Advances in Early HIV Detection

Early diagnosis is critical, but fraught with challenges including the widespread use of PrEP

Carolyn Yanavich, PhD

Carolyn Yanavich, PhD, has over 20 years of experience in laboratory science and clinical research. She has worked in the field of HIV and infectious disease for most of her...

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Published:Jun 03, 2020
|Updated:Jan 27, 2023
|4 min read

Acute HIV-1 infection disproportionately contributes to novel transmission and accounts for 24-50 percent of new infections. Despite global advances in antiretroviral treatment (ART) and prevention strategies, including the expanded access to pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP), approximately two million new HIV-1 infections occur annually. Early diagnosis is the key to ensuring patients are quickly aligned with both treatment options and prevention strategies to reduce the risk of further infections.

Acute HIV infection

Acute infection is defined as the disease period that occurs immediately following HIV-1 transmission. During this phase of disease, only the presence of HIV RNA and p24 antigen (an HIV core protein) are detectable; HIV-specific antibodies are not yet detectable. The acute phase of the disease typically lasts from two to four weeks. 

Historically, the standard for diagnosis of acute infection is typically performed using a combination of serological and molecular-based testing strategies. The US Centers for Disease Control and Prevention (CDC) and the Association of Public Health Laboratories currently recommend the use of a laboratory-based antigen/antibody serological assay, followed by an HIV-1/HIV-2 antibody differentiation assay and a nucleic acid test (NAT) for the detection of HIV RNA. 

Clinical Lab Manager Infectious Disease Research Survey  

The journey to fourth-generation assays

The landscape of HIV testing has significantly evolved since the development of the first-generation test, which tested for IgG and was used only as a blood screening tool rather than for HIV diagnosis. Later, the second-generation of testing added recombinant antigens, and the third-generation HIV tests added IgM detection and reduced the post-infection detection window to approximately three weeks. Currently, the preferred laboratory-based serological assays to screen for acute HIV-infection are those that are capable of detecting not only HIV 1-/HIV-2 antibodies but HIV viral proteins (p24 antigen), as the p24 antigen is detectable as early as 14 days post-exposure. These combination HIV antigen/antibody tests are often referred to as fourth-generation assays. If an antigen/antibody test is repeatedly reactive, the sample is reflexed for testing using an antibody differentiation assay. If the antibody differentiation assay is negative, a diagnosis of acute HIV infection is suspected. 

Fourth-generation tests can detect 80 percent of HIV infections in the acute phase that would otherwise only be detected using molecular-based testing strategies (e.g. HIV RNA PCR). Because of their high specificity, fourth-generation assays are unlikely to produce false positive results. Examples of fourth-generation assays include: Abbott Architect HIV Ag/Ab Combo, ADVIA Centaur HIV Ag/Ab Combo, Genscreen ULTRA HIV Ag-Ab, Bio-Rad Combo Manual, and Bio-Rad Combo Evolis. There are two additional tests on the market—Bio-Rad BioPlex 2200 HIV Ag/Ab and Elecsys HIV Duo—which are capable of independently reporting the result of each individual marker, rather than a combined result as seen with the other platforms.

Although rapid, point of care, fourth generation antigen/antigen testing is available, the current US Centers for Disease Control guidelines recommend the use of laboratory-based testing platforms using a blood sample if acute infection is suspected due to the increased sensitivity of the assays. 

While serological assays have the advantage of both a rapid turnaround time and a lower cost, molecular based assays have increased sensitivity, are virus specific, and can detect the presence of HIV earlier. For patients suspected of an acute HIV infection, NAT is often performed in conjunction with the serological assay as HIV-1 RNA is the earliest marker of infection and may be detected as early as five to 10 days post-infection. Within the US, there is only one FDA-approved NAT for HIV-1 diagnosis, which is the APTIMA HIV-1 RNA Qualitative Assay. 

Outside of the US, novel on-demand molecular-based diagnostic testing platforms have been developed for acute HIV-1 diagnosis. These platforms satisfy a global need for earlier, near real-time detection strategies, particularly for those global regions with a higher disease burden and incidence rates that need alternative testing strategies. Testing platforms such as the Cepheid GeneXpert HIV-1 Qualitative and the Alere q HIV-1/2 detect, offer total nucleic acid-based testing using small volume blood samples and diagnostic results at the bedside in about one hour. 

Diagnostic challenges with PrEP use

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Acute HIV infection is notoriously difficult to diagnose due to the non-specific disease symptoms. As the use of PrEP increases globally, so do the challenges of identifying acute HIV-1 infection. Although the risk of acquiring HIV-1 is significantly reduced with PrEP, it is suspected that in those who do acquire HIV, despite PrEP use, may have delayed seroconversion and atypical serology test results. The presence of the antiretroviral medications used in PrEP may alter the disease development cycle by suppressing viral replication and the development of antibodies. While ambiguous results seem to be rare, testing algorithms for those receiving PrEP should continue to be evaluated.

It is important to rule out acute infection prior to starting PrEP to avoid the risk of developing resistance to antiretroviral medications. Prior to initiating PrEP, current IAS-USA guidelines recommend testing with an antigen/antibody assay in combination with a NAT if acute infection is suspected with repeat serological testing performed within a month. Once PrEP is initiated, frequent HIV testing remains necessary. 


Identification of acute HIV infection is challenging, but a suite of diagnostic tests is available globally to aid clinicians in managing patient care. With the advent of PrEP, we can anticipate that our existing testing algorithms will evolve to further expedite the identification of novel infection. 

Carolyn Yanavich, PhD

Carolyn Yanavich, PhD, has over 20 years of experience in laboratory science and clinical research. She has worked in the field of HIV and infectious disease for most of her career in university, clinical research organizations (CRO), pharmaceutical, and government research settings. Her research interests are HIV-infection and associated co-morbidities and the role of the human microbiome.


DiagnosticsHIV diagnosisInfectious Diseasemolecular diagnosticsPublic HealthViruses