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B
Serologic and V~rolo~c Testing
The standard tests used to define individuals who have been exposed to
HIV detect antibodies to the virus in the serum. Antibodies to HIV can be
detected by several techniques, including enzyme-linked immunosorbent
assays (ELISA), immunofluorescent assays (IFA), and Western blot
analysis. Each of these techniques, when performed by expert techni-
cians, is very accurate at detecting antibody either to the whole virus or
to viral subcomponents.
In contrast to some viral infections, HIV induces antibodies that do
note in most cases, appear to effectively neutralize the establishment or
consequences of viral spread in an infected host. Therefore, most patients
with positive tests for HIV antibodies are considered to be simultaneously
and actively infected by HIV. The resulting concerns about the equation
of seropositivity with extant infection, continuing transmissibility, risk of
disease in an infected individual, and issues of social stigmatization have
caused HIV serologic testing to be very controversial.
The tests currently in use attempt to measure specific antibodies to
proteins or polyproteins produced as a result of infection with HIV. The
virus's gag and end genes encode for the predominant viral antigen to
which antibodies detected by today's tests are directed. The gag gene,
which encodes the protein constituents of the viral core, initially produces
a 55-kilodalton (kc) polyprotein that is present in large amount in
virus-infected cells. This nonglycosylated protein is subsequently cleaved
to form pl7 (a phosphoprotein), p24, and gag peptides. Although p24 and
ply are detectable in both extracellular virus and disrupted virus-infected
304
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APPENDIX B 305
cells, the 55-kd precursor protein is not present in significant amounts in
virus harvests used to prepare antigen. Antibodies to HIV core protein
p24 and its group antigen precursor are thought to appear earliest
following infection and are readily detected in a number of ELISA tests.
The ens gene encodes for a polyprotein of about 90 kd in its nongly-
cosylated form. Since it has numerous glycosylation sites, it migrates as
a glycoprotein of about 160 kd in electrophoretic analyses and is found as
such in infected cells. This glycoprotein gives rise to two principal
proteins, gpl20 and gp41. Both are present in infectious virus particles
and infected cells. Antibodies to these proteins are thought to appear
somewhat later than core antibodies and are present in most sera from
HIV-infected individuals.
Additional immunoreactive viral gene products include those nonstruc-
tural proteins encoded by sor, a short open reading frame of unspecified
function; tat, which is responsible for certain critical aspects of viral
expression; and 3'-orf, another open reading frame that encodes for a
27-kd protein of unknown function. The proteins encoded by the pol gene
of HIV, which catalyze essential enzymatic processes in viral replication,
are strongly immunogenic and recognized by sera from the vast majority
of infected persons. The distribution of antibodies against the proteins
encoded for by these genes is becoming better understood as newer and
potentially improved tests are being developed using recombinant DNA
technologies to produce specific viral proteins. While all are potential
substrates for improved serologic tests, so far no patterns of serologic
reactivity have been found to correlate with disease stage or prognosis.
The configuration of the ELISA test most frequently employed in
serologic analyses involves coating plastic microtiter wells or plastic
beads with HIV antigen and adding test serum in various dilutions. An
antigen-antibody reaction is detected by the use of so-called second-stage
antibodies, which react with any human antibodies remaining bound to
the viral antigens in the ELISA plate. The second-stage antibodies are
modified to facilitate their detection by conjugation with enzymes such as
horseradish peroxidase or alkaline phosphatase. If antibodies in the
serum tested are bound to viral antigens, then the antihuman antibody will
bind to the antiviral antibodies, if present, and the attached enzyme will
be free to catalyze a chemical reaction after the addition of the appropri-
ate substrate. The extent of the reaction is detected calorimetrically. A
control serum is used. "Positives" are distinguished from "negatives" on
the basis of the relative degree of absorbance of the test serum and
control. Where the cutoff point is set affects the sensitivity and specificity
of the serologic test.
While the ELISA test can identify the presumptive presence of anti-
bodies against HIV in a serum sample, another test, the Western blot
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306 APPENDIX B
analysis, permits the documentation of antibodies to specific viral pro-
teins and thus a more specific level of resolution of serologic reactivity.
The Western blot analysis is basically an immunoelectrophoretic test in
which viral proteins from purified disrupted virus are fractionated by size
using polyacrylamide gel electrophoresis. The fractionated viral proteins
are then transferred to nitrocellulose paper to permit subsequent immu-
nologic detection. Control molecular-weight standards are included to
identify migration of proteins of various sizes. Samples being tested for
antibody are added to the strip, followed by appropriate stages of
inoculation and washing. Enzyme-linked antihuman IgG globulin is added
and incubated. Then an appropriate substrate is added and the enzyme
catalyzes the calorimetric reaction, which detects bound antibody. Again,
the extent of the reaction is measured by the intensity of the color
produced.
There has been considerable variation in what different laboratories
have interpreted as positive in Western blots. In the past the presence of
antibody to the gag p24 alone was considered positive by the Centers for
Disease Control (CDC) criteria. With accumulating experience of HIV
Western blot determination, it became apparent that many patients who
have only this antibody appear to represent false positives. Recently, a
consensus has developed that serum specimens are considered specifi-
cally reactive with HIV if antibody to the following proteins is demon-
strated in the presence or absence of other bands: (1) p24 and p41, (2) p24
and pS5, (3) p41. If only p24 antibody is demonstrated, the reaction is
considered equivocal and must be repeated on the same serum sample.
Other tests for antibody detection include the radioimmunoprecipita-
tion and cytoplasmic or membrane immunofluorescence tests. Both tests
work well but are less well suited for screening purposes and appear to be
more appropriate for use in research laboratories. In good hands, im-
munofluorescent testing is as accurate as ELISA testing, but it is much
harder to standardize and therefore is not used as often.
In the first generation of the ELISA tests made available, the intact
virus was used to detect antibodies. In more recent versions, recombinant
antigens are employed. Even with the first generation of test kits,
accuracy has been very high. Sensitivity and specificity (see below) are in
the 95 to 99 percent range.
PERFORMANCE CHARACTERISTICS OF THE TESTS
In evaluating the utility of a test, the terms "sensitivity," "specificity,"
"prevalence," "predictive value," "gold standard," and "cutoff point"
are often used. The sensitivity of a test is the percentage of infected
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APPENDIX B 307
persons who will have a positive test. This often varies depending upon
the stage or severity of the disease. With HIV infection, seropositivity
appears to rise slowly after initial infection and then to remain relatively
stable.
Estimates of the true sensitivity of the HIV test are hampered by the
lack of a "gold standard" an independent verification of the presence of
infection. For example, if one could isolate virus in all cases, one would
be able to determine the exact sensitivity of a serologic test. In the case
of HIV, however, the virus isolation techniques are far from 100 percent
sensitive. Many investigators calculate sensitivity using small numbers of
samples that are not generally representative. Also, the use of sera from
AIDS patients to determine the sensitivity of a test may be very
misleading if the test is applied to low-risk populations to detect early
infections. Given the better appreciation of reactivity with certain partic-
ularly immunogenic viral proteins, it has become increasingly important
to determine the protein content of the different antigens used for the test
and to relate this to what is being learned about changes in the antibody
profile to various proteins over time.
Specificity is the percentage of uninfected persons who have a negative
test. One would like to have 100 percent specificity, especially for an
infection with the implications of HIV infection. The way the antigen is
prepared may affect specificity, in that one may be measuring antibodies
present in the test sera reacting with cellular products that contaminate
viral preparations. This may be particularly troublesome in patients who
use drugs or have chronic illnesses. Again, failure to use a large number
of broadly representative sera may give falsely high estimates of speci-
ficity. For example, measurements of specificity in well populations may
underestimate the problems encountered when the test is later used in
alternate populations.
The cutoff point is the point above which one calls a test positive and
below which one calls it negative. No test is 100 percent sensitive and 100
percent specific, so one is usually trading off either sensitivity or
specificity. One sets the cutoff point to maximize one or the other,
depending on whether it is important to detect all those who are infected
and then sort out true positives from false positives or whether one is
willing to miss a certain number of true positives in order to minimize the
risk of false positives.
Chapter 4 describes general aspects of the uses of these tests, their
applications in improving the safety of the blood supply, their emerging
use as an indicator of infection in individuals, and the problems associated
with these uses. Chapter 6 contains recommendations regarding desirable
future efforts in this area.
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308 APPENDIX B
BIBLIOGRAPHY
American Medical Association Council on Scientific Affairs. 1985. Status report on the
acquired immunodeficiency syndrome: Human T-cell lymphotropic virus type III testing.
JAMA 254:1342-1345.
National Institutes of Health. 1985. Workshop on experience with HTLV-III antibody
testing: Update on screening, laboratory and epidemiologic conditions. Bethesda, Md.,
July 31.
National Institutes of Health. 1986. Program and abstracts: Impact of routine HTLV-III
antibody testing on public health. Bethesda, Md., July 7-9.
Silberner, J. 1986. AIDS blood screens: Chapters 2 and 3. Science News 130:56-57.
Representative terms from entire chapter:
western blot
