Viral Diagnosis: Respiratory, Herpesvirus, Congenital, and CNS Syndromes

NAAT is the main method for most acute viral syndromes. Culture, direct fluorescent-antibody (DFA) staining, shell-vial culture, and antigen detection now have narrower, assay-specific roles.^{11 Miller JM, et al. Guide to utilization of the microbiology laboratory for… 2024.}

Respiratory viruses produce overlapping symptoms, so the clinical picture seldom identifies the virus. Testing is most useful when the result will affect isolation, antiviral treatment, antibiotic decisions, further testing, or public-health action.

Specimen selection and timing

Collect the specimen as early as practical in the illness, and use a specimen type accepted by the test manufacturer. A flocked nasopharyngeal swab is common, but it is not valid for every assay. Some products accept nasal, mid-turbinate, throat, saliva, nasopharyngeal aspirate, or combined specimens. The collection device, transport medium, storage conditions, patient age, and assay instructions all affect test performance.

An upper-airway specimen can be negative in lower respiratory tract disease. In severe pneumonia, especially in an intubated or immunocompromised patient, a validated sputum, tracheal aspirate, or bronchoalveolar-lavage test may add information when an upper-airway result is negative and a lower specimen is collected for clinical care. Never test a lower respiratory specimen with a panel that has not been validated for it.

The amount and duration of detectable respiratory-virus nucleic acid vary with the virus, specimen site, age, immune status, disease severity, vaccination, and prior infection. For SARS-CoV-2, NAAT can remain positive for weeks and sometimes up to 90 days after an infection, so a repeat NAAT during that interval may detect residual RNA from the earlier infection.^{33 Centers for Disease Control and Prevention. Overview of testing for SARS-CoV-2.}

Antigen tests

Rapid influenza antigen tests have limited sensitivity compared with molecular assays. A negative result during active community circulation does not exclude influenza, so test hospitalized patients with suspected influenza by a molecular assay. A positive antigen result is less reliable when influenza prevalence is low.^{44 Centers for Disease Control and Prevention. Rapid influenza diagnostic tests. 2026.}

RSV antigen tests are also less sensitive than NAAT, particularly in adults. Performance depends on the product, specimen, and patient age.^{55 Centers for Disease Control and Prevention. Diagnostic testing for RSV. 2026.} A negative SARS-CoV-2 antigen result is presumptive. Current Food and Drug Administration (FDA) instructions call for repeat testing 48 hours later when infection is still suspected, using the number of repeats specified by the product and current guidance.^{33 Centers for Disease Control and Prevention. Overview of testing for SARS-CoV-2.}

Some antigen assays are authorized for point-of-care or home use, but waived status is product-specific. Common direct antigen targets include influenza, RSV, SARS-CoV-2, and rotavirus. HSV and CMV antigen methods are not routine alternatives to modern NAAT.

Major respiratory targets

Human herpesviruses establish lifelong latency. A positive molecular result can therefore have different meanings at a lesion, in blood, or in cerebrospinal fluid (CSF). Consider whether the virus commonly reactivates, whether the specimen contains blood cells, and whether the host is immunocompromised.

Herpes simplex virus

Type-specific NAAT from a lesion is the preferred diagnostic test. When a vesicle is present, unroof it and rub the base firmly to collect infected cells. Viral culture is less sensitive, especially as a lesion heals. A negative lesion NAAT or culture does not exclude herpes simplex virus (HSV) because shedding is intermittent, and a blind swab collected without a lesion has low sensitivity. DFA, Tzanck preparation, and HSV IgM are not recommended for diagnosis.^{77 Centers for Disease Control and Prevention. Genital herpes.}

Type-specific glycoprotein G serology can establish prior HSV-1 or HSV-2 exposure in selected settings. It cannot identify the infected site or date the infection. HSV-1 serology is less sensitive than HSV-2 serology and does not distinguish oral from genital infection. When recent HSV-2 acquisition is suspected and the initial result is negative, repeat serology about 12 weeks after the presumed exposure. Low-positive HSV-2 enzyme immunoassay results require confirmation by a second method because false positives are common at low index values.^{77 Centers for Disease Control and Prevention. Genital herpes.}

Resistance testing at a reference laboratory may be requested for persistent or recurrent lesions during appropriate antiviral therapy. Phenotypic plaque-reduction testing requires a culture isolate. Genotypic tests can detect characterized resistance-associated mutations, usually in the HSV thymidine kinase gene, UL23, directly from clinical material. Some methods also examine the DNA polymerase gene, UL30. Genotyping is faster and does not require viable virus, but an uncharacterized mutation or a mechanism outside the sequenced region can limit interpretation.^{88 Wang H. Practical updates in clinical antiviral resistance testing. 2024.}

Varicella-zoster virus

PCR of lesion material is preferred for confirming varicella or zoster. Suitable specimens include cells from the base of an unroofed vesicle, vesicular fluid, a properly sampled maculopapular lesion, and a scab. Saliva, nasopharyngeal secretions, urine, bronchial washings, and CSF are less reliable for confirming a cutaneous eruption and can produce false-negative results. DFA, culture, and Tzanck preparations are less sensitive than PCR and are not recommended for routine confirmation.^{99 Centers for Disease Control and Prevention. Laboratory testing for varicella-zoster virus.}

A single varicella-zoster virus (VZV) IgG result indicates previous infection or vaccination and cannot distinguish the two. VZV IgM is less sensitive than lesion PCR, has limited specificity, and cannot separate primary infection from reinfection or reactivation.

Epstein-Barr virus and infectious mononucleosis

Primary Epstein-Barr virus (EBV) infection in early childhood is often asymptomatic. Infection in an adolescent or young adult can cause fever, pharyngitis, lymphadenopathy, fatigue, palatal petechiae, hepatosplenomegaly, and an aminopenicillin-associated rash. A complete blood count often shows leukocytosis with absolute or relative lymphocytosis and reactive lymphocytes. The historical Hoagland pattern of at least 50% lymphocytes and at least 10% atypical lymphocytes supports infectious mononucleosis when the clinical findings fit, but the smear does not establish EBV as the cause.

Heterophile assays detect nonspecific IgM antibodies that agglutinate animal red cells. Results are often negative early in illness and in young children. Positive reactions can also occur without EBV infection. The Centers for Disease Control and Prevention (CDC) does not recommend the Monospot test for general use. EBV-specific serology is appropriate when the presentation is atypical or a heterophile result is negative or discordant.^{1010 Centers for Disease Control and Prevention. Laboratory testing for Epstein-Barr virus. 2024.}

TORCH is a historical mnemonic for a group of congenital infections, and testing is ordered agent by agent. The grouped infections can produce overlapping findings, but their useful specimens, collection times, and interpretive rules differ. Broad TORCH (toxoplasmosis, other infections, rubella, cytomegalovirus, herpes simplex virus) IgM and IgG panels have low diagnostic yield and can produce misleading results. Select pathogen-specific tests by maternal history, exposure, and the infant's findings.

Meningitis is inflammation of the meninges. Encephalitis involves brain parenchyma and produces altered mental status or another sign of cerebral dysfunction. Aseptic meningitis is a clinical and CSF syndrome in which routine bacterial studies do not identify a cause. Aseptic meningitis is not always viral. A normal CSF white-cell count does not exclude encephalitis, especially early or in an immunocompromised patient.

Collect cerebrospinal fluid (CSF) into sterile tubes and deliver it promptly. Follow your laboratory's tube-allocation policy and provide enough volume for bacterial studies, molecular testing, and any additional tests selected from the syndrome and exposures. Viral culture of CSF has little routine diagnostic value.

Interpreting CSF molecular testing

CSF testing may use a targeted NAAT, a fixed multiplex panel, or both. A multiplex panel can return several common bacterial, viral, and fungal targets quickly, but it does not contain every important cause of meningitis or encephalitis. Arboviruses, lymphocytic choriomeningitis virus, acute HIV, JC virus, and several herpesvirus questions require separate tests. A not-detected result does not exclude an organism outside the panel, a low-level target, infection sampled too early, or infection better detected by another method or specimen.^{1515 US Food and Drug Administration. BioFire FilmArray Meningitis/Encephalitis Panel 510(k) decision…}

A detected result requires clinical interpretation. Latent-virus reactivation, chromosomally integrated HHV-6, blood contamination, or passage of DNA across an abnormal blood-brain barrier can produce CMV, EBV, or HHV-6 results unrelated to active CNS disease. When the syndrome and result disagree, review the raw laboratory data, specimen quality, immune status, imaging, and other microbiology findings. Repeat or targeted testing may be appropriate.