SARS-CoV-2 and Influenza Testing

Respiratory virus testing identifies the cause of an acute respiratory infection, which directs antiviral treatment, isolation and public health action. SARS-CoV-2 and influenza A and B are the most important targets, with respiratory syncytial virus (RSV) commonly tested in the same panel.

Methods are nucleic acid amplification tests (RT-PCR, including point-of-care and isothermal platforms), rapid antigen tests (lateral flow or fluorescence) and, for influenza, rapid molecular assays. Viral culture and serology have little role in clinical diagnosis.

Influenza causes 3 to 5 million severe illnesses and 290 000 to 650 000 respiratory deaths each year worldwide, with attack rates of 5 to 10% in adults and 20 to 30% in children. Severe disease is concentrated in those over 65 years, young children, pregnant women and people with chronic disease.

SARS-CoV-2 now circulates endemically with waves, with severity much lower than earlier in the pandemic because of population immunity, but it still causes severe disease in the elderly and the immunocompromised.

Indications. Acute respiratory illness with fever, cough, sore throat, myalgia or breathlessness, particularly in hospital admissions, patients at high risk of severe disease who qualify for antiviral treatment, immunocompromised patients, outbreak investigation in care homes and hospitals, pre-procedure screening in selected settings, and infection control decisions. Surveillance of severe acute respiratory infection.

Specimen. Nasopharyngeal swab (best sensitivity for both viruses), combined nose and throat swab, or nasal mid-turbinate swab, in viral transport medium. Saliva is acceptable for SARS-CoV-2 PCR in some platforms. Lower respiratory samples (sputum, endotracheal aspirate, bronchoalveolar lavage) in ventilated patients, since upper tract samples can be negative. Samples are kept at 2 to 8 °C and tested within 72 hours.

RT-PCR.

• Sensitivity over 95% and specificity over 99% when the sample is taken within the first 5 to 7 days of illness.
• Multiplex panels detect SARS-CoV-2, influenza A and B, and RSV, with results in 15 minutes to 4 hours on point-of-care platforms and 4 to 24 hours in laboratories.
• Cycle threshold (Ct) values reflect viral load inversely, but vary by assay and sample, and are not used for clinical decisions on infectiousness without local validation. Persistent RNA positivity can last weeks (up to 3 months) after recovery, with non-infectious fragments.
• Influenza subtyping (H1N1pdm09, H3N2) and lineage (B/Victoria, B/Yamagata) are done in reference laboratories for surveillance, and for novel avian influenza (H5N1, H7N9) in patients with exposure to poultry.

Rapid antigen tests.

• SARS-CoV-2: sensitivity 70 to 90% in symptomatic people early in illness, and 30 to 60% in asymptomatic people. Specificity over 98%. A positive result is reliable, and a negative result in a symptomatic person with high suspicion is confirmed by PCR. Repeat testing after 24 to 48 hours improves sensitivity.
• Influenza rapid antigen tests: sensitivity 50 to 70% (higher in children, who shed more virus), specificity over 95%. A negative result does not exclude influenza, and antiviral treatment is not withheld in a patient at risk.
• Digital immunoassay readers raise sensitivity to 80 to 90%.

Rapid molecular tests. Isothermal and fast PCR assays for influenza have sensitivity of 90 to 95% and are the preferred rapid option in hospital.

Timing. Viral shedding begins 1 to 2 days before symptoms, peaks at onset and falls over 5 to 7 days (influenza) or 5 to 10 days (SARS-CoV-2), and lasts longer in immunocompromised people (weeks to months).

Antiviral implications. Influenza: oseltamivir started within 48 hours of onset, and in hospitalised or high risk patients at any time. SARS-CoV-2: antivirals (nirmatrelvir with ritonavir, remdesivir, molnupiravir) are started within 5 to 7 days in high risk patients, with attention to drug interactions.

Critical values and findings needing immediate action.

• Positive influenza or SARS-CoV-2 in a patient with hypoxia (oxygen saturation under 92%), respiratory rate over 30 breaths/min, confusion or hypotension: severe disease, admission and antiviral treatment.
• Positive test in an immunocompromised patient or in pregnancy: early antiviral treatment.
• Positive test in a care home or hospital ward: isolation, contact tracing and outbreak control.
• Suspected novel influenza (avian exposure, severe pneumonia): notify public health, isolate, and send samples to a reference laboratory.
• Persistent positive PCR in an immunocompromised patient: consider prolonged infection and resistance (sequencing).

Interferences and pitfalls.

• Poor sampling technique is the main cause of false negatives.
• Rapid antigen tests lose sensitivity late in illness, in asymptomatic people and with low viral loads. A negative test does not exclude infection.
• Variants of SARS-CoV-2 with mutations in the target gene can cause PCR dropout, and multiple targets limit this.
• Co-infection with bacteria (Staphylococcus aureus, Streptococcus pneumoniae) causes severe pneumonia in influenza, and cultures are taken in severe cases.
• Detection of a virus does not prove it is the cause in a patient with another diagnosis, since asymptomatic carriage and prolonged shedding occur.
• Recent live attenuated influenza vaccine can cause a positive test within 7 days.
• Nasal swabs with blood, medicines or dyes can interfere with lateral flow tests.
• Contamination of PCR runs causes false positives, and positive results from a low risk population with a high Ct are repeated.

Special groups.

• Children: higher viral loads, and a higher sensitivity of rapid tests. RSV is the commonest cause of bronchiolitis.
• Pregnancy: higher risk of severe influenza and COVID-19, with early treatment and vaccination.
• Older adults: atypical presentations (delirium, falls) with fewer fevers.
• Immunocompromised patients: prolonged shedding and need for lower respiratory samples.
• Regions: influenza seasonality differs between temperate and tropical areas, and rapid molecular platforms extend access.

Follow up tests. Chest radiograph, oxygen saturation and arterial blood gas, full blood count, C-reactive protein, procalcitonin, creatinine and electrolytes, creatine kinase in severe disease, blood and sputum cultures if bacterial co-infection is suspected, D-dimer and troponin in severe COVID-19, and genomic sequencing for variants in surveillance.