CLN Article
Clinicians and laboratorians face several practical challenges when following the laboratory HIV testing algorithm (Figure 1A) recommended by the Centers for Disease Control and Prevention (CDC), which we have observed results in longer wait times for diagnosis and treatment. We instead recommend a two-step algorithm that would be easier for labs to follow and would shorten time to diagnosis. This could help to ensure that patients are not lost to follow up, while also decreasing the risk that they might unknowingly transmit the disease.
Figure 1. (A) Current CDC recommended laboratory HIV testing algorithm and (B) our proposed two-step automatically reflexed algorithm. Here, we compare the current CDC algorithm to our recommended two-step algorithm, which was developed based on our multi-institutional experience demonstrating incomplete testing, inconclusive Ab-differentiation results, and significant delays in time to diagnosis. Importantly, in both algorithms, the final step of HIV diagnosis prior to initiation of antiretroviral therapy is NAT confirmation, irrespective of the Ab-differentiation result.
The CDC’s HIV testing algorithm, established in 2014, involves a three-step testing process that uses a highly sensitive fourth-generation HIV-1/2 antigen/antibody combination screening immunoassay (Ag/Ab-screen) with high negative predictive value. It allows for rapid determination of negative cases in low-prevalence populations (1). Fourth-generation Ag/Ab-screening assays detect not only IgG and IgM antibodies to HIV-1, HIV-2, and group O, but also the HIV-1 p24 antigen present in acute infection (2).
Ag/Ab-screen positive cases are then tested by a supplemental antibody immunoassay that differentiates HIV-1 from HIV-2 antibodies (Ab-differentiation). The Ab-differentiation assay can be performed using the same serum or plasma gel separator sample tube (SST/PST) for Ag/Ab-screening. Commonly, the Ab-differentiation is automatically reflexed for Ag/Ab-screen positive cases. Ab-differentiation positive results are conclusive for either HIV-1 or HIV-2 infection. However, the Ab-differentiation supplemental assays cannot confirm acute infection prior to seroconversion, when only the p24 antigen is detectable. Therefore, Ab-differentiation negative, indeterminate, or undifferentiated cases require HIV-1 and/or HIV-2 nucleic acid testing (NAT) to adjudicate between these two entirely divergent outcomes of acute infection or biologic false-positive Ag/Ab-screen results. Adding to the complexity, HIV-2 indeterminate results by Ab-differentiation have a high likelihood of HIV-1 infection requiring NAT, despite the results (3).
There are currently only three NAT methods approved for the diagnosis of HIV infection (3). However, many laboratories use methods with indications for use limitations that preclude their use for diagnosis, or to confirm HIV infection following results that are indeterminate or Ag/Ab-screen positive but Ab-differentiation negative (4). These methods are technically intended only to determine baseline viral load prior to and throughout antiretroviral therapy (ART). The limited sample types and processing requirements for NAT assays also preclude the use of the initial SST/PST sample. Thus, patients are either required to return for another sample collection, or laboratories must collect, process, and store a second sample upfront for NAT testing, despite the fact that the overwhelming majority of Ag/Ab-screening results will be negative in low-prevalence testing populations.
In most settings across the United States, laboratory testing for HIV infection is performed for routine screening in primary care, obstetrics/gynecology, and infertility treatment where the pretest probability is low. Most inconclusive Ab-differentiation cases represent biologic false-positives. However, patients may not disclose high-risk behaviors that increase risk of infection, making it essential that NAT confirmation testing is performed so that the diagnosis and treatment of acute infection is not delayed. A reduced time to initiate ART has been shown to improve virologic suppression (5, 6) and immune recovery (7), while reducing long-term comorbidities (8−11) and rates of transmission (12, 13).
Unfortunately, this testing is commonly missed because of the complexity of the CDC testing algorithm and the inability of many laboratories to automatically reflex NAT. Internal studies at our respective institutions demonstrated that between 69%–84% of Ab-differentiation results are inconclusive in first-time Ag/Ab-screen positive patients, and 26%–29% of inconclusive Ab-differentiation results lack NAT confirmation. Additionally, we found that NAT confirmation can take more than 40 days on average due to the requirement for patients to return for a new specimen collection.
With these factors in mind, we recommend a single-tube, two-step testing algorithm that reflexes all Ag/Ab-screen positive samples directly to NAT confirmation and that skips the Ab-differentiation step (Figure 1B, above). Regardless of the HIV-1/HIV-2 Ab differentiation results, NAT testing is required in all Ag/Ab-screen positive cases for diagnosis to confirm biologic false-positives or establish a baseline viral load prior to initiating ART (Table 1). Based on this and our experiences at our respective institutions, we have determined that this step delays final diagnosis and treatment without any significant benefits to offset this.
Table 1. Simplified guidance for interpreting results and determining necessary follow-up testing for the laboratory diagnosis of HIV infection.
|
Test performed |
Result |
Final interpretation |
Follow-up testing |
|
HIV-1/2 Ag/Ab screen |
Negative |
Negative |
-- |
|
Positive |
-- |
HIV-1/HIV-2 Ab differentiation |
|
|
HIV-1/HIV-2 Ab differentiation |
Negative or Indeterminate |
-- |
HIV NAT |
|
Undifferentiated |
-- |
HIV NAT |
|
|
Positive |
-- |
HIV NAT (establish baseline) |
|
|
HIV-1 NAT |
Negative |
Biologic false positive |
Follow HIV NAT |
|
Positive |
HIV-1 positive |
Genotype, Follow HIV NAT |
The most significant challenge clinical laboratories will face if they use our algorithm is the need to either collect a dedicated tube upfront for quantitative NAT; reflex the Ag/Ab-screen SST for qualitative NAT; or validate the off-label use of an alternative sample type, such as PST, as a laboratory developed test now subject to dual oversight from the Food and Drug Administration and CLIA (14).
HIV testing commonly is routinely performed with other sexually transmitted infection and hepatitis screening tests in an automation-friendly tube containing gel. We therefore performed an internal validation of PST and demonstrated excellent correlation of viral copy number to results obtained from pristine EDTA tubes. In light of this, we urge manufacturers to broaden the constraints of the indications for use and sample type restrictions to allow clinical laboratories to use PST for NAT assays and more efficiently provide rapid diagnosis of HIV infection.
Although rare (less than 1% of people have HIV), it is also important to determine if HIV controllers (15), with detectable antibodies but low viral copy numbers, will be detected by current NAT methods. Further, although the likelihood of a spontaneous viral load below the limit of NAT is estimated to occur in approximately only 25% of the HIV controller population, it is important to prospectively study the incidence of this occurring at the time of initial testing and the potential for missing nonviremic controllers when not performing the Ab-differentiation.
It is critically important for the HIV testing algorithm to be easy to follow and involve the automatic reflex of the initial sample collected to rapidly provide a final diagnosis. We believe this will play a critical role in achieving UNAIDS 95-95-95 targets to end the AIDS pandemic (16).
Nicole V. Tolan, PhD, DABCC, is an assistant professor of pathology at Harvard Medical School and the medical laboratory director of clinical chemistry, mass spectrometry section, point-of-care testing, and home hospital program at Brigham and Women’s Hospital and Mass General Brigham, Boston. +Email: [email protected]
Nicholas Heger, PhD, NRCC, is an assistant professor at Tufts University School of Medicine and the director of clinical chemistry in the department of pathology and laboratory medicine at Tufts Medical Center, Boston. +Email: [email protected]
Gary L. Horowitz, MD, is a professor in the department of pathology and laboratory medicine at Tufts University School of Medicine and Tufts Medical Center, Boston. +Email: [email protected]