1) A study found that about half of patients treated for HIV early in infection failed to develop antibodies detectable by standard HIV tests or developed antibodies that later disappeared. 2) Current HIV diagnostic tests are designed based on the natural progression of HIV infection without treatment, but early treatment interrupts antibody development. 3) With increased emphasis on rapid treatment even in acute HIV, the limitations of diagnostic tests need to be addressed to accurately diagnose HIV in people on early treatment.

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© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.
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Timing is Everything - Shortcomings of Current HIV Diagnostics in the Early Treatment Era
Sheila M. Keating, Christopher D. Pilcher and Michael P. Busch
Blood Systems Research Institute and Departments of Medicine and Laboratory Medicine, University of
California San Francisco
Corresponding Author
Michael Busch
mbusch@bloodsystems.org
Clinical Infectious Diseases Advance Access published June 17, 2016
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In the current issue of Clinical Infectious Diseases, De Souza et al. [1] present an important study
documenting the challenges in HIV diagnosis using serological assays as ART is increasingly initiated
during acute or early HIV infection. The investigators studied samples from a large cohort of early ART
participants and rigorously characterized performance of 2nd, 3rd and 4th generation screening
immunoassays (IAs) and confirmatory western blots (WB), documenting both non-evolution of antibody
seroconversion and seroreversion due to viral suppression. Although this has been previously
documented in case reports and smaller case series (2), the current study is the first to provide clear
information on how different assays might be expected to perform in clinical practice—and the results are
cause for alarm. They show that about half (46%) of patients treated early in infection may fail to
seroconvert (or only transiently convert, and then revert to negative) on standard-sensitivity IgG antibody
tests for HIV. In the face of universal treatment for HIV infection, currently available tools for HIV
diagnosis need to be reevaluated to determine the impact both on accurate classification of infection on an
individual basis and also how this may impact public perception of and confidence in our ability to
diagnose HIV infection.
Diagnostic assays for HIV infection have evolved dramatically over the past 30 years with increased
sensitivity and specificity, faster turn-around time and ease of performance - all supporting earlier
diagnosis and better entry into continuum of care. Key technologic advances for HIV testing performance
in early infection have been 1) the availability of sensitive HIV nucleic acid tests for direct viral detection
in infant diagnosis and acute infection testing and blood screening programs; 2) the advent of 4th
generation antigen-antibody “combo” tests (which can also detect some pre-antibody conversion acute
infections); and 3) improvement in HIV antibody detection methods (used in 3rd
- generation and 4th
generation assays) so that these detect early IgM-class antibodies. In 2014, the Centers for Disease
Control and Prevention published strong recommendations that all HIV screening use a 4th
generation
test, and this is already common practice in much of the rest of the world (3).
As a result of these changes, most serological assays are expected to convert within 2-4 weeks of initial
viremia. The dynamics of viremia, antigenemia, the maturing antibody response, and test conversion have
been meticulously described by several laboratories in studies of samples from untreated seroconverters
(4). This detailed understanding has made it possible to design the diagnostic algorithms that are now
being used (5). However, there is an emerging understanding that the evolution of HIV biomarkers over
time is profoundly altered by early initiation of ART (Figure 1). As highlighted by the work of deSouza
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and colleagues, HIV tests simply do not work the way they should after HIV treatment has been initiated
very early in infection.
It is not entirely surprising that suppression of viremia during the early phases of infection should alter the
maturation of antibody responses against HIV. Induction and proliferation of pathogen-specific B cell
populations and long-term, stable antibody production from plasma cells may require some level of
continuous antigen exposure (reviewed in (6)). The data from de Souza and colleagues (1) demonstrate
that the induction of HIV antibody responses may require several months of active replication following
acute HIV infection. As illustrated in the Figure, this sort of interruption of antibody maturation explains
why tests requiring higher IgG antibody levels (2nd
generation assays, rapid tests) or greater antibody
diversification (Western blot) are most severely impacted by early treatment. Moreover, de Souza
observed that initial conversion was occasionally followed by seroreversion in the 24 weeks after ART.
The rate at which antibody responses may continue to revert over time is not at all clear.
How seriously should we take this problem? In the past, the issue of how one documents HIV status in
patients with immediately treated acute HIV infection would have been considered esoteric. But it has
now come front and center for clinicians handling new diagnosis of HIV infection, for several related
reasons:
1) Tests for acute and recent infection detection are now used by most testing programs, clinicians are
now commonly being asked to discriminate between patients with true acute infection and those with
false positive/unconfirmed screening tests.
2) Rates of acute HIV are increasing as a proportion of new cases. A recent study of 86,836 consecutive
HIV testers reported surprising rates of antibody-negative acute infections: New York, North Carolina
and San Francisco sites reported that acute infections accounted for 9, 10 and 18 percent of new cases
identified (7). This is almost certainly related to very frequent HIV testing by individuals at risk of HIV,
with 3-6 monthly testing now typical among men who have sex with men in San Francisco.
3) There is a new emphasis on rapid (even same day) treatment initiation, particularly in acute infection.
New guidelines by the International AIDS Society have recommended universal ART for all HIV infected
individuals regardless of CD4 count. In addition, public health programs are aiming to treat acute
infections aggressively and early (8). This is in part because acute HIV infection is a period of high
infectivity related to high genital viral load and may play a special role in the spread of HIV in
populations (9).
4) Rapid intervention in early infection is increasingly seen as important for clinical outcomes. The acute
phase of HIV infection is associated with extremely high viral loads initially infecting gut mucosal CD4
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cells (10), causing inflammation, gut permeability, microbial translocation and immune activation that
further drives viral dissemination (11). Identification and treatment of HIV infection in earlier stages of
acute infection (Fiebig stages I-III) results in preservation of gut-associated immune responses (12),
improves post-treatment CD4 counts and limits seeding of the long-lived viral reservoir (11) - all
suggesting that very early ART may help reduce inflammation and HIV-related co-morbidities over the
long term. For these reasons, ART is considered urgent in acute cases and may be initiated on the same
day of diagnosis, prior to HIV resistance genotyping and at the same time that other aspects of HIV care
are being initiated(13).
5) Adding to the potential for confusion, very large numbers of people at risk of HIV are using pre-
exposure prophylaxis (PrEP) for HIV. If a person is using PrEP while unknowingly infected (PrEP
breakthrough infection), this may also suppress or partially suppress viral replication in early infection,
with unknown impact on test performance in follow-up.
With all of these changes, HIV treatment programs in many places around the globe are being called on to
detect and confirm HIV infections in persons with acute/early infection in the context of rapid treatment
initiation. There is a new and urgent need for wider discussion (and ideally concrete public health
guidance) around the limitations of HIV diagnostic assays in the universal ART era. At the very least,
clinicians must understand that negative results on HIV diagnostic tests may not always be accurate in the
context of suppressive ART. In addition, awareness of the issue should spark discussion and further
research on how best to approach situations where confirmation or documentation of HIV status is needed
in the presence of treatment—where ultrasensitive nucleic acid tests, high-sensitivity antibody assays and
quantitative antibody assays applied to serial samples may each have an important role to play.
Notes
The authors have no reported conflicts of interest.
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References
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HIV-1 Infection leads to a High Rate of Non-reactive HIV Serology. Clin Infect Dis 2016: This issue.
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10. Ananworanich J, Schuetz A, Vandergeeten C, Sereti I, de Souza M, Rerknimitr R, et al. Impact of
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infection. PloS one. 2012;7(3):e33948. Epub 2012/04/06.
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infection. Journal of virus eradication. 2016;2:43-8. Epub 2016/02/19.
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12. Schuetz A, Deleage C, Sereti I, Rerknimitr R, Phanuphak N, Phuang-Ngern Y, et al. Initiation of
ART during early acute HIV infection preserves mucosal Th17 function and reverses HIV-related
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13. Jacobson KR, Arora S, Walsh KB, Lora M, Merjavy S, Livermore S, et al. High Feasibility of
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Immune Defic Syndr. 2016. Epub 2016/03/31.
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Figure 1. After HIV infection, p24 antigen and IgM and IgG antibody seroconversion occur which are
progressively detected by 4th
, 3rd
or 2nd
generation assays over the weeks post-infection. Diverse antigen-
specific responses can be differentially detected during this period by western blot (WB) and other
confirmatory assays. Sustained antigenic stimulation is required for maturation and maintenance of these
antibody responses. Early treatment with ART aborts the development of antibodies if treatment is
initiated very early and subsequent seroreversion may occur if treatment is initiated shortly following
seroconversion, making it difficult to detect or confirm HIV infection by standard diagnostic tests.
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