Nucleic acid amplification testing for diagnosis of acute HIV infection: has the time come?

Two articles, one by Stekler et al. in this issue of AIDS ^[1] and the second by Pilcher et al. in the New England Journal of Medicine, ^[2] are the most recent in a series of studies applying nucleic-acid amplification testing (NAT) to diagnose pre-seroconvesion window-phase HIV-1 infections ^[3-8]. We review key observations from these studies, and address issues impacting expanded use of NAT in donor screening, public health surveillance and diagnostic screening settings.

p24 antigen assays and subsequently RNA NAT, were implemented for blood and plasma donor screening in the early and late 1990s, respectively ^[9,10]. The yield of positive viral p24 antigen or RNA samples of US blood donors has been small (1 in 5 million donations screened for p24 antigen, and 1 in 2 million for HIV-RNA), confirming predictions based on incidence-window period models ^[9-12]. Despite poor cost-effectiveness in low HIV-1 incidence donor settings, HIV-1 (and hepatitis C virus) NAT of blood donors is now routine in most developed countries due to political and legal repercussions of 'breakthrough' transfusion-transmissions ^[13]. Several developing countries with serious HIV epidemics and residual transfusion risk are in the process of implementing NAT for donor screening as well ^[14-16].

RNA screening using NAT assays has played an important role in research studies of acute and early HIV infection ^[7,17-19]. For example, the US AIEDRP study group employs NAT screening as well as Sensitive/Less-Sensitive (S/LS) enzyme immunoassay (EIA) testing ^[20] to identify subjects for protocols investigating natural history, pathogenesis, and therapeutic strategies in early HIV infection (see http://www.aiedrp.org/publP.asp for publications from the group). Other groups have explored the public health importance of HIV-1 NAT as a supplement to antibody testing ^{[1,2,17,20-24]}. There is growing evidence that acutely infected individuals are very infectious due to high-titer viremia in plasma and genital fluids, and the absence of immune factors that may neutralize infectivity ^[17,21]. Several groups have estimated that a majority of new HIV infections result from individuals in the acute viremic phase ^[23,24]. As discussed by Stekler et al. ^[1] and Pilcher et al. ^[2], detection of subjects with acute infection may allow interdiction of transmission networks.

Early studies of diagnostic and public health utility of NAT used HIV-1 viral load assays that are expensive, not optimized for specificity, and not approved by the Food and Drugs Administration (FDA) for diagnostic testing ^[2-8,21,22]. Development of qualitative NAT systems for donor screening has dramatically changed this situation. These newer NAT systems are optimized for high-throughput, sensitivity and specificity, and designed and approved for application to individual samples or in minipool (MP) testing algorithms ^[25]. MP testing, which is used for donor screening, dramatically lowers the cost of NAT by decreasing the number of specimens that require testing. Use of MP-NAT seems appropriate for surveillance and diagnostic testing for acute infection, given accumulating evidence that most persons diagnosed with acute infections in high risk screening settings have high-level viremia ^[1-7]. This was clearly the case in the study by Stekler et al. ^[1], in which both minipools of 30 samples and individual samples were tested using a sensitive donor screening NAT assay; all seven viremic-seronegative samples were detected by the MP-NAT strategy.

Given these advances and potential benefits, should we be incorporating NAT into routine HIV testing? Although the recent reports are intriguing, we believe further study is needed to define when these tests are best used, the benefits of using such testing, and the cost-effectiveness of NAT compared to alternatives. In Stekler and colleague's study, 0.2% of persons with negative antibody tests were found to have HIV by NAT, representing 7% of those found to have HIV ^[1]; the corresponding numbers in Pilcher's study were 0.02% for positive tests, representing 4% of HIV infected persons ^[2]. The yield of NAT testing was low in both settings, although it represented a significant proportion of all persons tested who had HIV. The yield of NAT was 10 times higher in Stekler's study, which tested a higher risk population of men having sex with men. Over two-thirds of the NAT positive tests in Pilcher's study were in sexually transmitted diseases (STD) clinics. This finding suggests that strategies that target testing for acute HIV infection in high-risk settings should be considered.

Other testing approaches also deserve consideration. There have been substantial improvements in the sensitivity of serum-based serological assays. Third-generation EIA that are capable of detecting IgM antibodies, implemented by blood banks in the early 1990s, have recently been used for diagnostic screening. As the viremic pre-seroconversion window period is narrowed by these assays, the incremental yield of direct virus assays is reduced. This fact was demonstrated by Stekler et al. ^[1], in which two of four 'yield' samples evaluated were antibody positive by a third-generation EIA. Fourth-generation assays, which detect antibodies as well as p24 antigen, are likely to increase identification of acute HIV infection even further ^[14]. Thus, in the setting of increasingly sensitive serological assays, S/LS EIA strategies may prove more useful and cost effective than NAT for detecting acute infections ^[20]. Finally, with recent FDA-approval of rapid assays for testing presence of HIV antibodies using oral fluid, an increasing proportion of HIV diagnostic screening is being performed on non-blood specimens. NAT assays have not been optimized or approved for such samples, so it is unclear how RNA screening can be accomplished as diagnostic testing shifts to field-based rapid tests.

The benefits of detection of acute HIV infection also remain to be better identified. Picher's study found that detection of acute HIV helped protect 48 sex partners and one fetus from high-risk exposure to HIV ^[2], reinforcing the potential benefit in decreasing HIV transmission. Randomized, controlled trials as well as observational data in the AIERP network and other settings promises to define better the benefits of very early treatment on the subsequent disease course. The Centers for Disease Control and Prevention has recently launched a multicenter study to address other unresolved issues, including yield in various incidence setting, pros and cons of MP versus individual sample NAT, and adaptation of NAT to non-blood specimens. Together, these studies promise to define optimal approaches to detect and manage acute HIV infection in conventional HIV testing settings.