Early diagnosis of active bloodborne infections such as hepatitis C virus (HCV), hepatitis B virus (HBV), and human immunodeficiency virus (HIV) is crucial to improving health outcomes, reducing transmission, and preventing disease progression. Yet, a testing gap often hinders an effective, timely diagnosis. Patients fail to complete secondary confirmatory testing.
Many diagnostic pathways require patients to return for a second test after an initial positive screening. Unfortunately, many patients never complete this follow-up testing, leaving them unaware of their infection status. This leads to untreated infections and increased transmission risks. Reflex testing offers a powerful solution by streamlining the diagnostic process.
What is reflex testing?
Reflex testing is a laboratory testing protocol that automatically performs confirmatory testing on an existing specimen when an initial screening test is positive. One clinical encounter and one blood draw or specimen eliminates the need for a separate patient visit, making it particularly valuable for making a complete (and swift) diagnosis of bloodborne pathogens such as HCV, HBV, and HIV.
For example, when a patient tests positive for HCV antibodies, an HCV RNA viral load test is required to confirm active infection. Reflex testing ensures that the second test is performed automatically on the same sample or from the same blood draw. The results shared with the patient and their physician include the HCV antibody results and the HCV viral load.
This streamlined process leads to:
- Faster diagnosis
- Prompt access to treatment
- Reduced risk of loss to follow-up
- Better patient outcomes
- Lower transmission rates
Reflex testing can confirm the specific pathogen, its viral load, and its genotype, providing a more accurate diagnosis than traditional antibody-based tests. Molecular assays are particularly well-suited for reflex testing due to their high sensitivity and specificity, which reduces false positive and false negative results.
Shorter time from screening to treatment
A significant barrier to HCV and HBV treatment is delayed viral load measurement after a positive antibody test. A reflex viral load test ensures that when an HBsAg serological or anti-HCV screening test is positive, an HBV DNA nucleic acid test (NAT) or HCV RNA test is immediately performed on the same specimen or from the same blood draw. This allows physicians to confirm active infection within a single clinical encounter, reducing diagnostic delays from weeks to days.
Confirming an acute HIV infection (AHI) quickly is vital for prompt treatment and reducing transmission.
The earlier treatment can start, the greater the ability to slow the disease’s progression and maintain the patient’s immune function. A review of data from an HIV screening program in Chicago compared data before and after implementing HIV-1 RNA reflex testing and found that reflex NAT improved the timeliness of confirming AHI and significantly reduced the time to linkage to care from 12.7 to 2.6 days.1
Genotyping and viral load testing matter
Beyond diagnosing an infection, reflex testing provides critical insights into viral load and genotype, which is essential for developing the ideal treatment plan and managing the disease.
While each HCV genotype causes liver damage, specific genotypes may have a greater chance of developing cirrhosis or liver cancer.2 Determining the HCV genotype is also beneficial to determine the best treatment plan for a patient. HBV DNA testing provides insight into the severity of the infection and helps clinicians decide on appropriate antiviral therapy. For HIV, viral load measurement helps assess disease progression and determines the need for antiretroviral therapy (ART).
Reflex testing impact
A study published in early 2025 examining 10 years of data from an Atlanta, Georgia, healthcare system highlighted the risks associated with not implementing reflex HIV RNA testing. The study found that:
- One-third of patients with discordant HIV test results had AHI.3
- Nearly half of non-hospitalized patients with AHI either had confirmatory testing delayed by more than 30 days or never received confirmatory results.3
This delay significantly increases the likelihood of ongoing HIV transmission, as individuals with undiagnosed acute HIV infections may be highly infectious. In contrast, reflex NAT testing eliminates these confirmation gaps, ensuring patients are diagnosed and treated as quickly as possible.
Barriers to adopting reflex testing
Although the Centers for Disease Control and Prevention recommends reflex HCV RNA and HIV RNA testing, there are some studies that suggest these diagnostic algorithms are not always followed by clinicians.4 NAT assays are not always reflexed by a laboratory, meaning an additional physician order and blood draw may be required before this confirmatory testing is performed.
Implementing reflex NAT testing in a clinical setting can provide earlier diagnosis of HCV and HIV infection, which can decrease related illnesses and improve overall mortality. Laboratories, healthcare providers, and public health organizations should advocate for the broader adoption of reflex testing protocols to bridge these diagnostic gaps and improve patient outcomes.
Steps to improve reflex testing implementation:
- Increase provider awareness about reflex testing benefits and best practices
- Enhance laboratory protocols to integrate reflex testing into diagnostic workflows.
- Advocate for policy changes to include reflex HIV RNA testing in national testing guidelines
- Expand insurance coverage to ensure reflex testing is accessible without financial barriers.
- Improve patient education by communicating the importance of comprehensive testing.
Download LGC SeraCare’s white paper, Reflex Testing: Enhancing Diagnostic Efficiency and Patient Outcomes, to learn more about the expanding role of reflex testing, how it can improve patient outcomes, and considerations for implementing reflex testing in your laboratory.
References
- Reflex Human Immunodeficiency Virus (HIV) Type 1 RNA Testing Enables Timely Differentiation of False-Positive Results From Acute HIV Infection
- HCV Genotypes Fact Sheet
- Missed and Delayed Diagnoses of Acute Human Immunodeficiency Virus (HIV) Infection in a Southern Opt-Out HIV Testing Environment Without Reflex HIV RNA Testing
- Challenges in HIV Diagnosis Algorithm: Experience of the Confirmation Laboratory