Early diagnosis of HIV infection through analysis of oral fluid [Pre-Clinical]
1. A novel method – Antibody Detection by Agglutination-PCR (ADAP) – was developed to diagnose HIV in oral fluid samples.
2. ADAP is both highly sensitive and highly specific and may allow noninvasive early diagnosis of HIV in population-based screening efforts.
Study Rundown: Despite the high infectivity of the HIV virus during early infection, there is currently no rapid, non-invasive method for detecting viral infection at this stage. Current methods for diagnosing HIV infection rely on detection of HIV antibodies in blood samples. However, collection of blood samples is invasive and cumbersome, and requires special handling considerations. On the other hand, saliva is easily collected and is a non-infective fluid, thanks to low viral loads and the presence of natural inhibitors in oral fluid (OF). Historically, OF HIV detection has proven difficult due to low antibody titers in OF compared to blood. However, a new method – the ADAP test – represents a novel approach to detect multiple low-affinity HIV antibodies in OF with high sensitivity and specificity.
Like other HIV detection assays, ADAP relies on the formation of antigen-antibody complexes. However, the sensitivity of ADAP is much greater than other assays due to a subsequent DNA amplification step that is dependent on the formation of antigen-antibody complexes. The capability of these viral protein-DNA conjugates to identify HIV antibodies in OF samples was tested on OF samples from a California Public Health Screening program. ADAP was able to correctly classify HIV-negative and HIV-positive samples with 100% accuracy. Moreover, this test was able to identify HIV-positive samples that had been deemed inconclusive by other methods.
As a non-invasive and highly sensitive and specific test, ADAP has the potential to meet a crucial clinical need in HIV diagnostics. It remains to be seen whether ADAP will prove feasible and cost-effective in large population-level early HIV-detection programs.
Relevant Reading: How can we better identify early HIV infections?
In-Depth [in vitro assay]: Recombinant HIV proteins, p24, gp41, gp120, and gp160 were used as substrates for DNA conjugation. These antigen-DNA conjugates were generated through lysine modification and reaction with thiolated nucleotides, One antigen-DNA conjugate bears the 5' half of a PCR amplicon, while another conjugate bears the 3' half. These pooled conjugates were incubated with 1 µL OF and incubated to allow antibody binding. DNA ligase and a bridge oligonucleotide were subsequently added. Ligation into a full-length amplicon is dependent on the formation of an immune complex. A subsequent qPCR reaction allows the quantification of the amount of immune complex present in the OF sample.
The ability of these antigen-DNA conjugates to bind and detect antibody was tested on a panel of purified human p24, gp41, and gp160 antibodies. Using ADAP, a concentration-dependent increase in detection was observed. Compared with a common enzyme-linked immunoassay (EIA) used in many clinical setting, ADAP had over one thousand fold increased analytical sensitivity.
The accuracy of ADAP in correctly discriminating HIV-positive and HIV-negative samples was assessed through ADAP analysis of 22 EIA positive and 22 EIA negative samples. ADAP was found to be 100% sensitive and specific. When comparing the signal intensity between these two assays, a high correlation was noted (R= 0.80, p< 0.05). ADAP was then modified to simultaneously evaluate 3 antibodies in one test. This was accomplished by generating each antigen-DNA conjugate with unique DNA barcodes, resulting in antibody-conjugate discrimination. The same analyses as described above were performed, with a similar 100% sensitivity and specificity.
Finally, ADAP was tested on 8 samples that had previously been deemed inconclusive by EIA. ADAP was able to reclassify 6 out of 8 samples as HIV positive due to the presence of two or more HIV antibodies. One of these patients was confirmed HIV-infected by a follow-up blood test. These results suggest that ADAP might be superior to current methods for its ability to detect low levels of multiple HIV antibodies in OF samples.
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