Antibody detection by agglutination–PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis

Antibody detection by agglutination–PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis

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Oral fluid (OF) is a highly effective substrate for population-based HIV screening efforts, as it is noninfectious and significantly easier to collect than blood. However, anti-HIV antibodies are found at far lower concentrations in OF compared with blood, leading to poor sensitivity and a longer period of time from infection to detection threshold. Thus, despite its inherent advantages in sample collection, OF is not widely used for population screening. Here we report the development of an HIV OF assay based on Antibody Detection by Agglutination–PCR (ADAP) technology. This assay is 1,000–10,000 times more analytically sensitive than clinical enzyme-linked immunoassays (EIAs), displaying both 100% clinical sensitivity and 100% specificity for detecting HIV antibodies within OF samples. We show that the enhanced analytical sensitivity enables this assay to correctly identify HIV-infected individuals otherwise missed by current OF assays. We envision that the attributes of this improved HIV OF assay can increase testing rates of at-risk individuals while enabling diagnosis and treatment at an earlier time point.

Eliminating HIV from the human population will require innovative diagnostic and therapeutic strategies (1). Currently, large-scale population screening efforts remain the most effective public health mechanism to identify and funnel HIV-infected people to treatment (1). Early identification of newly infected individuals permits the timely initiation of antiretroviral therapy (ART) to reduce transmission rates and improve health outcomes (1). During this “acute” period immediately following infection, patients are up to 26 times more infectious and over 50% of new transmissions are thought to occur in this window (2, 3).

While blood-based assays efficiently diagnose HIV infection during the acute phase, these tests suffer from poor compliance rates due to their invasive nature (4, 5). In contrast, noninvasive assays such as oral fluid (OF) antibody tests have higher levels of compliance but lack the analytical sensitivity to detect very low levels of antibodies in the OF of acutely infected individuals (610). Currently, no existing test meets the pressing medical need to noninvasively detect HIV during acute infection, which is essential to maximize the number of people screened and to intervene at the earliest time.

HIV tests that analyze easily collected OF increase the numbers of individuals tested in situations where needle-mediated blood drawing is inefficient or unsafe (4, 5). The use of oral specimens has facilitated testing in many populations including (i) populations for whom it is inconvenient or unsafe to test using needles (e.g., prisons), (ii) patients whose veins are difficult to draw from (e.g., drug users, infants), and (iii) people who are averse to having blood drawn using needles (e.g., children, adolescents) (1012). Furthermore, antibodies in OF are stable for several weeks at ambient temperature, thus decreasing the likelihood of false results when cold chain shipping is not available (13, 14). Finally, OF is much safer to handle on a large scale, as HIV cannot be transmitted by OF thanks to significantly lower viral loads and the presence of naturally occurring enzymes and other inhibitors that deactivate the virus (15). OF therefore represents an ideal sample type for large-scale screening of HIV incidence in many groups, including those hard-to-reach populations.

Antibodies are the most reliable markers of HIV infection in OF (16). While HIV-derived RNA and proteins (i.e., p24) are considered powerful blood-borne markers for detecting early infections, these HIV components do not consistently appear in OF (16). Thus, assays that measure HIV RNA by quantitative PCR (qPCR) or HIV protein (p24) by enzyme-linked immunoassays (EIAs) are unsuitable for OF screening. By contrast, HIV-associated immunoglobulins are reliable markers of infection in OF (17, 18). Indeed, detection of OF IgG in EIA-type formats forms the basis of the FDA-approved OraQuick test. Unfortunately, however, this test cannot detect disease until at least 40 d after infection (18). This unacceptably long window period is attributed in part to the ∼1,000-fold lower antibody concentration in OF relative to serum/plasma (19). As a point of comparison, blood-based tests can detect infection as soon as 14–25 d after exposure (20). The diminished antibody titers along with much lower antibody production in the early phase of the disease pose significant analytical challenges for current HIV OF antibody tests (19).

Here we report an ultrasensitive OF HIV antibody detection method based on Antibody Detection by Agglutination–PCR (ADAP) technology (Fig. 1) (21). The ADAP platform, similar in nature to proximity ligation assay (PLA) (22), leverages multivalent binding of antibodies to drive the agglutination of antigen–DNA conjugates. The induced proximity enables ligation of DNA fragments to form a full-length DNA amplicon, which can then be quantified by qPCR. As reported previously, this amplification permits detection of antigen-specific antibodies at high zeptomole levels in 1-μL samples (21). Since ligation is only triggered following a productive antibody–antigen interaction, ADAP does not require washing steps to remove unbound antigen–DNA conjugates and is thus well-equipped to detect low-affinity antibodies. Furthermore, ADAP can detect antibodies of any isotype, including IgM, the earliest antibody marker of acute infection (20). Importantly, DNA barcoding allows multiplexing by linking the identity of each antibody to a unique DNA sequence. Thus, antibodies specific for multiple antigens can be detected in a single sample.