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

All major viral diagnostic modalities remain in use, but nucleic acid amplification now predominates. Culture, shell-vial antigen detection, rapid antigen immunoassays, and serology still have defined laboratory jobs. Antiviral treatment is out of scope except where it changes a laboratory action, such as retaining HSV culture for phenotypic resistance testing.

General principles

Several viruses grow in routine cell lines and are identified by cytopathic effect (CPE). Herpes simplex virus produces refractile ballooning or multinucleated giant cells within 1-3 days on many lines. Enteroviruses produce small cells with cytoplasmic tails plus large clearings in the monolayer. Adenovirus produces CPE in epithelial lines such as A549 over 2-5 days (up to 10-14 days); adenovirus 40/41 are fastidious and will not grow in routine culture, so stool PCR is used instead. Culture requires substantial technical skill and turnaround time and can propagate an emerging pathogen, so its use is largely limited to specialized settings.

Shell-vial culture uses centrifugation-enhanced inoculation of a cell monolayer, then immunofluorescence for early antigen before CPE develops. Turnaround shortens to 1-3 days but remains slower and less sensitive than molecular testing.

Direct or indirect fluorescent-antibody staining of a clinical specimen returns results in under 4 hours but is labor-intensive and quality-dependent. Rapid antigen immunoassays for influenza are CLIA-waived and suitable for point-of-care use. CDC currently describes RIDT sensitivity as about 50-70% with high specificity relative to molecular methods; some reader-device RIDTs reach about 75-80%, and FDA now requires RIDTs to achieve 80% sensitivity. A negative rapid influenza antigen test still cannot rule out infection, especially at peak community activity, and should be confirmed with a molecular assay when the result would change management. RSV antigen tests are less sensitive than NAAT; CDC does not assign them the same 50-70% figure. Routine antigen assays exist for only a few viruses: RSV, influenza, rotavirus, HSV, CMV, and hepatitis B.¹

Serology measures host antibody. IgM is the usual marker of acute infection; it is undetectable during the earliest window period, when the patient is already infected and infectious. For some pathogens a fourfold titer rise between acute and convalescent sera confirms infection.

NAAT is direct, reflects current infection, and detects infection during the window period. It is now the primary diagnostic method for most clinically relevant viruses. Target amplification (PCR, RT-PCR, qPCR, transcription-mediated amplification) and signal amplification (branched DNA, hybrid capture) both remain in use. Multiplex syndromic panels (8-21 targets) return results in as little as 1 hour.

Respiratory viral syndromes

Upper respiratory tract infection involves the nasal cavity, sinuses, nasopharynx, throat, larynx, conjunctiva, or inner ear. Lower respiratory tract infection involves the trachea and bronchoalveolar tree and includes bronchitis, bronchiolitis, and pneumonia. Clinical presentations overlap extensively, so etiologic diagnosis requires testing. Young children, older adults, patients with chronic pulmonary disease, and immunocompromised patients are at highest risk of severe disease. Viral load typically peaks within 2 days of symptom onset and falls to undetectable by day 5-8, with longer detection in severe or immunocompromised disease. Early collection matters. Nasopharyngeal aspirate gives the highest upper-airway yield, but a flocked nasopharyngeal swab in viral or universal transport medium is collected more often because it is easier to obtain. For isolated lower-tract disease, sputum, endotracheal aspirate, or BAL has higher viral loads.

Multiplex NAAT panels have replaced viral culture and antigen testing as the primary respiratory diagnostic in most U.S. clinical laboratories. CDC recommends influenza testing for all patients with suspected influenza who are being admitted to hospital; outpatient diagnosis during community activity does not always require a test.¹

Herpesviruses and infectious mononucleosis

All human herpesviruses are enveloped double-stranded DNA viruses that establish lifelong latency after primary infection and can reactivate. HSV-1/2 and VZV are alphaherpesviruses latent in sensory ganglia. CMV is a betaherpesvirus latent in myeloid-lineage cells. EBV is a gammaherpesvirus latent in memory B cells.

Primary EBV infection in early childhood is usually asymptomatic or nonspecific; primary infection in adolescence or young adulthood produces classic infectious mononucleosis. Transmission is via infected saliva or, less commonly, transfusion or transplantation. Incubation is 30-50 days. The classic triad is pharyngitis, fever, and lymphadenopathy. Other findings include fatigue, tonsillar enlargement, palatal petechiae, hepatosplenomegaly, and a drug-triggered rash, especially after a β-lactam. Hematologically, IM shows absolute lymphocytosis (>4,500/μL or >50% on differential), often with more than 10% atypical lymphocytes.

Heterophile (Monospot) testing detects IgM antibodies that agglutinate sheep or horse red cells. CDC does not recommend the Monospot for general use: heterophile antibodies are often absent in children, false positives occur with other infections, malignancy, or autoimmune disease, and a positive result does not confirm EBV itself. Heterophile testing cannot rule out primary EBV infection in young children. EBV-specific serology resolves heterophile-negative or ambiguous cases:²

CDC currently states that EBNA antibody (standard immunofluorescence) is not seen in the acute phase and slowly appears 2-4 months after onset. Textbook teaching that used a 6-12 week window for EBNA-1 IgG is therefore qualified to that later CDC interval.

EBV DNA NAAT is used to monitor EBV-associated malignancy and post-transplant lymphoproliferative disease. About 10% of mononucleosis-like illness has a non-EBV cause: primary CMV, human herpesvirus 6, HSV-1, adenovirus, acute HIV, group A streptococcal pharyngitis, or toxoplasmosis.

Primary VZV infection causes varicella, which is highly transmissible by contact and airborne routes. Its diffuse vesicular rash has lesions in multiple stages of healing simultaneously. After primary infection, VZV becomes latent in sensory ganglia; reactivation produces zoster. PCR of lesion fluid or a swab is preferred; saliva sampling can also be sensitive early. Complications occur most often in infants, adults, and immunocompromised hosts.

HSV-1 is classically transmitted via oral secretions in childhood; HSV-2 is classically transmitted sexually. Site overlap is now common, and most infections of either type are subclinical. NAAT is preferred. Culture is retained for phenotypic antiviral-resistance testing, currently the available format for HSV. Type-specific serology can remain negative for 2 weeks to 3 months after infection; it is used to establish past exposure or serostatus.

Congenital and perinatal viral infections

The historical TORCH mnemonic groups infections that share overlapping rash and ocular findings in the neonate. Toxoplasmosis and syphilis belong with parasitology and immunology; rubella, CMV, and HSV are covered here.

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Viral meningitis and encephalitis

Meningitis is meningeal inflammation with CSF pleocytosis; aseptic meningitis denotes a negative routine bacterial culture. Encephalitis is inflammation of the brain parenchyma itself.

Diagnosis for the arboviral encephalitis viruses listed here is usually serologic.