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RAPID COMMUNICATION
Silent Infections With Human Immunodeficiency Virus Type 1 Are Highly Unlikely
in Multitransfused Seronegative Hemophiliacs
By Joseph Gibbons, Joan M. Cory, lndira K. Hewlett, Jay S. Epstein, and M. Elaine Eyster
W e used the polymerase chain reaction (PCR) to determine
the frequency of silent human immunodeficiencyvirus type
1(HIV-1) infections in seronegative high-risk individuals
with hemophilia who had been exposed to contaminated
blood products more than 3 years previously. In a crosssectional study of a cohort of 57 prospectively followed
seronegative hemophiliacs who received multiple transfusions before 1986, HIV-1 proviral DNA was found tran-
M
OST PERSONS with hemophilia who received large
amounts of clotting factor concentrates from 1979 to
1985 have developed serologic evidence of infection with the
human immunodeficiency virus type 1 (HIV- l).'-5However,
a small minority of individuals treated with factor concentrates during this time have not developed antibodies to
HIV- 1. The polymerase chain reaction (PCR) is an in vitro
method that has been used to selectively amplify specific
HIV- 1 proviral DNA sequences in peripheral blood mononuclear cells (PBMC) of infected individuals! Recently, HIV- 1
DNA has been detected by the PCR in PBMC of high-risk
seronegative individuals as long as several years before
s e r o c o n ~ e r s i o n .The
~ ~ ~ proportion and duration of HIV- 1
viral infections that escape detection by current serologic
tests for HIV-1 antibodies are unknown. However, preliminary data suggest that these controlled or silent infections are
more likely to be found in seronegative homosexual men who
continue to engage in high-risk behavior than in seronegative
hemophiliacs with past exposure to contaminated blood
products. 'Ox'
The purpose of this study was to determine the frequency
of HIV-1 infections in a cohort of seronegative high-risk
individuals with hemophilia who had been repeatedly exposed to contaminated blood products before the implementation of blood donor education, screening, and viral inactivation procedures more than 3 years previously.
'
From the Division of Hematology. Department of Medicine, and
Department of Microbiology and Immunology, The Pennsylvania
State University College of Medicine. Hershey: and the Food and
Drug Administration, Bethesda. MD.
Submitted August 13,1990;accepted August 20. 1990.
Supported in part by the Delaware Valley Hemophilia Foundation, Grant No. AI25928-03 from the National Institutes of Allergy
and Infectious Diseases, and Contracts ME-88146 and ME-89054
from the Pennsylvania Department of Health.
Address reprint requests to M.Elaine Eyster, MD. The Milton S.
Hershey Medical Center, Division of Hematology/Department of
Medicine. PO Box 850, Hershey, PA 17033.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 I990 by The American Society of Hematology.
0006-49~1/90/7610-0035$3.00/0
1924
siently in only one patient. These data suggest that the rate
of HIV infection among high-risk antibody negative individuals with hemophilia is very low to absent, in the range of
0% to 2%. These findings should provide considerable
reassurance to seronegative persons with hemophilia and
their sexual partners.
0 1990 by The American Society of Hematology.
MATERIALS AND METHODS
Patient population. The Hemophilia Center of Central Pennsylvania located in Hershey provides comprehensive care for over 200
persons with hemophilia who are seen at 6-month intervals for
routine evaluation and testing. At each visit, aliquots of sera and
PBMC are stored frozen in -7OOC and -135OC repositories,
respectively.With informed consent, we studied all 57 HIV antibody
negative persons who had ever been exposed to clotting factor
concentrates as of September 1989, using frozen PBMC collected at
the time of the most recent visit. Forty-two of these individuals had
been repeatedly exposed to non-heat-treated factor VI11 concentrates before 1986. Fifteen had received only hat-treated concentrates since 1986, but had been repeatedly exposed to fresh frozen
plasma or cryoprecipitate before donor screening procedures were
implemented in 1985. Serial samples were tested retrospectively on
12 of the most heavily transfused HIV antibody negative individuals
and on four individuals with frozen cells available pre- and postseroconversion. Healthy blood donors and seropositive persons with
hemophilia served as negative and positive controls. All specimens
were coded and tests were performed in a blinded manner.
Serologic assays. HIV antibody and p24 antigen tests were
performed with commercially available enzyme-linked immunosorbent assay (ELISA) kits (Abbott Laboratories, Inc, North Chicago,
IL), and positive antigen tests were confirmed by neutralization with
specific antibody. Western blot analyses were done with the Biotech/
Dupont kit (E.I. DuPont de Nemours Co, Wilmington, DE).
HIV-1 cultures. PBMC were separated from whole blood using
Lymphocyte Separation Media (Organon Technika, Durham, NC).
Ten million patient cells were cocultured with 5 x lo6 phytohemagglutinin (PHA)-stimulated donor cells in multure medium
consisting of RPMI-1640 with 15% fetal bovine serum (FBS), 5%
interleukin-2 (IL-2) (Electronucleonics, Silver Spring, MD), 1%
penicillin-streptomycin, and 1% L-glutamine. One-third volume of
the culture supernatant was removed every 3 to 4 days and was
replaced with an equal volume of fresh multure medium. Fresh
PHA-stimulated normal donor cells were added to the cultures once
a week. Aliquots of culture supernatants were tested for HIV p24
antigen using the Enzyme Immunoassay (Abbott). Cultures were
terminated when two consecutive HIV p24 antigen determinations
r 3 0 pg/mL were obtained or when negative results were obtained
after 30 * 2 days.
Enzymatic amplification of HIV-1 DNA. Frozen, density gradient separated PBMC were thawed at room temperature, and washed
in phosphate-buffered saline (PBS). Cell lysates for PCR were
prepared by resuspending the PBMC pellet in 10 mmol/L Tris HCl
(pH 8.3), 50 mmol/L KCl, 4 mmol/L MgCl,, 0.5% NP-40, 0.5%
Tween-20, and Proteinase K 120 &mL. Proteinase K digestion was
performed for 1 hour at 55OC. The lysates were then heated to 95°C
for 10 minutes to inactivate the Proteinase K. The lysate from 150,000
cells (approximately 1 fig of DNA) was used for each amplification
Blood, Vol 76, No 10 (November 15). 1990: pp 1924-1926
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SILENT HIV-1 INFECTIONS IN HEMOPHILIACS
reaction. Cell lysates prepared in this manner ?outinely produced good
ampliation of PCR primer pairs for a region of DQ a gene.
Enzymatic HIV- 1 proviral DNA amplification was performed
using PCR essentially as described by Ou et a1.I2 Primer pairs for
conserved regions of the gag and env genes were amplified separatJy
for each sample. The primer pairs used were SK 38-39, which
amplifies as 114-bp gag segment (nucleotides 1551 through 1665),
and SK 68-69, specific for a 141-bp env fragment (nucleotides 7801
through 7942). Amplifications were performed in 10 mmol/L Tris
HCI (pH 8.3), 50 mmol/L KCI, 2.5 mmol/L MgCI,, 200 pmol/L of
each deoxyribonucleotide triphosphate, and 2.5 U of Thermus
aquaticus (Taq) DNA polymerase (Cetus Corporation, Emeryville,
CA). PCR was performed in a Perkin Elmer Cetus DNA Thermocycler for 35 cycles with denaturation at 94OC for 1.5 minutes, primer
annealing at 55OC for 1 minute to 32Pend-labeled oligonucleotide
probes specific for conserved internal amplified sequences of SK 19
(nucleotides 1595 through 1635) for gag or SK 70 (nucleotides7841
through 7875) for the env PCR product, and template extension at
72OC for 1.5 minutes. The hybridization mixtures were analyzed by
10% nondenaturing polyacrylamide gel electrophoresis, followed by
overnight autoradiography with Kodak XAR-5 film (Eastman
Kodak, Rochester, NY). The product was electrophoresed in an
agarose gel containing ethidium bromide to detect amplified DNA,
and the molecular weight of the product was estimated using
appropriate markers. Several PBMC lysate specimens from HIV-1
seropositive individuals and low-risk HIV-1 seronegative blood
donors were included in each set of assays as positive and negative
controls. An aliquot of lysis buffer was amplified concurrently to
confirm that the amplification reagents were free of HIV-1 DNA
contamination.
For a sample to be considered positive, both gag and env fragments
had to be detected by sequential amplification with each primer pair.
Samples were scored as negative if neither gag nor env signals were
detected by the same criteria. Samples with isolated gag or env
signals were considered indeterminate and retested. AU positive results
were confirmed by repeat testing of lysates from separate aliquots of cells.
Multiple well-characterized positive and negative controls,including a
negative buffer control, were run with each set of experiments.
RESULTS
In a cross-sectional study, HIV-1 proviral D N A was
detected in only 1 of 57 (1.8%) of the seronegative recipients
of clotting factor concentrates. This individual was an
asymptomatic child with a 5% factor VI11 level who had
received a total of 400 bags of cryoprecipitate from 1981
through December 1987. Two hundred of these bags were
given before donor screening was implemented in 1985.
Subsequently, he received 15,000 U of heat-treated factor
VI11 beginning in February 1988. Samples obtained in
November 1986 and July 1988 were repeatedly negative. A
sample obtained in February 1989 was repeatedly positive
using lysates of two separate aliquots of cells. A follow-up
sample obtained in March 1990 was again negative, a t which
time HIV-1 culture of fresh PBMCs was also negative.
HIV cultures were performed on 12 consecutive seronegative patients. All 12 were negative. Twelve intensively
treated PCR negative patients had two or more serial
samples available for testing over a 1- to 2-year period.
HIV-1 proviral D N A was not detected in any of these
samples, five of which were also culture negative. Serial
samples were also available on four patients before and after
seroconversion. HIV-1 proviral D N A was detected in one
p24 antigen positive patient before the detection of serum
1925
antibody, and in one p24 antigen negative patient 12 months
before the detection of serum antibody. HIV- 1 proviral D N A
was not detected in two other patients 8 and 14 months,
respectively, before the appearance of serum antibody. HIV-1
proviral DNA was consistently detected in all 21 HIV antibody
positive control individuals, 18 of whom were asymptomatic.
DISCUSSION
Theoretically, HIV-1 may remain latent in the form of a
nonreplicating provirus for undetermined periods of time
before the development of HIV antibodies. Infected individuals may harbor HIV-1 detectable by culture or PCR for
months to years after exposure to the virus before seroconversion as defined by commercially available antibody
assays.7.9.10.13
PCR has the unique ability to detect minute
quantities of HIV-1 specific nucleic acid independently of
viral replication or host immune response.
Several investigators have examined different groups of
high-risk seronegative subjects for evidence of HIV-1 D N A
by PCR. In 1989, Imagawa et a17first reported that HIV-1
infection in some homosexual men a t high risk could be
detected a t least 35 months before seroconversion. Subsequently, Wolinsky et a19 found that HIV-1 infection could be
detected by PCR 6 to 42 months before a diagnostic Western
blot assay in 20 of 24 homosexual men who continued to
engage in high-risk behavior. Hewlett et all4 reported that 11
(50%) of 22 seronegative female sexual partners of HIV-1
infected hemophiliacs were PCR positive. These studies
suggest that there may be a prolonged seronegative state
after acquisition of HIV-1 infection in some individuals.
However, Horsburgh et all0 found that infection for longer
than 6 months without detectable antibody was uncommon
in 27 homosexual and 12 hemophilic men for whom samples
were available before and after seroconversion. Jackson et
all' found none of 20 seronegative multitransfused hemophiliacs to be PCR positive, and Sullivan et a1 found no silent
infections among 20 seronegative female spouses with more
than 5 years sexual exposure to an HIV-infected hemophilic
male (reference 15 and personal communication, J.L. Sullivan, August 1990). Mariotti et all6 found no HIV-1 proviral
D N A in 76 high-risk seronegative individuals, including 20
polytransfused hemophiliacs. These findings concur with our
data, which suggest that the rate of HIV infection among
high-risk antibody negative individuals with hemophilia is
very low to absent, in the range of 0% to 2%.
These conflicting results must be interpreted with caution,
because PCR, although capable of detecting extremely small
quantities of HIV-1 genetic material (as few as 1 infected in
100,000 uninfected cells), is fraught with technical problems.
Many investigators have experienced problems with false
positive PCR results due to laboratory contamination. To
decrease the likelihood of carryover, we paid strict attention
to separation of pre- and postamplication processing, and
included multiple negative and positive patient samples as
internal controls with each set of assays. Positive results were
confirmed using separate blinded aliquots of cell lysates from
the same date.
The low frequency of PCR positive, seronegative hemophiliacs in our cohort could be due to lower sensitivity of our
PCR methodology. However, two things argue against lack
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1926
GIBBONS ET AL
of sensitivity having a significant impact on these data. First,
we were consistently able to detect all the HIV-1 seropositive
clinical specimens processed in a blinded manner in parallel
with our negative specimens. Second, we have not had any
seroconversionsduring the past 5 years in our study population, in contrast with the continuing seroconversionsobserved
in the Multicenter AIDS Cohort (MAC) study.
HIV-1 proviral DNA was demonstrated in two separate
aliquots of PBMC from a single subject. However, a follow-up specimen on this child was negative. Others have
noted that HIV-1 amplified products were not detected
consistently at all time points during long latency periods
preceding serocon~ersion.~.”
Either sampling error or episodic HIV replication in cells eventually cleared from the
circulation’8 could account for these results. Simmonds et
al” have reported that PBMC DNA from one seropositive
individual contained only five copies of provirus per lo6cells.
With only 1 positive test in 57, the Poisson model would
predict that nearly 1 million cells would be required to insure
a 90% probability of obtaining a positive test result at this
level of detection. Thus, considerably more than 1 pg of
DNA from 150,000 cells would be needed to insure reproduc-
ibly positive PCR results. Alternatively, the single positive
result in this individual who remains seronegative and
culture negative more than 2 years since his most recent
exposure to nonheat-treated products could have been due to
sample contamination. Continued follow-up will be required
to determine whether this isolated finding represents a false
negative due to decreased sensitivity or a false positive due to
lack of specificity.
The observation that only 1 of 57 (1.8%) seronegative
recipients repeatedly exposed to HIV-1 contaminated blood
products more than 3 years previously had possible evidence
of PCR reactivity in a cross-sectional cohort study indicates
that silent or controlled infections in multitransfused HIV- 1
negative hemophiliacs are infrequent, if they exist. These
findings should provide considerable reassurance to seronegative persons with hemophilia and their sexual partners.
ACKNOWLEDGMENT
The authors thank Jeffrey Sanders, Cynthia Gumbs, and Joseph
Bednarczyk for technical assistance; and Betsy Ohlsson-Wilhelm,
PhD, and Daniel Heitjan, PhD, for their valuable comments
regarding PCR and rare event analysis.
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From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
1990 76: 1924-1926
Silent infections with human immunodeficiency virus type 1 are highly
unlikely in multitransfused seronegative hemophiliacs [see
comments]
J Gibbons, JM Cory, IK Hewlett, JS Epstein and ME Eyster
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