HIV Diagnostics: New Developments and Challenges
Grosvenor Resort, Orlando, Florida
February 28 – March 1, 2005
Meeting Summary
The CDC/APHL-sponsored, HIV diagnostics meeting was attended by over
200 researchers, laboratorians, and industry representatives to share the
latest information on testing technologies and alternative methods to increase
the uptake of testing and diagnosis of persons with HIV infection, and
to discuss HIV incidence and drug-resistance surveillance. The two-day
meeting included over 50 poster and 39 oral presentations on the following
seven topics: (1) performance characteristics and use of recently approved
HIV enzyme immunoassays; (2) nucleic acid amplification testing (NAAT)
for acute HIV infection; (3) rapid HIV testing in the United States and
international settings; (4) options for confirmatory testing in different
settings; (5) dried blood spot (DBS) testing; (6) tests for recent HIV
infection; and (7) FDA requirements for use of tests for recent infection
for purposes other than surveillance. A brief summary of the content and
themes for each of these topics are presented below. 1. New HIV Enzyme Immunassays (EIA) This session included
presentations on (1) the performance characteristics of the bioMerieux
Vironostika HIV-1 Plus O and Bio-Rad Genetic System’s
HIV-1/2 Plus O, (2) an evaluation of Bio-Rad’s new EIA assay
by the Florida Bureau of Laboratories and by the Maryland Department
of
Health, and (3) a survey on HIV assays currently in use in public
health laboratories. Both of the two new HIV EIAs incorporate 3 (Vironostika)
or 4 (Bio-Rad) antigens including a group O specific antigen for
increased
detection of HIV-1 Group O. One of the antigens in the Bio-Rad assay
is a HIV-2 specific antigen that allows detection of HIV-2. Both
assays incorporate reagents that allow detection of IgG and IgM antibodies.
In the case of the Bio-Rad assay, IgG and IgM are detected by utilizing
the antigen-sandwich approach, while the Vironostika Plus O assay
uses
a goat anti-human immunoglobulin that binds both IgG and IgM. The
Bio-Rad HIV-1/2 Plus O has been available since 2003 and replaces
the Genetic
Systems HIV-1/HIV-2 peptide assay. bioMeriuex’s Vironostika
HIV-1 Plus O should be available in June, 2005 and will replace the
Vironostika
HIV-1 Microelisa system. Compared with their predecessors, both assays
have greater sensitivity to detect (1) lower antibody titers, (2)
persons earlier during seroconversion, (3) Group O infection, and
(4) HIV-2 infection
(Bio-Rad only). However, laboratories that have adopted the Bio-Rad
HIV-1/2 Plus O assay have observed an increase in the number of repeatedly
reactive
EIAs with negative or indeterminate Western blots. The cause of these
repeatedly reactive EIAs with negative or indeterminate blots could
not be confirmed for all specimens. They might be caused by the increased
sensitivity resulting in identification of samples that would be
missed
by less sensitive Western blots or indirect immunofluorescence assays,
or by cross contamination, cross-reaction, or non-specific binding
in the testing procedure. The Maryland Department of Health Laboratory,
for example, found decreased specificity of Bio-Rad’s Plus
O assay when testing cadaveric samples when compared to the Bio-Rad
HIV 1/2 peptide
assay. The Florida Bureau of Laboratories recommended that laboratories
that use Bio-Rad’s Plus O assay increase monitoring and adopt
procedures to prevent cross contamination which may produce higher
false-positive
rates. In a survey of 56 public-health laboratories, 63% of responders
currently use the Vironostika HIV-1 EIA, (the assay least sensitive
for early seroconversion); 10% use the Bio-Rad HIV1/2 Plus O, and
22% reported
that they intend to change EIAs in 2005.
2. Nucleic Acid Amplification Tests (NAAT) for Acute HIV Infection This
session included presentations on the application of NAAT for donor
screening and for publicly funded HIV testing in domestic and international
settings, and an overview on CDC’s new primary HIV infection (PHI)
study. The feasibility of screening for acute HIV infection using NAAT
on pooled seronegative specimens has been repeatedly demonstrated in
different settings and in multiple countries. However, the cost-effectiveness
of detecting acute HIV infection through NAAT varies considerably by
setting and is expected to change with adoption of third-generation,
highly sensitive EIAs and use of rapid HIV tests that obviate serum collection
for those who test negative. Over the first three years of NAAT screening
for U.S. blood donors, for example, only 12 acute HIV infections were
identified in 37 million donations, corresponding to >$1,000,000 cost
per quality-adjusted life year. In Maryland, NAAT screening (in-house
RT-PCR) of over 15,000 pooled specimens seronegative by the 2nd-generation
Genetic Systems HIV-1/HIV-2 assay has not identified any acute infections,
possibly because of increased availability and uptake of oral fluid and
rapid HIV testing by high-risk persons. However, NAAT screening of clients
tested in public-health clinics in San Francisco and North Carolina who
were antibody negative by the 1st-generation Vironostika identified 10%
and 4%, respectively, more HIV infections than would have been identified
by using the Vironostika assay alone. Identification of acutely infected
persons and referral of their partners may be a particularly important
prevention strategy if, as suggested by some studies, a high proportion
of HIV transmission is attributed to persons with PHI. CDC’s
new PHI study is expected to begin in the summer of 2005 and will
be conducted
by health departments in Florida and Los Angeles, in collaboration
with the Florida Bureau of Laboratories and the New York State Wadsworth
Laboratory
(respectively). The primary objective of this study is to determine
the yield and cost effectiveness of pooled NAAT in addition to antibody
screening,
coupled with partner notification and referral efforts to detect
HIV infections that would have remained undiagnosed with conventional
2nd
and 3rd generation EIA screening. 3. Rapid HIV Testing in the United
States and International Settings The session on domestic testing
included presentations on implementation barriers and outcomes of
rapid testing by several state and local
health departments. The implementation of rapid HIV tests in public-health
settings has varied considerably by state and local health department
due
to both
state regulatory requirements and system-development needs. State
regulations that require phlebotomy training for personnel performing
finger-stick
rapid-tests (California) or that CLIA-waived laboratories have
lab directors with a PhD (Pennsylvania) were barriers for implementing
CLIA-waived
rapid tests in community-based organizations. However, system
development needs such as revisions to state and local laboratory forms,
operator
training, and establishing effective quality assurance programs
were, by far, the most important implementation barriers. Addressing
these
needs requires considerable planning and resources, and as a
consequence, roll out of rapid testing in the United States has taken
time but
has been successfully implemented in several states. The number of
rapid tests performed by the Florida Department of Health (FDOH),
for example,
increased from approximately 4,000 in 2003 to approximately 24,000
in
2004. In 2005, FDOH expects to conduct between 40,000 and 50,000
rapid
HIV tests. Data presented from New York State and Florida, and
from CDC’s
OraQuick post-marketing surveillance project suggest that (1) most
persons seeking HIV testing services prefer rapid over conventional
tests; (2)
nearly all persons who are tested with rapid HIV tests receive
their results; (3) most health-department rapid HIV test programs have
instituted
effective quality assurance programs; and (4) rapid tests have
performed as expected with very few invalid and false-positive tests
reported.
As a consequence of the successful roll out of rapid testing, however,
some laboratories have had a reduction in the number of conventional
HIV tests performed. The potential reduced need for public health laboratory
HIV testing and corresponding loss in funding should be monitored
closely to mitigate any impact on requisite infrastructure.
Presentations
on
rapid HIV testing in international settings described the implementation
and performance of rapid testing in resource poor countries.
Rapid testing in CDC supported Global AIDS Program countries
has expanded quickly from a total of less than 250,000 tests conducted
in approximately
250 sites in 2002 to approximately 2,000,000 tests conducted
in 1800
sites in 2004. Of the 15 targeted President’s Emergency Plan
for AIDS Relief (PEPFAR) countries, 8 have implemented rapid testing
in over
600 sites.
In these 8 countries, a sequential rapid testing algorithm is currently
used in 5 and a parallel testing algorithm is used in 3. In 4 countries
with quality assurance programs that involved re-testing in reference
laboratories, concordance between on-site rapid testing and reference
laboratory re-testing
ranged from 95.7% to 99.5%. However, roll out of rapid testing
in PEPFAR and other resource poor countries has identified considerable
laboratory
infrastructure and training needs to develop and sustain effective
quality-assurance programs. For example, only one of the PEPFAR countries
has implemented
an external proficiency-testing program for rapid test operators.
In 6 external assessment exercises conducted in 29 Asian and Pacific
countries
by the national reference laboratory in Australia, rapid tests
produced
higher error rates than EIAs. Follow-up visits to participating
laboratories indicated insufficient documentation of laboratory procedures,
insufficient
use of external controls, and a lack of understanding and documentation
of quality-assurance principles and practices. 4. Options
for Confirmatory Testing This session included presentations
on potential alternative HIV testing algorithms that (1) reduce
the use of the Western
blot
(WB) by using
NAAT and highly sensitive
EIAs for supplemental confirmatory testing, (2) eliminate the WB
by using rapid tests in combination, and (3) use a combination
of
the Multispot
rapid test
and HIV-1 and HIV-2 EIA/WB assays to detect HIV-2 infection. Two
studies (CDC and American Red Cross) indicated that including
NAAT within
an HIV testing
algorithm increases sensitivity by detecting acute HIV infection
and reduces the need for the WB and the number of indeterminate
WB
results given to
non-infected persons. However, because some HIV-infected persons
have non-detectable viral
loads, both studies suggested that NAAT cannot completely replace
the WB as a supplemental confirmatory test. The CDC study, for
example,
found
that as
many as 3.3 % of serology positive individuals were NAAT negative.
Of 12.4 million American Red Cross blood donations from 1989
through 1999, 11,080
were EIA repeatedly reactive (RR), and of these, 7.1% were WB
positive, 46.6% were
WB indeterminate, and 46.3% were WB negative. The high-rate of indeterminate
and negative WB results is attributed, in part, to the FDA-required
interpretation
for blood donation screening (any WB background discoloration or
band must be reported, as opposed to only viral bands). Because
of the
high rate
of WB negative and indeterminate results, an alternative algorithm
for blood
donors
has been proposed to the FDA. The algorithm involves an initial EIA
screen; individual NAAT on donations that are EIA-RR; and pooled
NAAT on all
seronegative donations with discriminatory NAAT on deconstructed
pools to identify RNA-positive
donors. Donations that are EIA-RR/NAAT positive are considered infected
(WB is not performed). EIA-RR/NAAT-negative donations are tested
with a second
EIA using a different antigen. If the second EIA is negative, the
patient is considered to be non-infected (WB is not performed).
Western blots are only used to resolve donations that are EIA-RR
by the screening EIA,
NAAT-negative,
and EIA-RR by the second EIA. The new algorithm would improve sensitivity
by identifying donors with early infection, and eliminate 98.5%
of WB tests and
98.6% of indeterminate results (and unnecessary anxiety) given to
non-infected donors.
A prospective evaluation of rapid tests
conducted by the CDC and
the Los Angeles Department of Health Services demonstrated the
sensitivity and specificity of individual rapid tests. On the
basis of these results,
simulated algorithms were developed using up to 3 different rapid
tests. Each was as sensitive and specific as the conventional
EIA/WB algorithm
used for comparison. In this study, persons who tested negative
on the first rapid test were considered to be noninfected (no
further tests
performed).
If the first rapid test was positive, a second different rapid
test was performed. If both tests were positive, the patient
was considered to
be infected (no further tests performed). Finally, a third rapid
test (different
from the first two) was used if only the first two rapid test
results were discordant. For these discordant cases, persons
who tested positive
on
2 of 3 rapid tests were considered to be infected; persons who
tested negative on 2 of the 3 rapid tests were considered to
be noninfected.
The algorithm
was evaluated using 10 combinations of up to 3 different rapid
tests (2 evaluated using whole blood only, 1 using whole blood
and oral fluid).
Compared with the conventional EIA/WB algorithm, all 10 combinations
of
the rapid-test algorithm yielded sensitivities > 98% and all
were 100% specific (no false positives).
The final presentation
was on a
new HIV
testing algorithm used by the NYC Public Health Laboratory
(PHL), which was instituted because of increasing
concerns about HIV-2 infection. The average annual number
of HIV-2 infections by the NYC-PHL was 5 from 1988 through
1993
(mean 158,959
specimens tested/year),
12 from 1994 through 1997 (mean 156,020 specimens tested/year),
and 38 from 1998 through 2004 (mean 120,200 specimens tested/year).
Instituted
in April 2004, the new HIV-1/2 diagnostic algorithm involves
(1) screening all specimens with the Bio-Rad EIA HIV-1/2 Plus
O followed
by an HIV-1
WB on EIA-RR specimens; (2) using the MultiSpot rapid test
and HIV-2 EIA/WB algorithm on HIV-1 WB positive specimens,
and (3)
using a
second different HIV-1 EIA (Bio-Rad rLav) and HIV-2 EIA/WB
algorithm on WB
negative
or indeterminate specimens. 5. Dried Blood Spot (DBS) Testing This session included
presentations on the use of DBS testing for (1) the Newborn Screening
Quality Assurance Program (NSQAP),
(2) HIV surveillance
and research in Canada and internationally, (3) the diagnosis
of HIV
infection in infants through detection of HIV DNA and RNA,
(4) BED-CEIA-based detection of recent infection, and (5) HIV
subtype and
drug-resistance
testing. Since 1989, the NSQAP has conducted routine evaluations
of new filter paper lots to ensure uniformity of testing, and
produces, distributes,
and evaluates DBS quality control and proficiency testing
materials. Currently 49 laboratories in 18 countries participate
in the
NSQAP quality
assurance program for HIV testing. Multiple studies presented
suggest that, provided they are adequately collected, allowed
to dry, and
subsequently protected from heat and humidity, dried blood spots
are stable and
can be used for multiple applications for HIV surveillance (prevalence,
BED-CEIA-based
incidence estimation, and subtype determination) and clinical
management (viral load and drug resistance). Because of their
functional versatility,
low expense, and ease of use, transport, and storage, dried
blood spots offer considerable advantages over conventional serum
or plasma
collections.
In Canada, for example, DBS is used in multiple studies assessing
HIV prevalence, incidence, drug resistance, and subtype infection
among injection drug users and men who have sex with men. DBS
may also play
an
important
future role in the detection and clinical management of HIV-infected
infants in Africa and Asia. CDC has developed a magnetic
bead-based, real-time reverse transcriptase, polymerase chain reaction
(PCR) assay used to detect both HIV-1 DNA and RNA from dried
blood
spots
obtained
from infants. Test results of the real-time PCR were 99%
concordant with conventional NAAT (Amplicor). The per test cost of
the real-time
PCR is $5 (U.S.), considerably less than commercially-available
NAAT. Technology
transfer of the test is currently underway in Uganda, Kenya,
Thailand, and South Africa. 6. Status of Tests for Recent
Infection This session included presentations on (1)
preliminary results from a less sensitive (LS) incidence-assay
approach for a simple
and inexpensive
particle-agglutination assay, (2) a proposed weighting
mechanism for
assay-based HIV incidence estimation, (3) an evaluation
of the comparability
between two Vironostika LS assays, and (4) the calibration
of the BED incidence-assay for multiple HIV subtypes. The Serodia
HIV-1/HIV-2
particle-agglutination (PA) assay was modified to act as an
LS assay. At
a dilution of 1:58,000,
134/152 (88%) of samples from recently infected persons
were correctly
identified by the PA assay. The study demonstrated the
possibility of modifying an inexpensive, commercially available
rapid test
in resource poor countries to classify persons with recent
HIV infection
and
for incidence surveillance. However, use of routinely collected
sera for
incidence estimation is dependent, in part, on the completeness
of testing
from all HIV positive persons. In a study conducted in
the Caribbean, from 36 to 75% of all HIV-positive samples were
not
available
for incidence testing. A model constructed by the Institute
of Human Virology,
Baltimore
MD, suggests that incidence estimates are subject to a
significant downward bias when > 50% of positive specimens
are unavailable for incidence testing. To adjust for missing
incidence test results, a simple weighting
procedure was proposed that adjusts the denominator of
the incidence estimator by the proportion of positive specimens
available for testing
(i.e., # EIA-negative specimens multiplied by the proportion
of available positive specimens). The weighting mechanism is
consistent with that
in the CDC’s protocol for incidence calculations.
When reporting assay-based incidence estimates, however,
researchers
should describe
the number of HIV-positive specimens that were unavailable
for incidence testing and the potential downward bias of
reported estimates if
many positive specimens were unavailable. Because the Vironostika
Plus O
(VPlusO) assay is expected to replace the Vironostika MicroElisa
System in 2005,
CDC evaluated the comparability of the less-sensitive versions
of both assays (VPlusO-LS vs. V-LS) using 1009 specimens
obtained through
the
HIVNET Infected Participant Cohort study. In this study,
VPlusO was 90.2% concordant with V-LS (472 specimens classified
as
incident
by VPlusO-LS
vs. 459 specimens classified as incident by V-LS). Both
assays yielded
similar window periods for detecting recent infection (212
days for V-LS; 196 days for VPlusO). Finally, the BED capture
EIA
(BED-CEIA)
incidence
assay has been calibrated for several non-B HIV-1 subtypes
using seroconversion panels obtained from Thailand, Kenya,
Ethiopia,
and Zimbabwe. These
evaluations suggest small differences in the window period
with different subtypes
with overlapping confidence intervals, such that a consensus
window period of 153 days based on an optical density cutoff
of 0.8 can
be used for
incidence estimations. In three validation studies, BED-CEIA
incidence estimates were comparable to observed incidence
in 3 prospective
US and international cohorts. 7. FDA Requirements
for Use of Tests for Recent Infection This session
included discussion on FDA requirements for tests used
for recent infection for research purposes
and
for public-health
purposes
other than surveillance. Research purposes, for example,
would include assays used to identify persons eligible
for the National
Institutes
of Health Acute Infection and Early Disease Research
Program. Public-health purposes would include assays
used to concentrate
partner notification
efforts on persons who were identified as recently infected.
Results of assays used for these above purposes involve
clinical management
and reporting of results to individuals and providers,
and thus are required
to have an FDA investigational new drug (IND) or investigation
device exemption (IDE) designation. The current BED-CEIA
incidence assay manufactured
by Calypte is not subject to IND/IDE requirements when
used for population-based surveillance purposes. The
current version
of
the Vironostika LS-EIA
could be suitable for identifying individuals with acute
infection, especially if lower cutoffs are used to avoid
misclassification
of individuals who
are beyond the acute stage. Further validation would
be needed to calibrate the Vironostika Plus-O assay for
such
a purpose.
During the session,
both Calypte Biomedical and bioMerieux indicated they
would consider applying to the FDA for an IDE to permit
these
other uses of the assays. |
