Tradeoffs between targeted and expanded testing strategies for influenza A(H5N1)

ABSTRACT

In a recent article in ASM Case Reports, G. P. Higerd-Rusli, A. Karan, S. A. Hoffman, I. E. A. Morante, et al. (ASM Case Rep 2:e00165-25, 2025, https://doi.org/10.1128/asmcr.00165-25) describe one confirmed and one probable case of influenza A(H5N1) detected through universal subtyping of influenza A-positive specimens, including cases without known animal exposure. These findings highlight the limitations of exposure-based testing strategies and raise important questions about early detection of emerging pathogens. Drawing on lessons from prior pandemics, this commentary examines the tradeoffs between targeted and expanded surveillance and emphasizes the need for adaptive, flexible approaches to laboratory preparedness.

COMMENTARY

The epidemiology of highly pathogenic avian influenza A(H5N1) has shifted substantially in the past 2 years (1). Once largely confined to avian hosts, outbreaks have now been documented across multiple mammalian species, most notably among dairy cattle in the United States (2). Dozens of human infections have occurred through contact with infected animals (3), with the prospect of sustained human-to-human transmission an ever-present concern. To date, however, human testing has been limited to surveillance of individuals with known exposures to infected animals.

A recent study by Higerd-Rusli et al. in ASM Case Reports highlights concerns with this testing approach (4). The authors describe one confirmed and one probable case of influenza A(H5N1) identified through universal H5 subtyping of all influenza A-positive respiratory specimens at a large academic healthcare system in California. Sequencing of the confirmed case showed that it was phylogenetically related to clade 2.3.4.4b viruses associated with dairy cattle. The probable case showed reproducible detection of H5 at high cycle threshold values but could not be confirmed by the state public health laboratory or the Centers for Disease Control and Prevention (CDC). In both cases, the patients had no known exposures to infected animals, underscoring that neither case would have been captured by exposure-based testing strategies.

Current testing and surveillance strategies are targeted to individuals with the highest risk of disease. At present, the CDC recommends symptomatic monitoring for persons exposed to confirmed H5N1 infections in humans or animals (5). Those who develop compatible signs and symptoms should then undergo testing. While this strategy is practical and resource-conscious, it assumes that exposures are promptly identified and that transmission pathways remain stable and predictable.

The two cases presented here underscore the limitations of these assumptions and demonstrate the existence of edge cases that fall outside traditional testing paradigms. It should be emphasized that the detection of H5 in these two patients in itself does not establish community transmission, as these cases could simply reflect indirect/unknown exposures or environmental contamination. The authors even speculate whether consumption of dairy milk could have contaminated respiratory specimens with H5 viral RNA. Furthermore, the very low positivity rate reported by the authors (0.04%) is reassuring in that community transmission of H5N1 does not appear widespread. However, these two cases should nevertheless jolt readers into recognizing that the full extent of these and similar cases remains unknowable within current surveillance frameworks.

The debate about targeted vs. expanded surveillance for emerging high-consequence pathogens is not new. During the 2009 influenza A H1N1 pandemic, testing initially focused on severe cases, which led to under-recognition of milder infections (6, 7). Only after testing was broadened was the full scope of transmission more clearly appreciated. Similarly, early SARS-CoV-2 testing was limited and restricted to patients with known travel or exposures, delaying recognition of asymptomatic spread and community transmission (8, 9). Subsequent analyses demonstrated that widespread transmission had already occurred but went undetected due to restrictive case definitions for testing, which reduced the effectiveness of contact tracing and containment strategies (9–11).

What lessons can be learned then from previous spillover events, and how can these experiences inform future testing strategies for pathogens with pandemic potential like influenza A(H5N1)? Any expansion of testing must consider the diagnostic tradeoffs. The probable case reported by the authors illustrates the challenges of test interpretation in a low-prevalence setting. Detection of viral RNA near the limit of detection raises concerns about assay specificity, reproducibility, and the potential for false positives. Similar challenges were encountered during the SARS-CoV-2 pandemic, where high cycle threshold detections required careful interpretation in low-prevalence settings (12). Given the high sensitivity of molecular assays, expanding surveillance too early or in the wrong populations can potentially generate misleading results and complicate infection control efforts.

Broader testing also requires substantial resources and investment, which may be difficult to justify in the setting of workforce and funding constraints. There are also important questions of where and how expanded surveillance should be performed. While most clinical laboratories can detect influenza A, there are no widely available FDA-cleared assays for H5 subtyping. The authors of this report developed their own dual-target PCR assay (13), while the CDC also manufactures and licenses a four-target PCR assay that it provides to state public health laboratories (14). However, laboratory-developed tests (LDTs) like these, especially those with a higher number of targets, are limited in their scalability for high-volume testing (14). Their implementation also requires technical expertise, extensive validation, and ongoing quality assurance. Regulatory requirements may further limit adoption in certain states.

It is also notable that these cases were identified at a large academic medical center rather than through public health surveillance systems. The authors’ proactive approach, while commendable, may not fall under the purview of most clinical laboratories. Coupled with the limitations of scaling LDTs, broader testing in clinical laboratories is unlikely to meaningfully expand until high-throughput tests become available through FDA authorization pathways. Until then, subtyping is more likely to be scaled up within state public health laboratories. However, reliance on a single CDC-developed assay also brings its own vulnerabilities, as illustrated by the assay-related issues that led to delayed public health testing for SARS-CoV-2 (15).

Collectively, these considerations highlight the fundamental challenges and tradeoffs inherent to laboratory preparedness for emerging high-consequence pathogens (Table 1). Expanding testing too early risks overinvestment of resources with limited yield and a higher likelihood of misleading results (16, 17). Waiting too long, on the contrary, risks allowing transmission to become more firmly established before it can be recognized, which may render early containment strategies substantially less effective (8–11, 18). These competing priorities create an obvious dilemma: early cases of community transmission may only be detected once testing is broadened, but the decision to broaden testing depends on detecting these exact types of cases.

TABLE 1.

Tradeoffs between targeted and expanded surveillance for influenza A(H5N1)

	Targeted (exposure-based) surveillance	Expanded (universal or broad) surveillance
Testing population	Individuals with known animal exposure and compatible symptoms	All influenza A-positive specimens or broader populations, regardless of exposure
Operational setting	Primarily public health-driven	Requires closer coordination between clinical and public health laboratories
Resource utilization	Efficient; conserves laboratory and public health resources	Resource-intensive; requires staffing, reagents, and infrastructure
Scalability	Scalable within existing workflows	More limited by assay availability, throughput, and validation requirements
Sensitivity for early/atypical cases	Lower; may miss cases without recognized exposure	Higher; enables detection of unexpected or cryptic infections
Detection of community transmission	Delayed; may miss early spread outside known exposure networks	Earlier detection possible
Specificity/interpretability	Higher pretest probability; results easier to interpret	Lower pretest probability; increased risk of ambiguous or false positive results
Dependence on epidemiologic assumptions	High; relies on accurate identification of exposures and transmission pathways	Lower; less dependent on recognizing exposures in advance
Timing/trigger for implementation	Implemented early based on known risk pathways	Typically implemented later once broader transmission justifies expansion

In this context, geographically targeted or hybrid surveillance strategies, as suggested by the authors, may represent a practical middle ground (4). Focusing enhanced subtyping in regions with known animal outbreaks, high-risk agricultural interfaces, or early signals of atypical influenza activity could improve detection while preserving laboratory capacity and diagnostic specificity. Most countries have relied on exposure-based testing strategies for high-consequence infectious diseases, including Ebola, MERS-CoV, and early SARS-CoV-2 (11, 19). In contrast, expanded testing approaches in some countries during the COVID-19 pandemic demonstrated broader detection but proved resource-intensive and difficult to sustain (20, 21), supporting targeted or hybrid expansion as a more practical path forward.

Ultimately, the question is not whether all laboratories should adopt universal H5 subtyping, but how surveillance systems can remain sensitive to detect early outbreaks without overextending limited resources. Experience from prior pandemics suggests that testing often expands reactively, after transmission is already established. The findings presented here argue for a more proactive and adaptive approach, one that anticipates the limitations of exposure-based strategies and incorporates more flexibility in laboratory preparedness.

The views expressed in this article do not necessarily reflect the views of the journal or of ASM.