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Viral specimens, antigens and congenital infection

16 min

Skills you'll build4 skills
  • Choose a viral test and specimen for the syndrome and the day of illness
  • Decide when a negative rapid influenza antigen needs a molecular test
  • Read a negative CSF viral NAAT against the day of illness and its target list
  • Distinguish direct evidence of congenital infection from transferred maternal antibody

Try first

Try first

An intubated man with severe pneumonia had a nasopharyngeal swab tested on a molecular respiratory panel. Every target was not detected, and the internal control was valid. The team asks whether a respiratory virus is excluded. What do you advise?

Keep going to see the reasoning.

Keep going to see the reasoning.

Right. Keep going to follow the reasoning.

Keep going to see the reasoning.

Keep going to see the reasoning.

Get the idea

Use the explanation to check your first decision.

Choosing the specimen and the timing

A viral test result applies only to the specimen collected and the time it was collected. Nucleic acid amplification tests (NAATs) are the main method for most acute viral illness.1 A "not detected" result can come from a specimen collected too early or too late, from the wrong site, with too little material, or for a virus the assay does not target.1

QuestionSpecimen and testWhat a negative result can miss
Virus in severe pneumoniaTracheal aspirate or lavage on a panel validated for itA virus in the lower airways that an upper swab did not reach
Influenza in an admitted patientMolecular assayInfluenza that a rapid antigen test missed
Herpes simplex virus (HSV) encephalitisCerebrospinal fluid (CSF) HSV PCREarly infection, before the target is detectable
Vesicular rashSwab of a fresh lesion for HSV or varicella-zoster virus (VZV) PCRVirus in an old, crusted lesion
Congenital cytomegalovirus (CMV)Saliva CMV PCR within 21 days of birth, positives confirmed in urineCongenital infection, once the window has closed

Rapid influenza antigen tests

Rapid influenza antigen tests are less sensitive than molecular assays. During active circulation a negative result often misses influenza, so an admitted patient with suspected influenza is tested by a molecular assay.2

Early CSF HSV PCR

CSF HSV PCR is the standard test for HSV encephalitis, and it can be negative in the first days. When suspicion stays high, a second CSF collected 3 to 7 days later is tested.1 A negative HSV result on a multiplex panel is followed by a single-target HSV PCR on the same CSF.3 A multiplex meningitis panel reports only its listed targets, so a virus not on the list needs its own test.3

Congenital CMV testing

Maternal IgG crosses the placenta, so a newborn's CMV IgG may be the mother's antibody. Congenital CMV is shown by CMV PCR on saliva collected within 21 days of birth, with each positive confirmed by urine PCR. A first positive after 3 weeks of age can come from infection after birth.4

Lesion material is the specimen for a vesicular rash. PCR of a fresh lesion is more sensitive than culture.5,6

Follow the bench demonstration

Sources for this step
References
  1. Miller JM, Binnicker MJ, Campbell S, et al. Guide to utilization of the microbiology laboratory for diagnosis of infectious diseases: 2024 update by the Infectious Diseases Society of America and the American Society for Microbiology. Clin Infect Dis. 2024;ciae104. doi:10.1093/cid/ciae104
  2. Centers for Disease Control and Prevention. Rapid influenza diagnostic tests. Accessed September 27, 2026. https://www.cdc.gov/flu/hcp/testing-methods/clinician_guidance_ridt.html
  3. US Food and Drug Administration. 510(k) decision summary K160462: FilmArray Meningitis/Encephalitis Panel. Accessed September 27, 2026. https://www.accessdata.fda.gov/cdrh_docs/pdf16/K160462.pdf
  4. Centers for Disease Control and Prevention. Laboratory testing for CMV and congenital CMV. Updated April 15, 2024. Accessed September 27, 2026. https://www.cdc.gov/cytomegalovirus/php/laboratories/index.html
  5. Centers for Disease Control and Prevention. Laboratory testing for varicella-zoster virus. Accessed September 27, 2026. https://www.cdc.gov/chickenpox/php/laboratories/index.html
  6. Workowski KA, Bachmann LH, Chan PA, et al. Sexually transmitted infections treatment guidelines, 2021. MMWR Recomm Rep. 2021;70(4):1-187. Accessed September 27, 2026. https://www.cdc.gov/mmwr/volumes/70/rr/RR7004a1.htm

Watch one

Read each decision and its explanation.

A 34-year-old woman has fever and confusion that began yesterday. Her CSF, collected on illness day 2, is tested on a multiplex meningitis and encephalitis panel. HSV-1 is not detected, every other target is not detected, and the internal control is valid. The clinician still suspects HSV encephalitis. How do you interpret the negative result?

  1. Check the run: the internal control is valid, so the result is reportable as not detected.

    An invalid run gives no interpretable result.

  2. Check the target list: HSV-1 is on the panel, so the question is about HSV itself.

    A panel reports only the targets it lists.

  3. Check the day of illness: day 2 is early.

    HSV DNA can be below detection in the first days of encephalitis.

  4. Note the clinical question: suspicion for HSV encephalitis stays high.

    Clinical suspicion determines whether a repeat specimen is collected.

  5. Report the result with its limitations, test the same CSF with a single-target HSV PCR, and test a second CSF collected 3 to 7 days later when it arrives.

    A panel can miss HSV, and an early negative can miss it too.

HSV-1 is reported as not detected. An early negative does not exclude HSV encephalitis. The same CSF is tested with a single-target HSV PCR, and a second CSF collected 3 to 7 days later is tested by HSV PCR.

Practice this skill

Your turn

Problem 1 of 3 · Hints available

During peak influenza circulation, a hospitalized patient with suspected influenza has a negative rapid antigen test. What does the laboratory advise?

Incorrect. Rapid influenza antigen tests have limited sensitivity compared with molecular assays, so a negative result during active circulation does not exclude influenza.

Incorrect. The FDA instruction to repeat testing 48 hours later applies to SARS-CoV-2 antigen tests. A hospitalized patient with suspected influenza is tested by a molecular assay.

Correct. Rapid antigen tests have limited sensitivity compared with molecular assays, so during peak circulation a negative antigen result can be a false negative. Hospitalized patients with suspected influenza are tested with a molecular assay.

Hint
  1. Compare the sensitivity of rapid antigen tests with that of molecular assays.
  2. The patient is hospitalized and influenza is circulating.

Review Antigen tests

Problem 2 of 3 · Hints available

Congenital CMV infection is suspected in a 10-day-old infant. Which testing establishes it?

Incorrect. Maternal IgG crosses the placenta, and neonatal CMV IgG and IgM do not diagnose congenital infection.

Correct. Saliva is tested by CMV PCR within 21 days of birth, collected more than 1 hour after breastfeeding, and every positive is confirmed by urine PCR in the same window. Detection after 3 weeks of age can reflect postpartum infection, so a newly positive result after day 21 cannot separate congenital from postnatal infection.

Incorrect. A positive stored dried blood spot can confirm congenital infection retrospectively, but dried-blood-spot PCR is less sensitive, so a negative result leaves congenital infection possible.

Hint
  1. Consider whose antibody a newborn's IgG may be.
  2. Congenital CMV is shown by finding the virus within a set number of days after birth.

Review Congenital and perinatal viral infections

Problem 3 of 3 · On your own

A 5-week-old infant fails a hearing screen repeated at an outside clinic. No newborn CMV test was done. Urine CMV PCR collected today is detected. What can the laboratory say about congenital infection?

The urine was collected after 21 days of age. A first positive at 5 weeks can come from infection after birth.

After 21 days a new positive cannot separate congenital from postnatal infection. A positive dried blood spot from birth shows the virus was already present then.

Late testing leaves the timing unknown. It does not exclude congenital infection.

Infant IgG can still be the mother's antibody at 5 weeks, and infection after birth also raises it.

Reported neonatal IgG as congenital infection

Maternal IgG crosses the placenta, so a newborn's IgG reflects the mother's antibody. Congenital CMV is shown by a positive saliva PCR within 21 days of birth, confirmed by urine PCR. A first positive result after 3 weeks can reflect postnatal infection.

Review Congenital and perinatal viral infections

Use it

Read the case evidence, then make each decision. The step is complete without it.

  • Rosa Dillon, 67, a retired school bus driver who still coaches Little League, is admitted in January with fever, cough and low oxygen. Influenza is circulating widely.
  • The emergency department's rapid influenza antigen test is negative.
  • A nasopharyngeal swab on a molecular respiratory panel is then not detected for every target, with a valid control.
  • On day 3 she is intubated with bilateral pneumonia. A tracheal aspirate is collected for bacterial culture.
Decision 1 of 3

When the antigen test came back negative, what was the laboratory's advice?

Antigen tests miss many infections during peak circulation.

Reported influenza excluded on a negative antigen test

Rapid antigen tests have limited sensitivity compared with molecular assays, so during peak circulation a negative result often misses influenza. For a hospitalized patient with suspected influenza, a molecular assay is needed before influenza is excluded.

Repeating a test with limited sensitivity gives the same limitation. The 48-hour repeat applies to SARS-CoV-2 antigen tests.

She is admitted with suspected influenza during active circulation, so a molecular assay is needed before influenza is excluded.

Review Antigen tests

Decision 2 of 3

On day 3 the team asks whether a viral cause is ruled out. What do you advise?

Her disease is in the lower airways, and the upper swab can miss it. The aspirate samples the site of disease.

A swab from the nasopharynx can be negative in lower respiratory disease.

Relied on an upper-airway swab in lower respiratory disease

An upper-airway specimen can be negative in lower respiratory tract disease. In severe pneumonia, a sputum, tracheal aspirate, or bronchoalveolar lavage specimen tested on a panel validated for it can detect a virus the swab missed.

A new swab samples the same upper airway that was already negative.

Review Choosing and interpreting a viral test

Decision 3 of 3

The in-house panel is validated only for nasopharyngeal swabs. What happens to the aspirate?

A panel's performance is known only for the specimens it was validated with. A comment does not make an unvalidated result reliable.

The aspirate goes to an assay validated for it, so its result is reliable.

Diluting the aspirate does not validate it and can lower the amount of virus below detection.

Review Choosing and interpreting a viral test

The answer depends on where the specimen came from. Her disease was in the lungs, and the swab sampled the nasopharynx. A lower respiratory specimen tested on a validated assay answers the team's question.

For reference

Sources checked