The evolving global landscape of reflex testing

In this guest editorial, Eric Morreale, PhD explores global reflex testing trends, strategies, and evolving policies

16 Oct 2025

In well-funded healthcare systems, lab automation allows seamless reflex testing within centralized laboratories. Point-of-care (POC) diagnostics, such as portable PCR devices, support rapid reflex testing in rural and remote settings.

Evolving guidelines and policies for reflex testing are becoming more specific and standardized. Regulatory bodies such as the WHO and national health agencies continuously update these policies to align with technological advancements and emerging healthcare needs. In 2022, WHO revised its HCV guidelines to include explicit recommendations for reflex RNA testing following a positive antibody result. Its 2024 HBV guidelines recommend reflex HBV DNA testing for those testing positive for HBsAg, using either an existing laboratory sample or a clinic-based sample collected immediately following a positive HBsAg rapid diagnostic test¹.

In addition to guideline updates, regulatory changes in some countries are removing barriers to reflex testing. Historically, some healthcare systems required specific physician orders for confirmatory tests, leading to delays. Recent policy shifts allow initial test orders to authorize necessary reflex tests, reducing ambiguity and improving efficiency.

Regional reflex testing strategies and practices

Europe — at the forefront:

HCV: Many European laboratories implement reflex HCV RNA testing as standard practice. The European Association for the Study of the Liver (EASL) strongly endorses reflex RNA testing, citing its ability to significantly increase the proportion of antibody-positive patients who receive confirmatory testing and subsequent care².

Reflex RNA testing is integrated with dried blood spot (DBS) sampling and one-step point-of-care RNA tests in outreach and resource-limited settings. This widens access to a diagnosis, especially for marginalized populations who struggle with multiple healthcare visits³.

HBV & HDV: A notable European reflex testing practice is hepatitis D (HDV) screening for all HBsAg-positive patients. This recommendation stems from EASL guidelines, which recognize the severe clinical implications of HDV coinfection. Although many European laboratories have implemented this recommended reflex anti-HDV antibody testing, it needs to be more widely adopted as HDV infection continues to be underdiagnosed^{4, 5}.

HIV: European HIV testing protocols mandate confirmatory reflex testing following any reactive screening result. European regulations also emphasize the importance of verifying a new HIV diagnosis with a second specimen before initiating treatment. This quality control measure ensures diagnostic accuracy and prevents false positives due to sample handling errors^{6, 7}.

Asia-Pacific — expanding access:

HCV: Reflex HCV RNA testing is being incorporated into national hepatitis programs across Asia. Countries such as India, Japan, and Australia have adopted this approach to enhance diagnosis and linkage to care. In rural and resource-limited settings, point-of-care RNA assays are being explored as alternatives to traditional laboratory-based testing^8,9.

HBV: Diagnostic algorithms in Asia vary by country, but reflex testing is gaining momentum. In some areas, reflex HBV DNA testing is available immediately following an HBsAg-positive result. In lower-resource areas, confirmatory testing may require additional clinician authorization or referral to centralized laboratories¹⁰.

Despite the Asian Pacific Association for the Study of the Liver (APASL) recommendations for universal HDV screening in HBsAg-positive individuals, HDV testing remains underutilized in parts of Asia. Expanding reflex HDV testing is a critical area for improvement¹¹.

HIV: Asia’s HIV testing algorithms typically follow WHO’s multi-test strategy. Many countries utilize serial rapid tests, while high-resource settings adopt laboratory-based reflex confirmation. National programs are working to standardize protocols. Korean clinical guidelines note that, once diagnosed, patients should be tested for HIV RNA viral load, HCV, HBV, and hepatitis A before starting treatement¹².

Africa — focused on a unified system

HCV: While the WHO recommends reflex HCV RNA testing for individuals who test positive for HCV antibodies, testing is expensive and not widely available in some African countries. Point-of-care HCV RNA testing has helped improve same-day diagnosis and linkage to treatment, and self-testing kits are used to increase screening rates with positive tests confirmed through laboratory-based testing¹⁵.

Africa CDC and the Ministry of Health and Population of Egypt created a training program in 2022 to replicate Egypt’s successful 'screen and treat' HCV and HBV elimination initiatives across the continent¹⁶.

HBV: HBV remains a significant public health issue in Africa, with many individuals unaware of their status. Reflex testing is often integrated into HIV testing programs due to the high co-infection rate between HIV and HBV. Some African nations incorporate multiplex rapid tests capable of detecting HIV, HBV, and HCV in a single sample to increase efficiency¹⁷.

HIV: HIV testing in Africa has improved, primarily due to the widespread use of rapid diagnostic tests and routine screening programs. Countries have implemented the WHO-recommended HIV testing algorithm, including sequential testing with different test kits to minimize false positives and negatives¹⁸. Self-testing has gained traction, allowing individuals to collect samples at home and confirm results through laboratory-based reflex testing.

The Africa CDC has worked to integrate HIV reflex testing with TB and hepatitis programs to enhance efficiency and reduce missed diagnoses. False positives in rapid HIV tests remain a concern in low-prevalence settings, highlighting the importance of using highly sensitive confirmatory tests¹⁵.

Canada, Central and South America — mixed implementation:

HCV: In Canada and parts of Latin America, reflex HCV RNA testing is increasingly incorporated into national protocols. The Pan American Health Organization (PAHO) has urged countries to implement reflex RNA testing to improve diagnosis rates and treatment initiation¹³.

Many Latin American countries lack sufficient laboratory capacity, meaning confirmatory RNA testing often requires a separate visit or referral to a centralized laboratory. Countries like Brazil and Argentina have been working to improve reflex testing capabilities, mainly through national hepatitis elimination programs. In Brazil, professional nurses are trained to help expand viral hepatitis tracking and diagnosis and can conduct rapid tests¹⁴.

HBV: Reflex testing practices in the Americas vary. While Canada follows a model similar to Europe, where neutralization assays and HBV DNA reflex testing are becoming common, many Latin American countries still require separate test orders. Despite growing evidence supporting routine HDV screening, HDV testing is not universally performed on HBsAg-positive samples.

HIV: In Canada and Brazil, reflex confirmatory testing is well-established. HIV-1/HIV-2 differentiation assays and HIV RNA reflex testing are standard for resolving indeterminate results. Some Latin American countries still require a Western blot or line immunoassay as a confirmatory test, despite WHO’s recommendation to phase out these older methods in favor of faster, more reliable strategies.

Governing bodies and their stances on reflex testing

World Health Organization (WHO): Recommends reflex HCV RNA testing following a positive antibody result and added HBV DNA reflex testing in its 2024 HBV guidelines. For HIV, WHO mandates confirmatory testing with at least two different assays before diagnosis.
European Centre for Disease Prevention and Control (ECDC): Supports reflex RNA testing for HCV, automatic confirmatory assays for HIV, and integrated reflex testing strategies for at-risk populations.
APASL (Asian Pacific Association for the Study of the Liver): Advocates routine HDV screening in HBV-positive patients and HCV RNA reflex testing as a best practice.
African Centres for Disease Control and Prevention (Africa CDC): Encourages governments to use point-of-care screening and other diagnostic tools integrated with existing one-stop testing services.
Pan American Health Organization (PAHO): Encourages wider adoption of reflex HCV and HBV testing to strengthen diagnosis and linkage to care.

Future of reflex testing

While reflex testing offers significant advantages, several challenges remain:

Laboratory infrastructure: Many low- and middle-income countries lack the capacity for widespread nucleic acid testing, limiting the feasibility of reflex testing.
Cost and reimbursement: Some health systems do not cover reflex confirmatory tests, requiring policy changes to ensure financial sustainability.
Regulatory barriers: In some regions, laboratories cannot perform reflex testing without a separate physician order, delaying diagnosis.

Reflex testing enables faster and more accurate disease confirmation while reducing loss to follow-up. As global health organizations continue to refine guidelines and new technologies emerge, the implementation of reflex testing is expected to expand across more laboratories worldwide. Ensuring patients receive timely and accurate diagnoses is a shared global goal, and reflex testing plays a crucial role in achieving this.