As laboratorians working in microbiology or core clinical laboratories, we tend to dread the phrase that creeps up every fall: flu season. Not only do we know that it signals our workloads are about to ramp up, but also that we’ll be fielding countless inquiries from providers about appropriate specimen collection, turnaround times, and test utilization. These pertinent and timely questions will remain so as influenza viruses keep their status as a top driver of respiratory illness with potentially severe consequences in immuno-suppressed or compromised individuals.

A number of different techniques are available to diagnose influenza, including viral cell culture, direct fluorescent antigen (DFA), rapid influenza diagnostic tests (RIDT), and molecular methods. Since appropriate cohorting and treating of patients with influenza depends on accurate and timely diagnosis, many hospitals and laboratories have opted for point-of-care or in-laboratory rapid diagnostic testing methods, including RIDTs or molecular methods.

Many Food and Drug Administration (FDA)-approved RIDTs detect influenza A and/or B. These methods are quick (<30 minutes), easy to perform in either physicians’ offices or laboratories, and specific. However, they lack sensitivity (50%-70%) compared to viral culture. The primary FDA-approved rapid molecular testing methods use either reverse-transcription polymerase chain reaction (RT-PCR) or loop-mediated isothermal amplification (LAMP) technologies.

Molecular methods are much more sensitive than RIDTs, but depending on the assay, compromise slightly on turnaround time, cost, and ease of use. Molecular methods also usually detect both influenza A and B together, and often subtype the influenza A viruses. Clinical laboratories handle the majority of molecular testing as opposed to doctors’ offices or inpatient units. However, this is now changing with the availability of LAMP testing, which has been designed to be faster than RT-PCR and used in a point-of-care testing environment.

Regardless of method used and site of sample collection or testing, in my experience, the following are three of the most common influenza testing questions clinical laboratories handle during flu season.

What specimen type should I collect, and how should it be collected?

For any rapid influenza assay, appropriate specimen collection and handling is key to accurate diagnosis. While FDA-approved specimen types vary by assay, nasopharyngeal swabs and nasal washes/aspirates are the most sensitive for detecting influenza virus. Throat and nasal swabs are FDA-approved for certain, but not all, rapid diagnostic tests, and they typically have inferior recovery of the virus.

Requests for testing other, non-FDA approved sample types such as bronchoalveolar lavage samples or endotracheal aspirates are handled differently depending on the testing laboratory. These specimen types require a separate validation or a disclaimer if being tested. Some laboratories may prefer to send these requests to a reference laboratory that has validated these specimen types.

The specific swabs to be used for specimen collection vary depending on the rapid assay performed. Some assays may provide or recommend a specific swab for specimen collection. I can’t overstate the importance of using the recommended swabs for each assay, as certain swab materials can be inhibitory to certain assays.

To preserve their integrity, specimens should be placed in the appropriate, assay-specific, universal or viral transport medium and sent to the testing laboratory immediately or refrigerated if a transport delay is expected. Inappropriate sample types include swabs placed in dry containers or any transport medium other than those listed in the manufacturer’s instructions for the assay.

How quickly will I get my patient’s test result?

The answer to this question depends on the type of test being performed, where it is being performed, and which turnaround times hospitals and laboratories have agreed upon. RIDTs and LAMP assays have the fastest turnaround times (<30 minutes), whereas RT-PCR assays generally take 1–1.5 hours. In reality, turnaround times can be much longer depending on how quickly the specimen makes it to the lab, laboratory staffing, and whether or not the assay is a part of a lab’s STAT testing menu.

Is the reported result reliable?

Since the performance characteristics of RIDTs and molecular methods differ dramatically, appropriate patient management depends on proper interpretation of test results. Due to the low-to-moderate sensitivity of RIDTs, false negatives are common, especially during peak influenza season. Negative results do not exclude influenza infection, and providers should consider another diagnostic method—DFA, culture, or molecular—for confirmation if clinical suspicion of influenza is high. Due to the high specificity of most RIDTs, a positive result is considered reliable. Molecular methods, on the other hand, are both sensitive and specific, with both negative and positive results typically reliable. However, an important limitation of molecular methods is the possibility of detecting non-viable or non-replicating virus.

In addition to the performance characteristics of the specific test used, several other factors influence reliability of rapid test results, including specimen type, specimen collection and handling, and how soon after symptom onset the specimen was collected—ideally between 12 hours and 3 days afterwards. Assay-specific manufacturers’ recommendations should be followed to minimize the possibility of inaccurate results.

Armed with answers to these questions, labs will be ready to go when flu season strikes.

Tanis Dingle, PhD, D(ABMM), FCCM, is a clinical microbiologist at the Provincial Laboratory for Public Health in Edmonton, Alberta, Canada. Email: