polymerase that is
6
important for the replication of this virus, and
7 there is an x-gene here
which is very important for
8
initiation and probably maintenance of the
9
infection. It is probably also
weakly oncogenic as
10
well. Now, once one understands
these things, you
11
can then start to see what happens after an
12
infection.
13 Next slide, please.
This slide shows what
14
happens in the typical course--now, I am going to
15
talk about typical things here.
Certainly all of
16
us know about the exceptions and we have all seen
17
them at one time or another, but I think it is
18
important to deal right now with the typical
19
changes that can occur. Again
what happens, you
20
have a hepatocyte that is infected.
There is a
21
very short eclipse phase in which we would not see
22
virus in the hepatocyte. Then
viruses start to be
35
1
produced and are transported out of the cell.
2
Then, one sees initially HBV DNA in the
3
bloodstream. It precedes the
expression of
4
hepatitis B-surface antigen which then starts to
5
occur here very early in the course of the disease
6
and peaks usually during the acute phase of a
7
clinical illness.
8
What happens then in this
non-cytolytic
9
infection is that there are usually some holes that
10
are punched in the plasma membrane of a hepatocyte.
11
ALT, which is in the cytosol, aminotransferase
12
enzyme which is in the cytosol of hepatocytes, are
13
released into the bloodstream.
So, the ALT is
14
elevated in these patients and then follows the
15
clinical symptoms of anorexia, fatigue, jaundice,
16
etc.
17 Usually about the time the ALT begins to
18
appear, HBeAg and IgM anti-HBc are found. IgM anti
19
HBc appears in high titer or high concentration in
20
individuals as a response to the synthesis of
21
nucleocapsids and, therefore, represents active
22
viral replication. The IgM rises
during the early
36
1
courses of infection and then starts to disappear.
2
Usually by 4-6 months or so, or 6-9 months, the IgM
3
anti-HBc disappears, although it may be present for
4
up to 2 years in individuals, if you use very
5
sophisticated research tools.
6 Usually the HBV DNA disappears in the
7
acute infection at the time that a hepatitis
8
B-surface antigen disappears.
So, you have a
9
pattern here. HBV DNA first; HB
surface antigen,
10
and the HBsAg disappears and usually about that
11
time HBV DNA is gone, or at least is
12
non-detectable.
13 Again, another pattern appears. When
14
HBsAg disappears it is replaced by its antibody,
15
anti-HBs. Now, we know that
probably anti-HBs is
16
produced very early in the course of the infection,
17
even probably back in this area here, which results
18
in serum sickness, vasculitis or other things, but
19
you don't detect it because there is so much
20
hepatitis B surface antigen available so it is
21
non-detectable. But eventually
the surface antigen
22
goes; anti-HBs then is detected.
When HBeAg
37
1
disappears in the active infection, then anti-HBe
2
replaces it. Anti-HBe lasts in
most patients for a
3
long period of time but in about a third of the
4
patients disappears after about 6 months of
5
follow-up.
6 Of course, the IgM anti-HBc--there is a
7
switch-over between the IgM anti-HBc and IgG
8
anti-HBc. What is important to
recognize is that
9
the assay, the total anti-HBc assay, detects not
10
only IgM but IgG antibodies. It
is not an IgG
11
test; it detects both IgG and IgM.
So, whenever
12
IgM is present you ought to see the total anti-HBc
13
test positive.
14 Next slide, please.
This is an older
15 slide
I have but I think it helps us a little bit
16
understand the relationship of all these assays
17
that we are looking at. The EIA
assay and those
18
that are being developed can detect down to about a
19
tenth of a nanogram of HBsAg.
HBsAg circulates up
20
to 200 mcg or more per ml of blood.
These tests
21
really have an upper level of sensitivity of about
22 1
mcg/ml. So, usually they are at their
upper
38
1
limits of detection for most infections.
2 The second test that came along them were
3
the hybridization assays, the liquid phase and the
4
filter hybridization assays.
These could detect
5
down to a tenth to 1 pg of genomic equivalence in
6
the blood. At least, at 24 hours
if you have the
7
autoradiographs stay for maybe up to 5 days, you
8
can detect anywhere from 2- to 10-fold more virus
9
in the blood. So, they are
detecting about this
10
level here, somewhere between here and here, and 1
11
pg is equivalent to about 300,000 copies/ml. You
12
will see 280,000, 330,000 but let's just talk about
13
300,000 copies/ml. So, 1 pg is
here, 300,000,
14
30,000, 3,000 and 300 down at the femptogram
15
levels.
16 The PCR assays, which are somewhere
17
between 700-7,000 times more sensitive than
18
hybridization assays, can detect down to 50 copies
19
or less per ml. So, they are
down in this range,
20
here. As I said, this is
300,000, 3,000 and 300
21
copies/ml. This would be 30
copies/ml. These are
22
usually with nested PCRs or other assays, nucleic
39
1
acid--the tests that are available today.
2 So, you see the differences between these
3
assays. One might ask why is it
then that the EIA
4
tests are detecting HBsAg very closely to the HBV
5
DNA. Part of the reason for that
is that there are
6
anywhere from 1,000 to 10,000 times more--at least
7
10,000 times more hepatitis B surface antigen
8
particles per infectious virion, and there may be
9
even more naked HBV particles.
So, you have a lot
10
more HBsAg produced initially than you do
11
infectious virions. That is the
reason I think
12
that there is not a great deal of difference, at
13
least initially, in the detection of these
14
particles.
15 Next slide, please.
So, during the course
16
of infection HBV DNA is detected from 2-5 weeks
17
after infection and up to 40 days before the HBsAg
18
is detected. However, there is a
wide coefficient
19
here of variation but the mean of that is around
20
6-15 days, and there are some exceptions to this.
21
It rises slowly during the course of infection at a
22
relatively low level, say 10
2 to 104
copies/ml
40
1
during the seronegative period, that is, before the
2
HBsAg appears.
3 Next slide, please.
HBsAg, on the other
4
hand, appears 1-3 weeks before the ALT becomes
5
abnormal or 3-5 weeks before the onset of symptoms
6
or jaundice. It reaches a peak
during the acute
7
stage of the disease and then it declines to
8
undetectable levels within 4-6 months.
9 Next slide.
IgM anti-HBc is indicative,
10 as I said, of ongoing viral replication and appears
11
at the onset of ALT abnormality at high
12
concentration. It is present but
undetectable in
13
some chronic infections and may reappear,
14
therefore, during reactivation of HBV during
15
chronic infection.
16 Now, one of the questions that often
17
arises is when you have acute hepatitis how do you
18
determine that this is not a reactivation of
19
chronic disease compared to an acute disease? That
20
has always been an issue. For
all practical
21
purposes, the IgM anti-HBc test can be utilized to
22
determine that. It is at high
concentrations.
41
1
Whereas, patients who have reactivation of their
2
hepatitis B virus infection from chronic disease
3
often have a relatively low concentration of IgM
4
anti-HBc in their test. We use a
sample to cut-off
5
ratio and it is usually somewhere between 1-3 in
6
those cases, whereas in acute disease it is at its
7
maximum.
8 In addition, the other way, if you have
9
the means to do this, is that the IgM has a 19S
10
sedimentation coefficient during acute infection.
11
It has a 7S sedimentation coefficient during
12
chronic infection. That is
another way that one
13
could use to determine acute from chronic.
14 Next slide, please.
The anti-HBs is a
15
neutralizing antibody occurring during recovery and
16
after infection, and in most cases indicates
17
protection against reinfection.
However, it may
18
become undetectable in up to 20 percent of patients
19
after several years.
20 Next slide.
This slide sort of shows what
21
happens in a patient who gets acute disease and
22
gets over it. The anti-core is a
very strong
42
1
antibody, high concentration of antibody compared
2
to anti-HBs. Over time, as I
said, in about 20
3
percent of patients this antibody will disappear.
4
Then you are left with a person who has only an
5
anti-HBc response.
6 Next slide.
One of the ways that one can
7
tell that that is present is you can do an HBsAg
8
vaccine challenge. You can give
them a vaccine.
9
About 2-4 weeks later you have them come back in
10
for a quantitative anti-HBs test and if the
11
response is greater than 10 milli-international
12
units/ml, that is indicative that this person
13
probably had a prior infection and was immune. It
14
is unusual for a susceptible person or a person
15
with a low-level infection to respond to the
16
hepatitis B surface antigen by 2-4 weeks. Probably
17
less than 3 percent in 2 weeks will respond to the
18
vaccine at this particular level.
19 Next slide. I
have talked a little bit
20
about chronic infection. The
difference is that
21
arbitrarily, say, if the HBsAg is still positive
22
after 6 months or the person has ALT abnormalities
43
1
for a period of time, then they probably have
2
chronic infection. In addition,
the HBsAg is at
3
very high concentrations through that period of
4
time. It doesn't start to
decline. So, if you
5
could dilute these samples as a patient is being
6
followed along, you will find that the HBsAg
7
concentration is declining. Just
remember that the
8
upper level of their reference is about 1 mcg/ml so
9
you do have to do some dilutions in order to see
10
this happening. The IgM does
disappear but remains
11
usually positive at very low levels.
With research
12
tools you can detect it in most patients. It just
13
isn't positive in the way the test is done, which
14
is to dilute the sample about 1:1000 or 1:2000.
15
That is why you don't see it in these individuals.
16
That is how the test is configured.
Part of that
17
is because of prozone effects and other things
18
which could make a positive test negative if you
19
did not dilute.
20 E antigen is usually present initially,
21
but over time about 5-10 percent of patients will
22
seroconvert from HBeAg positivity to anti-HBe
44
1
positive. During that period of
time they usually
2
show reactivation of their disease.
Often it is
3
just an elevation of the liver enzymes but they may
4
become jaundiced and they may develop symptoms that
5
look just like acute disease.
Until we understood
6
this, this was one of the reasons we used to think
7
that 10 percent of patients who had acute disease
8
would become chronically infected.
Now that we
9
know that many of these patients were chronically
10
infected to start with, we now feel that probably
11
less than 3 percent of the immunocompetent patients
12
who develop acute disease will actually become
13
chronically infected. Of course,
the anti-core and
14
HBsAg remain positive in most patients.
15 Next slide, please.
Let me just go
16
through, finally, some of the findings that we just
17
talked about. This is HBVd and a
surface antigen,
18
anti-HBc and anti-HBs. You can
see in the
19
pre-seroconversion window period--we have a couple
20
of windows here because we used to talk about a
21
window period between the end of the HBsAg phase
22
and the development of anti-HBs in which the IgM
45
1
anti-core is present during acute disease. But
2
here I am talking about the pre-seroconversion of
3
antibodies or the seronegative, if you will, window
4
period.
5 Then, in the early acute infection both
6
the HBVd and a and HBsAg are positive.
The
7
anti-core and the anti-HBs are negative. The
8
presence of HBsAg is indicative of a hepatitis B
9
infection, either acute or chronic.
Just the HBsAg
10
alone--you can't tell. But the
presence of all
11
three of these together in the absence of anti-HBs
12
indicates an HBV infection, either acute or
13
chronic. Eventually you will
lose these markers
14
and you are left with anti-HBc and anti-HBs, which
15
means a previous infection with immunity.
16 Next slide.
The other findings are in
17
individuals who have a solitary anti-HBc test
18
present. They have no anti-HBs
and no HBsAg. Some
19
of them may contain HBV DNA and these represent
20
low-level carriers which do not have enough surface
21
antigen present in the blood to be detectable by
22
the current assays.
46
1 The other thing is that if this is
2
negative, this could also indicate an early
3
convalescent period in which this anti-HBc will be
4
IgM. It could represent an HBV
infection in the
5
remote past, as we talked about, in which the
6
anti-HBs has disappeared and you could do a vaccine
7
challenge, or it could be in many cases a
8
false-positive reaction. Most of
these reactions
9
show anti-core at a very low level, again, a sample
10
to cut-off ratio somewhere between 1-3.
The
11
anti-HBe is usually absent in these individuals as
12
well, and they don't respond to the vaccine
13
challenge.
14 Then you have a group that have no
15
markers. This usually excludes
an HBV infection in
16
this case. Then you have
individuals with anti-HBs
17
only. This represents a vaccine
type response. It
18
is really unusual to see something else happening,
19
or you shouldn't see anything other than anti-HBs
20
in a patient who gets the vaccine because it only
21
contains HBsAg.
22 Next slide.
This is my final slide. It
47
1
looks at discordant or unusual hepatitis B
2
serologic profiles requiring further evaluation. I
3
have probably seen all of these at some time or
4
another in my career. For
example, I have a
5
patient right now who has HBsAg positive only, who
6
is anti-core negative, with 900 million copies or
7
IU of virus per ml of blood. He
has been like that
8
most of his life. It happens in
some young
9
children who get hepatitis B and become chronically
10
infected. It is seen in other
adults, particularly
11
in Asians. But it is a really
unusual phenomenon.
12
So, that is a possibility but really rare.
13 Finding of all these three markers in one
14
patient does occur in about 5-10 percent of
15
patients. It is mostly in drug
users or people who
16
have been exposed to different genotypes of the
17
virus. It could be found in
people who have been
18 vaccinated and have made antibodies to a
different
19
genotype, and it usually is in patients who have
20
more serious liver disease.
21 Anti-HBs or anti-HBc positive only--we
22
have talked about those phenomena.
The are not so
48
1
uncommon. HBsAg negative
individuals who are HBeAg
2
positive--you shouldn't really see that but I have
3
seen at least two cases that have been HBeAg
4
positive, HBV DNA negative but do not have the
5
presence of surface antigen present.
6 You could have patients who are positive
7
for both e antigen and anti-e at the same time.
8
That has to do with the nuances of the test and
9 probably nothing else. And, you can find patients
10
who are anti-core negative--I mean, you should not
11
find a patient who is anti-HBc negative but IgM
12
anti-HBc positive.
13 Well, I hope now you are a little bit more
14 familiar with the serology of this disease.
15
Antigens are followed by antibodies.
HBV DNA
16
precedes the development of antigens very early in
17
the course and usually remains throughout the
18
disease entity itself. Thank you
very much.
19 DR. NELSON:
Thanks, Blaine. Are there
20
any questions? Harvey?
21 DR. KLEIN:
Blaine, there have been a
22
number of reports of people who have low levels of
49
1
HBV DNA and have anti-HBs. Do
you know if any of
2
those are infectious? Have you
ever seen one?
3 DR. HOLLINGER:
Well, it is not whether we
4
have ever seen one. The issue I
guess is that we
5 have
this question all the time about occult
6
infection in HBV DNA even though there are no
7
markers, usually in people who are very sick and
8
would not be donors, for example, liver cancer
9
patients and immunocompromised individuals. So,
10
you wouldn't find these as donors anyway. It is my
11
belief that most of these issues of finding nucleic
12
acid in the presence of antibodies probably means
13
it is neutralized and it is not infectious. You
14
see that with RNA found very late in the course of
15
the disease. In the presence of
antibody it
16
doesn't appear to be infectious biologically,
17
clinically or in trials.
18 So, I think that finding anti-HBs at a
19
reasonable concentration and HBV DNA would probably
20
not indicate somebody is infectious.
But you just
21
have to do these studies, and no one has really,
22
say, taken a chimpanzee and looked at these. I
50
1
think Prince did so a few years ago and the data
2
was that they were not infectious.
3 DR. NELSON:
You didn't mention the
4
genetic variations in the surface antigen. Could
5
those lead to a false-negative surface antigen test
6
in people who were actually infectious?
7 DR. HOLLINGER:
Yes, Kenrad, it is a good
8
question. There are about six or
seven genotypes
9
of this disease, from a to h, but because they all
10
have a group a antigen present, they all seem to be
11
detectable by the usual tests which are available
12
today. Now, could it
happen? Sure, I think it is
13
possible but I think it would be unusual.
14 DR. NELSON:
One other comment too, we
15
have studied hepatitis B in injection drug users
16
and it is extremely common for them to have
17
hepatitis B core antibody only, without surface, at
18
very high levels. They are
actually truly infected
19
and it is up to 20 percent of injection drug users.
20
It is possible that co-infection with hepatitis C
21
may play a part in that because they are all
22
infected with hepatitis C as well.
It is a very
51
1
complex interaction. But seeing
hepatitis B core
2
antibody only in somebody who actually is a drug
3
user and comparing drug users with others without a
4
history, it is like 20 percent of the drug users,
5
maybe 1 or 2 percent in gay men who have had
6
hepatitis B infection.
7 DR. HOLLINGER:
Yes, you see it not only
8
in drug users but you see it in volunteer donors
9
who are found to be anti-HCV positive and then you
10
go do some other tests and they are found to be
11
anti-HBc positive only. They
don't respond well to
12
the vaccine. And, we know that
HCV interferes with
13
the replication of hepatitis B virus so that is a
14
possibility.
15
DR. NELSON: Other comments?
16 [No response]
17 Thanks. Dr.
Herman, from Roche?
18
C. Preclinical and Clinical Data for HBV MP NAT
19 DR. HERMAN:
Thank you very much. It is a
20
pleasure to be here to describe to you the results
21
of our non-clinical and clinical performance
22
studies on our COBAS AmpliScreen HBV test.
52
1 [Slide]
2 I am going to begin the talk with a
3
description of our non-clinical performance
4
studies, and Dr. Frank will conclude with a
5
description of our clinical study results.
6 [Slide]
7 Here is an outline of the talk. I am
8
going to give an overview of the COBAS AmpliScreen
9
system and then I am going to describe the
10
different portions of the non-clinical performance
11
studies and give you their results.
12 [Slide]
13
The COBAS AmpliScreen
system is designed
14
for screening of minipool samples or individual
15
donor samples. The samples are
put onto the
16
Hamilton microlab pipetting robot which prepares
17
the pools. Then the pooled or
individual samples
18
are processed to extract nucleic acid with the
19
generic sample processing method.
There are two
20
variations of the method, the multiprep method for
21
the pooled samples and the standard method for
22
individual donor samples, and I will describe them
53
1
in the next slide.
2 Aliquots of the extracted material can be
3
processed in parallel with our three different
4
COBAS AmpliScreen tests, the test for HBV and the
5
licensed tests for HCV and HIV.
The analysis is
6
done on the COBAS Amplicor analyzer which automates
7
the nucleic acid amplification and detection
8
processes. Then, the software
for the analyzer and
9
the software for the pipetting machine interact
10
with the data output management system.
11 [Slide]
12 This slide describes the two sample
13
preparation methods, the multiprep method which is
14
used for the minipools, and the standard method
15
that is used for individual donor samples. For the
16
minipool method, 1 ml of the pool is centrifuged at
17
23,500 g to enrich for the viral targets, HCV, HIV
18
and HBV. After the centrifugation,
900 mcL of the
19
supernatant is discarded, leaving behind the pellet
20
and 100 mcL of the supernatant.
The nucleic acids
21
are extracted by adding a k-atropic lysis reagent
22
which contains the internal control.
After a brief
54
1
incubation, isopropanol is added to precipitate the
2
nucleic acids. The nucleic acids
are collected by
3
centrifugation. The pellet is
washed with ethanol
4
and resuspended in a specimen diluent, and then an
5
aliquot of the specimen diluent can be used in each
6
of the AmpliScreen tests.
7 The standard specimen processing method
8
for individual samples is almost identical. All
9
the green steps are identical and the differences
10
are highlighted in red. So, the
starting specimen
11
volume is 200 mcL instead of 1 ml, and there is no
12
concentration step. The
k-atropic lysis reagent
13
with the internal control is added directly to the
14
200 mcL specimen and then the remaining steps are
15
the same.
16 [Slide]
17 Now I am going to describe the analytical
18
sensitivity studies that were performed. These are
19 the results using the multiprep procedure. We used
20
the World Health Organization HBV DNA international
21
standard and we prepared dilutions of it between
22
100 and 3 IU/ml and analyzed 120 replicates of each
55
1
dilution, at Roche, using 2 different kit lots.
2
This shows the results. So, we
had 100 percent hit
3
rate at 100, 30 and 10 IU/ml and a 95.8 percent hit
4
rate at 5 IU/ml. The predicted
95 percent limit of
5
detection, using the PROBIT statistical method, is
6
4.4 IU/ml.
7 [Slide]
8 This slide shows the results using the
9
standard specimen processing procedure.
In this
10
study we analyzed dilutions of the World Health
11
Organization standard from 300 to 10 IU/ml. There
12
was 100 percent hit rate on 100 replicates at 300
13
and 100 IU/ml, and 99 percent on 30 IU/ml, and 97
14
percent at 20, and 95.8 percent at 15 IU/ml. The
15
predicted 95 percent limit of detection, using the
16
PROBIT method, is 16 IU/ml for the standard
17
specimen processing method.
18 [Slide]
19 This slide describes the results on a
20
panel prepared by CBER. The
original panel
21
contained 3 members at 100, 10 and 0 copies/ml, and
22
at Roche we prepared 4 additional dilutions from
56
1
the 100 copy/ml member at 50, 25 5 and 2.5
2
copies/ml.
3 This slide shows the results using the
4
multiprep specimen processing method and on 6
5
replicates, 100 percent hit rate at 100 copies and
6
at 10 copies/ml, then 67 percent and 42 percent
7
hits at 5 and 2.5 copies/ml.
8 Using the standard specimen preparation
9
method, 100 percent hits at 100 copies/ml with 6
10
replicates and 92 percent and 75 percent at 50 and
11
25 copies/ml.
12 [Slide]
13 This slide shows the results of our
14
analysis of the performance of the test on HBV
15
genotypes A through H. These
samples were obtained
16
from commercial sources, and we analyzed up to 25
17
individual isolates for each genotype.
The samples
18
were quantified using the COBAS Amplicor HBV
19
monitor test or, in the case of genotypes G and H,
20
monitor tests that we have in development. Then
21
the samples were diluted to approximately 2-3 times
22
the limit of detection for the multiprep method and
57
1
the standard prep method, and then they were
2
analyzed using the COBAS AmpliScreen test. So,
3
with the multiprep method and the standard method
4
all the isolates yielded positive results. So, the
5
test detected HBV genotypes A through H.
6 [Slide]
7 This slide summarizes the results on 40
8
commercially obtained seroconversion panels using
9
the multiprep method and the standard prep method.
10
The slide shows the number of days that DNA was
11
detected prior to detection of surface antigen
12
using a licensed surface antigen test, the Ortho
13
surface antigen test system 3.
The orange bars
14
show the results with the multiprep test, and for
15
that analysis the samples were diluted 24-fold to
16
simulate minipool testing. The
blue bars show the
17
results with the standard prep on undiluted
18 samples. The panels are sorted by the number of
19
days prior to antigen that DNA is detected with the
20
multiprep method. So, using the
multiprep method,
21
in 38/40 panels HBV DNA was detected prior to
22
surface antigen, and in 2/40 panels HBV DNA was
58
1
detected in the same bleed as surface antigen.
2 Using the standard prep method on neat
3
samples in 39/40 panels HBV DNA was detected prior
4
to surface antigen, and in 1 panel HBV DNA was
5
detected on the same day as surface antigen. I am
6
going to show you the results on one of the panels,
7
this panel, 39.
8 Before that, with the multiprep method, on
9 the 38 panels on which
DNA was detected prior to
10
surface antigen, DNA was detected an average of 17
11
days earlier, and with the standard method DNA was
12
detected an average of 22 days earlier.
13 [Slide]
14
Here are the results on panel
39. This
15
panel had 14 bleeds. This column
shows the results
16
with the Ortho Surface Antigen Test System 3, and
17
you can see that this turned positive on bleed 12
18
at day 143. This column shows the
results with the
19
minipool test, and you can see that it was positive
20
on the first and third bleed and then positive
21
again at the 11th bleed on day 113.
22 On the previous slide, on this panel it
59
1
was described as having DNA detected prior to
2
antigen 30 days in advance this one bleed. Even
3
though DNA was detected intermittently earlier than
4
that, we made a conservative interpretation and
5
only counted the number of bleeds that DNA was
6
continuously detected prior to surface antigen.
7 In this column, the results with the
8
standard preparation method on undiluted samples is
9
shown. There were 11 bleeds
prior to detection by
10
surface antigen and the HBV DNA was positive in 7
11
of them but did not yield a positive result on
12
bleed 11. So, in the previous
graph the standard
13
prep method was described for this panel as being
14
detected on the same day as surface antigen.
15 [Slide]
16 This just illustrates that even though DNA
17
was detected intermittently earlier, on multiprep
18
method we only counted 1 bleed, which was 30 days
19
in this case.
20 [Slide]
21 This slide summarizes the results on the
22
40 seroconversion panels. Using
the multiprep
60
1
method on samples diluted 24-fold to stimulate
2
pooling, DNA was detected prior to surface antigen
3
in 38/40 panels and on the same bleed in 2/40
4
panels an average of 17 days prior to surface
5
antigen. Using the standard
procedure on undiluted
6 samples, DNA was detected prior to antigen in
39/40
7
panels an average of 22 days earlier, and on the
8
same bleed on the 40th panel.
9 [Slide]
10 This slide summarizes the results of the
11
analytical specificity testing and the testing of
12
potentially interfering substances.
For the
13
analytical specificity we looked at 38
14
microorganisms and cell lines, and all these
15
samples yielded negative results with the
16
AmpliScreen and valid internal control results.
17
There was no cross-reactivity observed.
18 For the potentially interfering
19
substances, we looked first at 95 clinical
20
specimens with different diseases in patients
21
infected with these viruses or with autoimmune
22
disease or with yeast infections, and multiple
61
1
specimens of each. These
specimens were tested
2
both with the addition of HBV target and without.
3
So, the testing with the addition of HBV target was
4
looking for interference. No
interference was
5
observed. Testing without HBV
target was looking
6
for cross-reactivity and no cross-reactivity was
7
observed.
8 Then we looked at these various
9
potentially interfering substances.
Again, they
10
were tested with and without the addition of HBV
11
target. On the tests with HBV
target there was no
12
inhibition and on the tests without HBV target
13
there was no cross-reactivity.
14 [Slide]
15 This slide and the next look at our
16
reproducibility study. This
slide shows the
17
results of the multiprep procedure.
The study was
18
conducted at 3 clinical sites using 3 lots of
19
reagent. The study was conducted
over 5 days with
20 2
operators at each site. Each day the
operators
21
analyzed the 6-member panel, blinded 6-member panel
22
consisting of 2 negative samples and 4 samples at
62
1
low and moderate target concentrations.
2
Essentially the same results were obtained with the
3 3
kit lots and at the 3 sites. So, there
was 1
4
false-positive result with lot 3 and that happened
5
at site 3. At the low copy
samples the percent
6
hits were nearly identical among the 3 lots and
7
among the 3 sites.
8 [Slide]
9 This slide shows the reproducibility study
10
with the standard procedure and the same outcome.
11
Essentially the same results between the 3 lots and
12
between the 3 lights on the negative and the
13
positive samples.
14 [Slide]
15 Finally, this slide describes the
16
performance of the AmpliScreen test alone or in
17
combination with the licensed anti-core test on 918
18
antigen-positive clinical specimens.
I am showing
19
the results with the multiprep test on samples
20
diluted 1:14 to simulate pool testing and with the
21
standard method on undiluted samples.
So, looking
22
at the results of the AmpliScreen test alone with
63
1
the minipool method, 871 of the 918 samples were
2
DNA positive for sensitivity of 94.9 percent.
3
Using the standard method on undiluted samples, 898
4
samples were positive for sensitivity of 97.8
5
percent. These 20 samples that
were not detected
6
by the standard test were among the 47 that were
7
not detected by the minipool test.
All the NAT
8
negative samples were sent out for anti-core
9
testing and all of them were positive by the
10
anti-core test. So, using the
multiprep method, 1
11
sample had insufficient volume for the testing, a
12
sample that was positive with the standard sample
13
prep and negative with multiprep so we couldn't get
14 a
result on that so it was excluded from the
15
sensitivity analysis. So, for
the 917 samples with
16
NAT positive results and anti-core results on the
17
NAT negative samples the sensitivity was 100
18
percent for NAT and core combined relative to
19
antigen. On the 1900 samples
with evaluable
20
results using the standard method, again, the
21
sensitivity was 100 percent.
22 [Slide]
64
1 To summarize the non-clinical performance
2
studies, the analytical sensitivity was determined
3
on the World Health Organization standard. With
4
the multiprep method the limit of detection at 95
5
percent probability is 5 IU/ml and with the
6
standard method the limit of detection at 95
7
percent probability is 15 IU/ml.
The performance
8
on the CBER panel with the multiprep method, 100
9
percent hits at 10 copies/ml and with the standard
10
method 92 percent hits at 50 copies/ml.
Genotypes
11 A
through H were detected by the test.
12 [Slide]
13 On the 40 seroconversion panels using the
14
multiprep method on samples diluted 24-fold, HBV
15
DNA was detected prior to antigen in 38/40 panels
16
an average of 17 days or earlier, and with the
17
standard method on undiluted samples DNA was
18
detected in 39/40 panels prior to antigen an
19
average of 22 days earlier, and there were no
20
panels in which antigen was detected prior to DNA.
21
There was no cross-reactivity or interference
22
observed in the analytical specificity and
65
1
potential interfering substances studies. And, the
2
sensitivity on antigen-positive specimens, in
3
combination with the anti-core assay, was 100
4
percent.
5 [Slide]
6 I would like to acknowledge all the people
7
who did this work, Yuanfeng Yang and his group at
8
Roche Molecular Systems did an excellent job
9
developing the assay and performing these studies.
10
Larry Pietrelli coordinated the clinical studies
11
and, in this part of the talk, he coordinated the
12
reproducibility studies. They
were conducted at
13
the Community Blood Center of Greater Kansas City,
14
Royal Blood Centers, Minnesota and the Gulf Coast
15
Regional Blood Center.
16 Now I think I will turn the microphone
17
over to Dr. Frank who will describe the results of
18
the clinical performance study.
19 DR. FRANK:
Thank you, Dr. Herman.
20 [Slide]
21 It is my pleasure to be here today to
22
describe to you a prospective study to evaluate the
66
1
screening of plasma pools from volunteer blood
2
donations for the presence of HBV DNA.
3 [Slide]
4 A clinical trial summary will focus today
5
on two areas of primary focus of improved safety of
6
blood donations by HBV minipool-NAT.
The key focus
7
in that particular area will be looking at the
8
potential window period or pre-seroconversion areas
9
of HBV. A secondary focus will
be replacement of
10 surface
antigen by HBV minipool-NAT. To focus
on
11
that, we will look primarily at the areas of
12
samples that were surface antigen positive that
13
were picked up by minipool-NAT and surface antigen
14
positive picked up by anti-core.
15 This clinical study was initiated in
16
August of 2002 at 5 U.S. sites, and was completed
17
in April of 2003. A total of
704,902 specimens
18
were tested with HBV minipool-NAT, of which 581,790
19
were included in the sensitivity and specificity
20
analysis. The differential
between the two numbers
21
occurred because specimens were excluded if no
22
electronic results for surface antigen and/or
67
1
anti-core were available from the site.
2 [Slide]
3 If you look at this chart, which is a
4
schematic of the results listing the analytes
5
anti-core, surface antigen and DNA--if you can't
6
see in the back of the room, the red means they
7
were HBV negative for that particular analyte; the
8
green means they were positive for that particular
9
analyte. The totals are on the
far right-hand
10
column, the numbers that had that criteria. As you
11
can see, the majority of the donors were, as
12
expected, negative for all three.
13 [Slide]
14 We were primarily interested in looking at
15
this group down here, and those were enrolled in
16
our follow-up study, the protocol for which I will
17
be describing briefly.
18 [Slide]
19 Of these 4 eligible for the follow-up
20
study, the 2 areas we wanted to look at are
21
primarily the last row, which is the potential
22
window cases, that is, those cases which were HBV
68
1
minipool DNA positive but negative for core and
2
surface antigen. This represents
the type of
3
pattern one would expect in pre-seroconversion
4
window cases, and then the potential surface
5
antigen false-positive results, that is, surface
6
antigen positive but core negative and HBV minipool
7
negative as well.
8 [Slide]
9 I mentioned the follow-up protocol that
10
was utilized and this is the follow-up protocol for
11
those columns that we followed up, the test index
12
donation was tested by alternate NAT, provided by
13
the National Genetics Institute.
If alternative
14
NAT was positive, the result was quantitated and
15
then the subject was offered enrollment in a
16
6-month follow-up study for weekly draws times 4
17
then monthly draws to complete 6 months of
18
follow-up. The analytes tested
during follow-up
19
are as listed on your screen.
20 [Slide]
21 The primary objective was to improve the
22
safety by use of HBV minipool-NAT for detection of
69
1
window period cases.
2 [Slide]
3 As stated previously there were 23 donors
4
that were HBV DNA positive, surface antigen
5
negative and core negative. Of
these 23, 14 were
6
enrolled in the follow-up study.
It should be
7
noted that 9 donors declined follow-up and for the
8
purposes of our calculations for sensitivity and
9
specificity these were presumed to be false
10
positive and were negative on additional index
11
testing as well.
12 Of the 14 enrolled donors, 2 were
13
confirmed window period cases, the details of which
14 I
will be showing next, and 12 were shown to be
15
valse positive on HBV due to persistently negative
16
anti-core, persistently negative surface antigen
17
and persistently negative HBV DNA, and negative by
18
alternate NAT index specimens.
19 [Slide]
20 This window period case is a 26 year-old
21
male repeat blood donor with no known risk factors.
22
His HBV DNA was positive for a quantitative level
70
1
of 2000 on index donation. On
day 17 post index
2
donation the surface antigen was positive, and on
3
day 48 anti-core was positive.
4 [Slide]
5 The next window case is a subject who is a
6
49 year-old female repeat blood donor, healthcare
7
worker, with a history of HBV vaccination. This is
8
an interesting case because she had a negative
9
anti-surface antibody result 2 months prior to her
10
index donation when she donated blood previously.
11
What is interesting is between that time, where
12
this result 2 months prior was negative, and here
13
index donation was 2,340--now, this normally
14
wouldn't be done during her blood screening but she
15
was HBV DNA positive and she quantitated out at 200
16
copies/ml, which would be an infectious dose. So,
17
this is a window period case of an individual who,
18
on follow-up, never did convert for surface antigen
19
and did, on day 22, convert for anti-core. What
20
this likely represents is that somewhere between
21
the 2 weeks prior to her index donation when this
22
was negative and the result of her index donation,
71
1
she became infected with HBV and had a nice immune
2
system response to her previous HBV vaccination.
3 [Slide]
4 There are additional window cases I would
5
like to show to you today, and these were detected
6
by sites continuing to use HBV NAT following
7
conclusion of our clinical study.
The dates for
8
those 3 sites were April, 2003 to the present, and
9
there are 3 additional window period cases that
10
were detected in the one million donations
11
screened.
12 [Slide]
13 The first case is a 27 year-old male
14
repeat blood donor who reported high risk male
15
sexual partners. He had HBV DNA
positive on
16
donation with a quantitative level of 61,000. On
17
day 7 his surface antigen turned positive and on
18
day 36 his core was repeat reactive.
19 [Slide]
20 This case is a window period case of a 29
21
year-old male repeat donor who has no appreciable
22
risk factors for hepatitis B, other than a possible
72
1
acupuncture treatment for 8 weeks prior to his
2
donations, weekly treatments.
His HBV index was
3
positive at a quantitative level of 2,300. His
4
surface antigen turned positive 7 days later and
5
his core turned positive 28 days later.
6 [Slide]
7 This last case is still on follow-up but
8
this is a 50 year-old male repeat blood donor,
9
positive on index donation with 37,000 copies/ml.
10 On follow-up he remains positive on HBV DNA
and the
11
traditional blood banking serologies are still
12
pending.
13 [Slide]
14 In summary, I thought it was interesting
15
to look at the 5 cases that HBV minipool-NAT picked
16
up. If you look at the left-hand
column, and as I
17
have noted in the footnotes of your slides, all of
18
these are repeat blood donors who could normally be
19
expected to be donating blood regularly to the
20
blood pool, and all donated blood units that would
21
have been missed by the current blood banking
22
algorithms. All of them had
infectious dose
73
1
concentrations on their index donations. In fact,
2
even the lowest dose at 200 copies/ml, if the unit
3
were divided appropriately, would be sufficient to
4
infect everyone in this room.
5 [Slide]
6 Well, what is the yield for HBV
7
minipool-NAT? If you look at the
clinical study
8
yield alone we had 2 cases that I showed you out of
9
approximately 0.7 million donations for a yield of
10
1/350,000. If we look at the
continuing site data,
11
which I also showed you for the 3 cases in
12
approximately 1 million donations, the yield is
13
similar, 1/330,000.
14 Well, what does this mean in perspective
15
of what we are already doing in blood banking to
16
protect the nation's safety of the blood supply?
17
The yield for HBV minipool-NAT is approximately
18
equal to the yield for hepatitis C minipool-NAT and
19
the yield for HBV minipool-NAT is far greater than
20
the yield for HIV minipool-NAT.
21 [Slide]
22
If we look at this data
that was presented
74
1
by Mike Busch in Paris of this year, this
2
emphasizes what I have just stated, that for HBV
3
the yield is approximately 1/340,000, based upon
4
his presentation. For hepatitis
C, 1/230,000, but
5
for HIV, 1/3.1 million. Although
these aren't
6
quite comparable, West Nile Virus was included for
7
because there aren't a plethora of antibody and
8 antigen tests available yet implemented for
West
9
Nile Virus but that yield is 1/5,000.
10 [Slide]
11 This brings us to a sensitivity and
12
specificity calculation. You can
see how we
13
assigned HBV status, on the screen here, for
14
positives, negatives, the HBV status unknown for
15
those that were surface antigen negative but core
16
positive.
17 [Slide]
18 So, again, in summary, here are the totals
19
and the results of the initial testing.
These are
20
the potential surface antigen false-positive
21
results. These are the potential
window cases.
22
This last row I have gone into detail on, the row
75
1
above, the 4 and the 3 will be going into the
2
secondary objective.
3 [Slide]
4 The final status determination is here, on
5
the fir right-hand column. The
ones that could not
6
be determined are yellow, for those of you in the
7
back of the room. The green is
HBV positive. The
8
red is HBV negative. The window
cases that were
9
proven in the clinical study are shown here as 2.
10
The remaining 21, which will be accounted for in
11
sensitivity and specificity calculations, were
12
counted as HBV positive but final status HBV
13
disease negative.
14 [Slide]
15 To that point, our specificity
16
calculations, if you look at the total number of
17
subjects for which we have data--the difference
18
between the numerator and the denominator is the 21
19
cases that I just spoke about a second ago to yield
20 a
specificity calculation of 99.9964 percent, with
21 the
ranges noted on your screen. For
sensitivity
22
we looked at the positive AmpliScreen minipool HBV
76
1
results in specimens with positive HBV status and
2
divided by the total number of specimens with
3
positive HBV status. Again,
these calculations are
4
for HBV minipool-NAT alone and the sensitivity here
5
is 89 out of 105 or 84.7 percent, with a range of
6
74.4 to 91 percent.
7 [Slide]
8 So, the primary objective conclusion is
9
that COBAS AmpliScreen HBV test has identified
10
individuals in
pre-seroconversion window period
11
that would otherwise have been undetected by the
12
blood banking system. The COBAS
AmpliScreen HBV
13
test is suitable for blood screening with minipool
14
strategies presented today, and these data indicate
15
that HBV minipool-NAT will increase the blood
16
safety.
17 [Slide]
18 Let's turn now to the secondary objective,
19
and that is consideration for the HBV minipool-NAT
20
and anti-core for replacing surface antigen and
21
anti-core.
22 [Slide]
77
1 The index donations of interest for this
2
discussion then would be those that were anti-core
3
negative/ surface antigen positive and we will
4
discuss those first. Those
donors fitting this
5
category were 7 out of the total number for which
6
we have data, or 0.0012 percent.
Those that had
7
core-negative/surface antigen positive HBV DNA
8
negative were 3. Of those 3, 1/3
enrolled and
9
converted to anti-core. The
other 2 remaining
10
donors declined the follow-up study.
These were
11
determined to be HBV final status positives. For
12
DNA negatives, that is DNA negative, core negative,
13
surface antigen positive donors, 2 out of these 4
14
enrolled in follow-up. Two of
the remaining
15
declined to enroll in follow-up but we do have
16
additional data that has been available and I will
17
make available and show to you today.
All 4 of
18
these were assessed to be HBV negative.
Let's look
19 at
some of those details.
20 [Slide]
21 This first subject was HBV DNA negative,
22
surface antigen positive, core non-reactive. On
78
1
follow-up this subject remained core non-reactive
2
throughout the follow-up study.
On surface antigen
3
the subject has non-reactivity on follow-up, and
4
HBV DNA remained negative, as did the other
5
follow-up indicators.
6 [Slide]
7 This would indicate a case of an initial
8
surface antigen test which did not repeat on
9
follow-up, and all other negative serology results
10
indicate this patient had an initial surface
11
antigen positive that is consistent with
12
contamination or carryover.
13 [Slide]
14 The next case is a little bit more
15
interesting. This case is
surface antigen positive
16
again on index donation, HBV negative, core
17
non-reactive. It should be noted
that the surface
18
antigen was positive and repeat reactive throughout
19
follow-up but that the surface antigen was negative
20
on neutralization for all of these testing time
21
points. The donor also states a
history of HBV
22
vaccination. Of note, the core
never did react
79
1
throughout the course of follow-up and HBV DNA was
2
negative throughout the course of follow-up. The
3
anti-surface antibody was positive and that we
4
attribute to his vaccination status, and the repeat
5
reactive, again, was negative on neutralization
6
throughout.
7 In summary for this case, the anti-surface
8
antibody is explained by the donor's vaccination
9
history. And, the persistent
surface antigen EIA
10
repeat reactivity, which is negative on
11
neutralization, combined with all the other
12
negative results such as anti-core, is consistent
13 with a false-positive surface antigen result
due to
14
cross-reactivity.
15 [Slide]
16 We have additional data for the 2 donors
17
who declined the follow-up protocol.
Of the
18
remaining 2 donors, 1 donor retested 1 month
19
following his index donation and was negative for
20
surface antigen, anti-core and anti-surface
21
antibody. The other donor was
retested a year and
22 a
half post index and was negative for surface
80
1
antigen and for core antibody.
2 [Slide]
3 This slide may look somewhat familiar.
4
This is a look at the total HBV infected, HBV
5
surface antigen positive specimens.
In our study
6
there were 103 specimens fitting that
7
categorization. If we look at
sensitivity from
8
that perspective alone, HBV detected 87 of the 103
9
specimens and was negative on 16, for a sensitivity
10
as a stand-alone of 84.5 percent in this clinical
11
study. However, the proposal of
HBV minipool-NAT
12
plus anti-core antibody yielded positive in all 103
13
of 103 HBV positive cases and missed none of the
14
cases, for a sensitivity of 100 percent. This
15
parallels the non-clinical data shown to you
16
earlier today by Dr. Herman.
17 [Slide]
18 What are the secondary objective
19
conclusions? All 105 HBV
positive donors were
20
identified by anti-core and HBV minipool-NAT
21
combined. These 105 include 2
window case donors
22
that would have been missed by anti-core and
81
1
surface antigen combined, and 103 surface antigen
2
positive cases that were identified by the HBV
3
minipool-NAT and anti-core as an alternative.
4
These data then suggest that HBV minipool-NAT
5
combined with anti-core provide an alternative to
6
combined surface antigen and anti-core screening.
7 [Slide]
8 In summary conclusion today, I offer that
9
the COBAS AmpliScreen HBV test improves the safety
10
of blood transfusion. I offer
that COBAS
11
AmpliScreen HBV test combined with anti-core may be
12
considered as replacement for surface antigen
13
combined with anti-core. This is
supported by the
14
clinical trial data, and also supported by the
15
non-clinical trial data, specifically
16
seroconversion panel data shown here today and the
17
other HBV positive clinical specimens shown here
18
today by Dr. Herman.
19 [Slide]
20 With that, I would like to acknowledge the
21
blood centers that participated in this large
22
study, as well as the Roche Molecular personnel.
82
1
Thank you.
2 DR. NELSON:
Thank you very much.
3
Questions or comments? Yes.
Harvey?
4 DR. KLEIN: On
the two window period cases
5
that you detected with HBV alone, what was the
6
antigen assay that was used for those?
7 DR. FRANK: In
the two in the clinical
8
study, the first window period case was Ortho Test
9
System 2, and all remaining four window cases that
10
were presented were Abbott Auzyme.
11 DR. KLEIN:
And, do you by any change have
12
any data with head-to-head comparisons with PRISM?
13 DR. FRANK: I
don't believe we ran this
14
test against any test that was not approved by the
15
FDA.
16 DR. KLEIN:
Even in Europe?
17 DR. HERMAN: We
analyzed the
18
seroconversion panels against the PRISM and the
19
Ortho and I can show that data if you would like to
20
see it. That was the European
version of the PRISM
21
test that has the Paul Ehrlich Institute approval.
22
Do you want to see that data?
83
1 DR. NELSON:
Yes, I think so.
2 [Slide]
3 DR. HERMAN:
These are the same 40
4
seroconversion panels that I showed you previously
5
and the same NAT results but compared to the Paul
6
Ehrlich Institute licensed Abbott PRISM test, and I
7
guess this is the lot number.
Again, these are
8
sorted by the results with the multiprep test on
9
samples that were diluted 1:24-fold.
Those are the
10
orange bars. The blue bars
represent the results
11
with the standard test.
12 [Slide]
13 With the multiprep method DNA was detected
14
an average of 14 days prior to antigen, and with
15
the standard method DNA was detected 19 days prior
16
to antigen.
17 [Slide]
18 This slide has the summary. Compared to
19
the European PRISM test, with the multiprep method
20
DNA was detected prior to antigen in 34 of the 40
21
panels, and with the Ortho DNA was detected prior
22
to antigen in 38 of the 40 panels.
On the other 6
84
1
panels DNA and antigen were detected in the same
2
bleed. Using the standard method
compared to the
3
PRISM test, DNA was detected prior to antigen in 38
4
of the 40 panels, and on the same bleed in the
5
other 2 panels.
6 DR. NELSON:
Thank you.
7 DR. HOLLINGER:
I just have a question.
8
We talk about the mean and the median between the
9
appearance of one antigen for nucleic acid before
10
the other one. But these are not
tests that are
11
done every day so the panels do not include--and to
12
say 14 days or 19 days is a little misleading I
13
think in some cases because theoretically if you
14
draw blood today and you drew one 200 days from
15
now, and that is all you had, you would say, well,
16
it would detect things 200 days before one was
17
positive and one was negative.
So, you know,
18
unless samples are collected every single day for a
19
period of time, this is somewhat misleading. So,
20
give me some feeling about the days between each
21
one of these sample collections.
22 DR. HERMAN: It
varies between the panels.
85
1
So, the only data that I have available on the
2
slides here is the one panel I showed you where the
3
interval between bleeds I think was as few as a few
4
days and as large as 30 or 40 days.
5 DR. HOLLINGER:
It is not your fault in
6
doing this; it is just that, unfortunately, that is
7
how panels are set up. But I
think all of us have
8
to understand that it may be much less than that.
9
It won't be more than that but it certainly could
10
me much less than that.
11 DR. HERMAN:
That is absolutely correct,
12
and that is a limitation of these studies so one
13
shouldn't interpret this to conclude that if
14
testing was done every day these would be the
15
numbers. This is an
approximation, subject to the
16
limitations of the interval between the bleed dates
17
and the different panels.
18 DR. NELSON:
Actually, often
19
epidemiologists would assume a median--a mid-point
20
seroconversion between the two days.
So, if you
21
had a sample that was taken on day zero and day 14,
22
you would assume that the one that the one that was
86
1
positive on day 14 seroconverted on day 7. That
2
would be one way, a different analysis of the data
3
than probably what you have done.
4 DR. EPSTEIN:
Could I get you to clarify
5
whether the apparent window period samples
6
themselves were tested with HBsAg assays with known
7
sensitivities of 0.1 ng/ml?
8 DR. HERMAN:
Let me defer to my
9
colleagues. The window period cases
from the
10
clinical study?
11 DR. EPSTEIN:
Correct, and you say you had
12
five of them. Right? Two from the trials that
13
analyzed sensitivity and specificity and the
14
additional three--you have five available samples
15
that could have been retested with the most
16
sensitive available HBsAg assays.
17 DR. STRONG: I
have to declare my conflict
18
here because I am one of the clinical test sites,
19
but we have been able to test four of the five.
20
The most recent one hasn't been tested, but the
21
four have been tested on the Ortho system 3, the
22
new licensed test. It picked up
one of the four
87
1 but missed three.
2 DR. KUEHNERT:
Could you clarify what
3
those are? You said five.
4 DR. STRONG:
The five window cases that
5
are described here. The first
four were tested and
6
it picked up the one that you would expect it to
7
pick up, which is the high copy number sample. The
8
fifth sample, which hasn't been tested, also has a
9
relatively high copy number and you might expect it
10
to be picked up as well, with 37,000 copies.
11 DR. NELSON:
Are there blood donor
12
policies or recommendations for the interval
13
between receiving hepatitis B vaccine and donation?
14
Because that is a source of a false-positive
15
infection for the surface antigen.
Are there any
16
policies on that?
17 DR. EPSTEIN:
FDA has no policy for donor
18
deferral after HBV vaccination.
I am not certain
19
whether AABB has a voluntary policy, but I think
20
for non-live vaccines they would also not recommend
21
deferring. Someone else would
need to clarify
22
that.
88
1 DR. KLEINMAN:
I think that is right, Jay,
2
except that there have been some instances where
3
people have had false-positive surface antigen so
4
it is sort of unspoken that you might want to defer
5
people for a week or so, but there is no absolute
6
AABB requirement to do that.
7 DR. LEITMAN:
Can I ask a different
8
question. I am not sure I am
following what is
9
going on in the 16 donors who were HBsAg positive,
10
anti-core positive but multipool-NAT, COBAS
11
negative. Were those individuals
individual donor
12
NAT-negative? In other words,
are they infectious
13
or are they not infectious? If
they are not
14
infectious, do they just happen to have a lot of
15
HBsAg particles circulating but not infectious
16
virion?
17 DR. FRANK: I
have to apologize, I can
18
only hear parts of the question because of where I
19
am standing.
20 DR. LEITMAN: I
don't think I can do that
21
again. The 16 donors who were
HBsAg positive,
22
anti-core positive but NAT-multipool negative, were
89
1
they retested in individual donor NAT to see if
2
they have low level viremia or DNA nucleic acid, or
3
are they, in fact, true HBsAg positive but not
4 infectious
because they don't have intact virion
5
but just have particles circulating?
6 [Slide]
7 DR. FRANK: You
are talking about these
8
16?
9 DR. LEITMAN:
Those 16, yes.
10 DR. FRANK: The
final status here is that
11
they were HBV positive. The
question you are
12
posing is are these 16 patients infectious. They
13
have surface antigen positivity and core
14
positivity. I have to defer to
my colleague as to
15 whether or not we tried to quantitate with NGI the
16
HBV minipool NATs.
17 DR. PIETRELLI:
I am Larry Pietrelli, from
18
Roche Molecular Systems. Yes, on
those 16 donors a
19
sample was supposed to be tested by alternate NAT
20
and also by individual donation.
One donor was not
21
tested by either assay. Of the
15 remaining, 10
22
were positive by ID-NAT, of which 7 sere also
90
1
positive by alternate NAT. Two
were negative. One
2
was not done. Of the 5 that were
negative by
3
ID-NAT, 2 were positive by alternate NAT; 2 were
4
negative; and 1 was not tested.
The viral
5
concentration was low, as expected.
6
DR. LEITMAN: Thank you.
7 DR. KUEHNERT:
I have a follow-up question
8
about those 16. They were core
antibody positive.
9
Is there any way to quantitate that level of core
10
antibody positivity? Were any of
these at an
11
equivocal level? I am just sort
of getting at, you
12
know, whether you had greater numbers whether some
13
of these might have been close to a threshold for
14
missing them by core antibody positivity.
15 DR. FRANK: I
don't have that data. I
16
don't know whether my colleagues have access to
17
that data today.
18 DR. PIETRELLI:
No, we did not collect
19
that information but we could go back to the sites
20
to see what the S to CO was.
21 DR. HOLLINGER:
I have a technical
22
question. Is there are a reason
you used 24,000
91
1
times g for pelleting? I know
others have used
2
40,000 times g for pelleting for an hour at the
3
enhancement stage, so to speak.
Also, why did you
4
use 24 samples in your minipool or multipool?
5 DR. HERMAN: I
can answer the first
6
question about the centrifugation.
It is a
7
limitation of the instrument that is widely
8
available, which is a table-top centrifuge. It is
9
not quite an ultracentrifuge so it is more
10
convenient and easier to use.
That was a choice of
11
what equipment would be the most suitable for the
12
multiprep procedure for all three virus targets.
13 DR. HOLLINGER:
And the other question?
14 DR. HERMAN:
The choice of pool size, I
15
will defer to Mike.
16 DR. STRONG:
The pool size is one that was
17
selected for the HIV, HCV trials which were
18
concluded a couple of years ago and were licensed.
19
So, it was just to continue with the logistical way
20
in which laboratories operate to be consistent with
21
the other testing, and it is what the software does
22
for us.
92
1 DR. NELSON: It
is interesting that there
2
are many places, in the Orient particularly, where
3
core antibody testing is not done because of very
4
high rates of positivity. So,
that would cast some
5
limitation. Even though the NAT
assay would be
6
useful under those circumstances, it wouldn't
7
perhaps be as useful as continuing using the core
8
antibodies as is done in the U.S. and Europe. You
9
had a question, Donna?
10 DR. DIMICHELE:
Thank you. I was just
11
wondering if anybody can estimate, based on these
12
data or based on your data, what the residual
13
risk--in using your assay, if your assay was to be
14
implemented with core antibody testing, what do you
15
estimate the residual risk of transmission of HBV
16
would be through transfusion?
17 DR. FRANK:
That is a good question, and I
18
can say that we have shown the yield today but in
19
terms of discussion of residual risk, Dr. Kleinman,
20
would you like to comment?
21 DR. KLEINMAN:
Yes, Steve Kleinman. I am
22 a
consultant to Roche Molecular Systems on this
93
1
project. The residual risk
estimate is dependent
2
upon what you think the risk is now prior to
3
initiating HBV NAT. We have
estimated that risk
4
through the REDS study on two occasions, one that
5
was published in 1996 and then we kind of confirmed
6
the same numbers about six years later as about
7
1/60,000. The risk estimate is
based on the
8
incidence window period model and the data for that
9
model with HBV are not as strong as they are for
10
HIV and HCV. So, other people
have estimated the
11
residual risk now prior to NAT as being lower than
12
1/60,000, being about 1/200,000.
13
At any rate, if you accept
the 1/60,000
14
risk estimate, and then you take a look--the
15
question is how much does this new assay decrease
16
the window period. If you draw
on data that was
17
published by Robin Biswas and colleagues on their
18
panel testing of decrease of the window period, you
19
get about a 10-day decrease of the window period in
20
that study, and in this study it looks like the
21
window period decreases by 17 to 20 days based on
22
the panel. But, as Dr. Hollinger
mentioned, that
94
1
is a maximum case because you don't have closely
2
spaced samples.
3 At any rate, if you take a 10-day
4
lessening of the window period and you apply it to
5
what we think the current window period is, about
6
59 days, and you work this through with the window
7
period and the incidence of HBV, you would probably
8
get that the residual risk is still 1/200,000 to
9
300,000 even with this test being implemented.
10
Actually, it may even be 1/100,000.
So, you are
11
removing some of the risk but because you are only
12
cutting the window period back 10 days out of a
13
potential 59 days, the likelihood is that most of
14
the risk still remains even with minipool-NAT and
15
that in order to decrease that risk further you
16
have to go to either smaller minipools or
17
individual donation NAT, which would buy you more
18
in terms of lowering the window period.
So, you
19
are making an incremental step in decreasing the
20
risk; you are not decreasing the majority of what
21
we think is the residual risk.
22 DR. NELSON:
These data are heavily
95
1
dependent on what the window period is, which may
2
vary in different populations.
3 DR. STRONG:
Yes, they are dependent on
4
the incidence and what you think the current window
5
period is, and that is something that there hasn't
6
really been good data on. We
have estimated the
7
current window period as 59 days based on some
8
older work from the TTVS study and then, through
9
REDS, we made a revised estimate of the window
10
period through mathematical modeling to be about 45
11
days now, the infectious window period.
So, these
12
are estimates. We don't really
know what the
13
current window period is so it is a little bit
14
difficult. We don't know exactly
how much we
15
shortened it so that is why I think you have to say
16
that we can't make the kinds of precise estimates
17
that we have made about HCV and HIV.
18 DR. DIMICHELE:
Can I just ask you a
19
follow-up question then? What is
the
20
estimate--let's say given that residual risk, what
21
is the estimate of the impact of vaccination,
22
widespread vaccinations now going on in the younger
96
1
population with respect to when they become blood
2
donors? Does anybody know how
the risk of
3
transmission will be decreased just by vaccination,
4
widespread vaccination alone?
5 DR. KLEINMAN:
I don't know the answer to
6
that. I will answer kind of a
related question,
7
which is what is the risk to recipients? As more
8
recipients get vaccinated, then they should be
9
immune to a challenge, you would think
10
theoretically even if they were exposed. However,
11
most of our recipients are in the older age group,
12
60 and above, and some have compromised immune
13
systems. So, I don't think that
vaccination is
14
going to protect much of the recipient population.
15
Your question is different, whether it makes donors
16
less likely to acquire new HBV infection and I
17
don't know that anybody has worked that through
18
mathematically.
19
DR. ALTER: Harvey Alter, NIH. The
20
mathematical models have been extremely useful but
21
there has always been a little discomfort with HBV
22
in that you just don't see the cases as frequently
97
1
as you would have predicted from the model.
2 It seems to me now that if you make the
3
assumption that the real risk would be coming from
4
HBV DNA positive donors, you could actually
5
determine what the actual risk is now by taking the
6
best or maybe all the different HBV DNA assays and
7
testing these samples as single donations. If you
8
are picking up 1/350,000 now in a minipool format
9
you will pick up even more in an individual donor
10
format. That theoretically would
be the risk. If
11
we can exclude those donors, that risk would be
12
removed. It seems too simplistic
to be true but I
13
would like to hear a counter argument.
14
DR. BUSCH: Mike Busch.
I wanted to
15
comment on the vaccine issue. In
this study, I
16
think of the yield cases one of the yields that is
17
in the original clinical trial, clearly by history
18
vaccinated person, had an anamnestic anti-surface
19
response, low viremia in the setting of the index
20
sample. I think one of the
additional yield cases
21
also had anti-surface on index.
It is interesting
22
that the data from Taiwan is showing that when they
98
1
have applied NAT to donor screening--this is a
2
massively vaccinated population--they are seeing as
3
these young people, you know, enter adolescence and
4
in their 20s, they are seeing a fairly high rate of
5
the same kind of phenomenon.
These are essentially
6
vaccine breakthrough infections.
So the vaccine,
7
rather than being sterilizing--still people are
8
getting exposed and infected and, in a sense,
9
boosted from a wild exposure. In
their setting the
10
vast majority of their NAT yield are people who
11
have been vaccinated and have anti-surface and
12
never develop surface antigen, never develop
13
chronic infection. So, the critical
question of
14
whether these could transmit I think is still on
15
the table.
16 DR. STRONG:
Another point to follow-up on
17
what you were commenting on, on the value of
18
anti-core, I think this clinical trial also
19
demonstrated, as has been published, a small yield
20
of DNA positives from the anti-core positive group,
21
roughly in the same area of 5.5 percent or
22
thereabouts. Also, I think it
demonstrated a
99
1
difference in specificity with the anti-core assays
2
between the two licensed tests.
So, there is
3
clearly a large number of false-positive anti-core
4
donors that we have excluded on the basis of
5
false-positive reaction. I think
those of us who
6
have been involved in this trial, we have been
7
anxious to try to push the sensitivity of the assay
8
to allow us to reenter a lot of those donors
9
because it is a very large number.
10 DR. HOLLINGER:
I just want to follow-up
11
briefly on what Mike said. I
mean, this shouldn't
12
really surprise us very much, if you look at most
13
of the vaccine trials and follow patients for ten
14
years or so you will see anti-core appearing in
15
these patients. I just mentioned
that anti-core is
16 a
marker of viral replication. So,
somewhere along
17
the line these patients will probably have DNA in
18
the bloodstream. The issue is
whether it is
19
infectious or not and that is, of course, I think a
20
very critical issue with it.
21 Then I just wanted to ask a question
22
either of Harvey or Steve about how many cases are
100
1
recorded now of transfusion-transmitted hepatitis
2
B? I don't see very many. I don't want to get
3
back to this argument we had about 20 or 30 years
4
ago when we said there was no hepatitis C or
5 non-A/non-B hepatitis when really there was a lot.
6 DR. KLEINMAN:
No, I think that is exactly
7
right, Blaine. There are very
few post-transfusion
8
hepatitis B cases reported. As
you know, it is a
9
question about whether those people become
10
symptomatic. So, there may be
asymptomatic cases.
11
Some may go on to be chronic carriers.
That is
12
certainly possible. Secondarily,
even if a case
13
occurs, is it recognized by the clinician?
14
It is certainly a correct
statement that
15
we don't see the number of HBV clinical cases
16
reported as post-transfusion cases as the
17
mathematical models would predict.
So, then you
18
have two potential explanations.
One, the
19
mathematical models are wrong and these cases don't
20
occur or, two, the cases occur and they are not
21
recognized and reported. I don't
think we have a
22
way to sort out which one of those hypotheses is
101
1
correct. Harvey's point is that
we would have to
2
take a different approach to estimate risk, that
3
is, we would have to use the most sensitive DNA
4
assay available, hoping we could pick up one
5
copy/ml or even less. By that,
we could basically
6
make the equivalence that if we have a DNA-emic
7
specimen that equates to an infectious specimen,
8
and we could do that kind of study that was done 15
9
years ago or I guess 17 or 18 years ago for HIV,
10
but that would be an extremely laborious and
11
expensive study to do.
12 DR. NELSON: I
think we will take a break
13
at the moment and then come back for the open
14
public hearing. Maybe 20 minutes.
15 [Brief recess]
16 DR. NELSON:
The meeting is now in session
17
again and we will start with an open public hearing
18
on the topic presented of HBV DNA testing. Before
19
we start I will read this statement.
20 Both the FDA and the public believe in a
21
transparent process for information gathering and
22
decision making. To ensure such
transparency at
102
1
the open public hearing session of the advisory
2
committee meeting, FDA believes that it is
3
important to understand the context of an
4
individual's presentation.
5 For this reason, FDA encourages you, the
6
open public hearing speaker, at the beginning of
7
your written or oral statement to advise the
8
committee of any financial relationship that you
9
may have with the sponsor, its product and, if
10
known, its direct competitors.
For example, this
11
financial information may include the sponsor's
12
payment of your travel, lodging or other expenses
13
in connection with your attendance at the meeting.
14
Likewise, the FDA encourages you at the beginning
15
of your statement to advise the committee if you do
16
not have any such financial relationships. If you
17
choose not to address this issue of financial
18
relationships at the beginning of your statement,
19
it will not preclude you from speaking.
20
With that, Mike Busch?
21 Open Public Hearing
22 DR. BUSCH:
Thank you.
103
1 I wanted to just briefly touch on three
2
items. One is just one slide
sort of summarizing
3
the different context of some of the issues that
4
were raised in the questions as to the value of HBV
5
minipool-NAT both relative to the other NAT systems
6
and ID-NAT. Then in most of my
comments I want to
7
focus on the issue of can we drop either anti-core
8
surface antigen and, if not or if the data doesn't
9
support it at this point, what further studies
10
would be useful to address that.
I have Roche's
11
computer here--
12 DR. NELSON:
That sounds like a conflict
13
of interest!
14 [Slide]
15 DR. BUSCH: The
committee does have this.
16
This is a published analysis of cost-effectiveness
17
and I am not going to talk at all about sort of
18
cost-effectiveness but I just wanted to show one
19
slide that sort of emphasizes the relevant clinical
20
impact of these three viruses.
We tend to talk
21
numbers as if each of these viruses is equivalent
22
and the NAT yield is equivalent, but what this is
104
1
showing is the number of quality life years lost
2
per transmission.
3 What you can see is that for HIV it is
4
about 7 quality life years to the average 60 years
5
old transfusion recipient.
Transmission of HIV
6
would cost that person 7 quality life years; HCV,
7
0.6 and HBV, only 1.6. This is
because most
8
recipients are older people who naturally resolve
9
the acute infection of HBV, and even if chronic it
10
rarely evolves to significant morbidity/mortality
11
over the persistent life span of the average
12
recipient. So, you can see that
essentially HBV is
13
one-quarter as clinically important as HCV and 44
14
times less important than HIV.
15 The other point I wanted to make has to do
16
with this issue of going from current serologic
17
testing to minipool versus serologic testing to
18
ID-NAT, and how much of the yield that would be
19
accomplished by going to ID-NAT is detected by
20
minipool-NAT. For HIV we had
very sensitive
21
antibody tests in place so NAT by minipool closes
22
the window by about 5 days and ID closes it by
105
1
another 5. So, we picked up
about 50 percent of
2
what could be picked up but the risk is very low.
3 With HCV, because of the long plateau
4
phase, 95 percent of the window period is detected
5
by minipool-NAT. With HBV,
because of the slow
6
ramp-up, at least the model estimates and some of
7
the empiric data like in the Biswas paper would
8
suggest that minipool is only picking up about 23
9
percent or a quarter of what could be detected by
10
ID.
11 Next slide.
The issue of anti core and
12
could we get rid of anti core--there are three
13
studies that have been either fully published or in
14
abstract form, actually the Roche data, that have
15
looked at high numbers of donations that were
16
anti-core only reactive--so surface antigen
17
negative, anti-core positive--and subjected then
18
HBV NAT. Each of these studies,
as you can see,
19
has given almost identical rates both on a per
20
anti-core and, if you extrapolate, on a
21
per-transfused unit basis of detecting DNA in
22
donations that were surface antigen negative. So,
106
1
about a quarter to a half percent in all these
2
studies.
3 What is interesting is that in each of
4
these studies there was quantitation done in one
5
context or another, and all of these viremic
6
anti-core reactive units, surface antigen negative
7
have very low viral loads. In
Roche's data, for
8
example, 92 percent based on previously presented
9
work of their viremic anti-core only were negative
10
by minipool but only detected by ID-NAT. So, these
11
are very low viral loads that really would require
12
ID-NAT to detect.
13 [Slide]
14 This is just a summary and I am not going
15
to walk through this but with respect to low
16
viremic anti-core reactive donations or anti-core
17
only's there are a number of studies that have
18
documented transmissions, rare but there are
19
transmissions from these low viremic units. Again,
20 I
don't have time to go into these but we also know
21
that in liver transplantation from anti-core
22
reactive, surface antigen negative donors very
107
1
frequently transmits the virus.
So, these people
2
who have low viremia do harbor infectious virus
3
and, obviously, the anti-surface status is a
4
factor. But I personally don't
think there is any
5
prospect for dropping anti core.
6
[Slide]
7 Next slide.
This was alluded to earlier.
8
There was one study where purposeful transfusion of
9
these anti-core reactive, DNA positive, surface
10
antigen negative units into chimps was done. This
11
was by Fred Prince and was published.
These did
12
not transmit. Three patients who
had remote
13
hepatitis B, persistent anti-core, negative
14
antigen, low-level DNA. However,
the caveat is
15
that all three of these donors had anti-surface and
16
the volume inoculated into chimps was quite small,
17 1
ml. So, I think larger studies with
larger
18
volumes may be indicated to potentially explore the
19
infectivity of these units.
20 [Slide]
21 Next slide. In
terms of dropping antigen,
22
which I think is the more likely and I believe
108
1
optimistically we will get there, I think clearly
2
for these viruses like HIV, HCV, HBV where there is
3
not only the acute phase but there are chronic
4
carriers and some of these chronic carriers have
5
low-level infectious viremia the optimal paradigm
6
really is the combination of a front-end sensitive
7
direct assay and a serologic test for antibody.
8
For HBV we have both antigen and NAT which could be
9
viewed as redundant, analogous to P24 antigen in
10
HIV NAT. Clearly, on the front
end we know, and
11
saw from Roche and other studies, that HBV NAT is
12
clearly more sensitive than antigen during the
13
acute phase.
14 Next slide.
But the problem is, as Blaine
15
summarized, HBV is really an unusual virus in that
16
it produces in chronic infections amazingly high
17
levels of circulating antigen in the absence of
18
infectious particles. Therefore,
this excess
19
antigen can occasionally be detected in the absence
20
of detectable DNA by minipool or even ID-NAT. I
21
want to present a recent study on that.
Therefore,
22
to really be sure that we are not taking a step
109
1
back and dropping antigen when we have NAT, we need
2
to do large studies and I think Roche's data is
3
very impressive but whether it is enough is for the
4
committee to decide. But these
studies need to
5
look at these issues in a different context,
6
obviously, the front-end window period anti-core
7
negative as well as chronic carriers.
But also I
8
think they need to reflect populations with
9
different endemnicity and different routes of
10
transmission; populations that have been vaccinated
11
because we are, as I mentioned earlier, seeing
12
vaccine breakthrough infections, and how would they
13
play out in terms of antigen versus DNA; and also
14
genotypes and mutants.
15 Next slide.
Just to contrast, one of the
16
big differences in acute phase versus chronic is
17
the relationship between the DNA load and the
18
antigen reactivity. This is one
of a number of
19
studies. This is a Japanese
study. During this
20
acute phase these were all anti-core negative
21
front-end infections. They were
actually all
22
detected, 181 units detected as negative by the
110
1
Japanese particle agglutination antigen test.
2
Actually, 105 of these were reactive by the PRISM
3
assay. So, only a subset, about
40 percent of
4
these remained negative. But
what you are seeing
5
here is a very nice linear regression relationship
6
between the reactivity in the antigen test and DNA
7
load. That is typical of the
front-end acute
8
viremic phase.
9 Next slide. I
want to summarize now a
10
paper that is in press in collaboration with Mary
11
Kuhns and Steve Kleinman from the REDS group, where
12
we took 200 antigen positive, anti-core positive
13
donations in REDS. We first
subjected them to
14
quantitative HBV DNA using the Amplicor Roche
15
assay. This assay has a
sensitivity of 400 copies,
16
and 64 percent of the antigenic units had DNA above
17
400 copies. Those samples that
were negative, the
18
72, were taken through a more sensitive assay that
19
had a sensitivity of about 65 copies, which is
20
probably comparable to minipool type NAT, and 12
21
remained negative even at that level.
Those 12
22
samples were taken to a very high sensitivity,
111
1
essentially 1 copy/ml assay, high volume, and still
2
there were 6 that were negative.
So, we ended up
3
with still 3 percent negative even after taking it
4
through sequential increasing sensitivity assays.
5
So, these are the chronic carriers in whom DNA is
6
undetectable with at least one time point and one
7
high sensitivity assay.
8 Next slide.
This is showing the
9
relationship between antigen reactivity and viral
10
load. Unlike that nice linear
relationship, here
11
there it is essentially a scatter plot.
These are
12
the 3 that were negative by all NAT.
These are the
13
ones that were detected only by the high
14
sensitivity. These are the
Amplicor negative but
15
single input, not as sensitive, positive. And,
16
these are the quantifiable. You
can see that
17
relationship is not observed in chronic carriers.
18 Next slide.
That is people who have
19
anti-core. I think Roche did a
nice job of sort of
20
saying that if we keep anti-core the issue is not
21
that problem with anti-core positives, antigen
22
positives having absence of DNA.
It is really are
112
1
there donations that are antigen positive that lack
2
anti-core that are truly infectious and not
3
detectable by NAT?
4 In the REDS program and in other studies
5
varying 1-5 percent of antigen positive donations
6
lack anti-core. Now, when these
are worked up most
7
of these are window phase seroconverters so they
8
have high-level DNA very consistent with acute
9
infection. But in a proportion
of DNA negative,
10
and we saw those cases in Roche, the issue is
11
studying enough of those cases to sort out are they
12
true HBV infections, that either there is a
13
mutation not detected by NAT or individuals who
14
have failed to form anti-core, chronic carriers who
15
have low-level DNA and have failed to form
16
anti-core. Do they lack
contamination, which I
17
think most of them are? Are they
persistent,
18
non-specific antigen reactivity, and Roche showed
19
us an example of that? Or, are
they possibly
20
recent vaccinees?
21 Next slide.
This is three studies, two
22
published papers and some unpublished data from
113
1
REDS that have looked for these people who are
2
antigen positive, anti-core negative and either
3
lack or have very low-level DNA.
I am not going to
4 go through these in detail but in Japan one study
5
identified three such donors who had no history of
6
vaccination and who remained low-level PCR-reactive
7
with only a very high sensitivity enhanced assay;
8
remained anti-core non-reactive, so never
9
converted; and, again, remained antigen positive
10
and very low-level PCR-reactivity for over a year.
11 The French group, Couroce's group reported
12
two donors from endemic countries as well, with 0.1
13
percent of their antigen-positive donors who had
14
this pattern of persistent antigen without
15
anti-core and very low-level HBV DNA, and no
16
history of vaccination or immunosuppression and no
17
evidence of mutations to explain the failure of
18
this person to form anti-core.
19 In REDS--this is a cross-sectional study
20
so we weren't able to do follow-up, but we had 20
21
antigen-positive donors who lacked anti-core.
22
Sixteen of these were positive for DNA and were
114
1
probably typical acute phase, but 4 were negative.
2
Three of these were probably contaminations. They
3
had no other serologic markers, probably represent
4
surface antigen carryover. But
one had anti-e and
5
could represent an atypical carrier.
6 So, just a caution that there are some
7
studies, particularly from endemic settings where
8
there may be people who have antigen in the absence
9
of anti-core and low-level or absent DNA, and I
10
think we just need to study these further.
11 Next slide.
Just in conclusion, I do
12
think that we would see a small incremental yield
13
by minipool-NAT but the clinical impact of that I
14
think needs to be considered as well as the yield,
15
and we also need to view the context of what would
16
we get were we to get all the way to ID-NAT.
17 I am optimistic that we will eventually be
18
able, with ID-NAT or very small pools or high
19
sensitivity HBV DNA, to get rid of antigen,
20
however, I think this is going to require further
21
studies. Particularly, we are
capturing units
22
daily that could be worked up to accrue large
115
1
numbers with high volume of the plasma components
2
from these various sort of atypical patterns.
3
Particularly relevant is that I do think the
4
combination of NAT anti-core is the ultimate goal.
5
Particularly relevant are these samples that are
6
antigen positive, anti-core negative, and really
7
both studying these samples and particularly
8
enrolling and following the donors, as Roche did,
9
to determine whether these are false positives or
10
contamination. Thank you.
11 DR. NELSON:
Thank you. Comments? Yes,
12
Harvey?
13 DR. KLEIN:
Mike, we heard from Dr. Alter
14
earlier that, with the problems in trying to
15
calculate what the residual risk is, it might be
16
reasonable as a start to do a study simply by
17
taking 400,000, 600,000 specimens and using a
18
single unit detection system along with the
19
serology. I would just like to
know what your
20
opinion on doing that kind of a study might be.
21 DR. BUSCH:
Yes, I think that is a good
22
idea. I mean, we have the Biswas
paper included
116
1
comparing assays on window period panels neat
2
versus minipool. That is kind of
these model
3
estimates that minipool picks up only a quarter of
4
what might be detected neat.
But, yes, I think
5 such a large-scale
study either with neat--you
6
know, one of the issues with Roche's system is
7
their multiprep high extraction volume assay--we
8
saw the data that was comparing the multiprep pool
9
of 24 versus the standard prep neat, but had they
10
applied their high sensitivity multiprep neat,
11
which is what was done in the Biswas paper, they
12
would have seen a greater incremental closure than
13