Parainfluenza Virus Workup

• The CBC count is usually within the reference range. Lymphocytosis may be present.
• Virus antigen detection and isolation

  • Collection and preparation of clinical specimens: Nasopharyngeal aspirations, nasal washings, and nasal aspirations are optimum specimens for collection. Throat swabs and nasal swabs can also be used. Specimens should be collected and placed in viral transport media (VTM), preferably at 4°C; if a delay of more than 24 hours is anticipated, specimens should be frozen. Nonrespiratory specimens such as CSF, rectal swabs, and stool, though rare, can be used. Paired sera (acute and convalescent phase) should be collected, separated quickly, and stored at either -20°C or -70°C; both samples should be tested simultaneously.
  • Electron microscopy: Microdrops of secretions or garglings are placed directly on carbon-coated electron microscopy grids and stained with phosphotungstic acid. Virions typical of the Paramyxoviridae may be observed. However, this study yields poor sensitivity.
  • Immunofluorescence: Indirect immunofluorescence is normally used with antisera against each serotype of parainfluenza virus. However, immunofluorescence findings cannot be used as the sole diagnostic criterion, as it also yields poor sensitivity. A study carried out in Arizona between 1983 and 1985 showed that immunofluorescence revealed human parainfluenza virus (HPIV)–1 in only 63% of throat specimens from which this serotype was isolated in tissue cultures. HPIV-3 was revealed in only 31% of virus-culture–positive throat secretions.
  • Direct detection of viral antigens: HPIV antigen can be detected with enzyme-linked immunoassay (ELISA), radioimmunoassay, fluoroimmunoassay, and immunofluorescence. Synthesized recombinant HPIV-1 and HPIV-3 nucleocapsid proteins in the yeast Saccharomyces cerevisiae are used as a source of antigens.^[6] The latter two tests are both rapid and specific. Shell vial assay is another method for rapid identification of HPIV. Shell vials have yielded an average sensitivity of 84% in testing against standard tissue culture–positive HPIV cases.
  • Virus isolation: HPIV grows best in primary monkey kidney (PMK) cells (rhesus monkey kidney cells, cynomolgus, and African green monkeys). The LLC-MK2 strain is also excellent for continued passage and is almost as good as PMK cells for primary isolation. HPIV-2 induced host ADAM8 expression in human salivary parotid adenocarcinoma cell line (HSY) during cell fusion. ADAM8 is responsible for cell-to-cell fusion and formation of multinucleate giant cell formation, especially in osteoclast formation.^[7] Trypsin (2-3 mg/mL) must be added to the maintenance medium of LLC-MK2 cells to recover all HPIV serotypes. Proteases are necessary for parainfluenza virus to replicate, and it is hypothesized that proteases present in primary cell cultures are absent in continuous cell lines.
  • Detection and typing: Cytopathic effects (CPEs) are rarely demonstrated during primary isolation of HPIV in tissue culture except with HPIV-2, which, when cultured, shows syncytia formation. All HPIVs demonstrate greater cytopathic effects upon adaptation to a particular cell line, with HPIV-3 being the most aggressive, destroying more than 50% of tissue culture monolayer by the third day. Virus growth is detected with hemadsorption inhibition using guinea pig erythrocytes within 3-10 days of incubation. However, the prolonged incubation time (3-10 d) required for virus isolation severely restricts the usefulness of virus isolation in short-term management.
  • Serologic diagnosis: A 4-fold rise or drop in titer is generally thought to signify acute infection if the testing is performed at the same time on paired acute- and convalescent-phase serum pimples. Hemagglutination inhibition, neutralization, complement fixation, ELISA, radioimmune assays, and Western blotting are frequently used antibody-based serological tests for diagnosis of HPIV infections.
  • Genomic detection: HPIV RNA can be detected directly with Northern hybridization or a dot blot analysis using virus-specific DNA probes. Polymerase chain reaction (PCR) assay is sensitive and specific in detecting HPIV. A multiplex reverse-transcriptase PCR (RT-PCR) assay for detecting HPIV-1, HPIV-2, and HPIV-3 has been developed. RT-PCR enzyme hybridization assay (RT-PCR-EHA) is available for detecting HPIV types 1-4, respiratory syncytial viruses A and B, and influenza A and B virus, with a reported sensitivity of 95-100% and specificity of 97-100% when compared with results of tissue culture. PCR yielded twice the sensitivity of cultures and 4 times the sensitivity of fluorescent antibody staining.^[8] The RT-PCR-EHA yields results in approximately 7 hours. A multiplex RT-PCR assay kit under dual priming oligonucleotide system (DPO) that is used to detect 12 common viruses causing respiratory tract infections in children has recently become available.^[9]

• Radiographs of the neck or chest are important if epiglottitis, croup, or pneumonia is a possibility.
• Anteroposterior views of the neck may demonstrate subglottic swelling with narrowing of the air shadow of the trachea (steeple sign) with croup; in patients with epiglottitis, lateral views may demonstrate enlargement of the epiglottis and ballooning of the hypopharynx.

The epithelium of the respiratory tract may show inflammation and necrosis. Subglottic tissues in particular may appear to be involved.