Human Parainfluenza Viruses (HPIV) and Other Parainfluenza Viruses Workup

Updated: Mar 19, 2019
  • Author: Subhash Chandra Parija, MD, MBBS, PhD, DSc, FRCPath; Chief Editor: Mark R Wallace, MD, FACP, FIDSA  more...
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Workup

Approach Considerations

In human parainfluenza virus (HPIV) infection, the complete blood count (CBC) is usually within the reference range. The white blood cell (WBC) count is usually normal; however, lymphocytosis may be noted.

The diagnosis of HPIV infection can be confirmed in either of the following 2 ways:

  • Isolation and identification of the virus in cell culture or direct detection of the virus in respiratory secretions by means of immunofluorescent assay, enzyme-linked immunosorbent assay (ELISA), or polymerase chain reaction (PCR) assay

  • Demonstration of a significant rise in specific immunoglobulin G (IgG) antibodies between appropriately collected paired serum specimens or in specific immunoglobulin M (IgM) antibodies in a single serum specimen

On histologic examination, the epithelium of the respiratory tract may show inflammation and necrosis. Subglottic tissues in particular may appear to be involved.

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Viral Testing

Collection and preparation of clinical specimens

Nasopharyngeal aspirations, nasal washings, and nasal aspirations are the optimal specimens, though throat and nasal swabs can also be used. Specimens should be collected and placed in viral transport media, preferably at 4°C, as the infectivity is lost at temperatures above 4°C; if a delay of more than 24 hours is anticipated, specimens should be frozen. In rare situations, nonrespiratory specimens (eg, cerebrospinal fluid [CSF], rectal swabs, or stool samples) may be used.

Paired sera (acute and convalescent phase) should be collected, separated quickly, and stored at either –20°C or –70°C; the 2 samples should be tested simultaneously.

Electron microscopy

Micro drops 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, the sensitivity of this study is poor.

Indirect immunofluorescence

Indirect immunofluorescent assay is normally used with antisera against each of the HPIV serotypes. However, the findings from this assay cannot be used as the sole diagnostic criterion, because its sensitivity, like that of electron microscopy, is poor.

Direct detection of viral antigens

Synthesized recombinant HPIV-1 and HPIV-3 nucleocapsid proteins in the yeast Saccharomyces cerevisiae are used as a source of viral antigens. [23] HPIV antigen can be detected with ELISA, radioimmunoassay, fluoroimmunoassay, or immunofluorescent assay; the last two tests are both rapid and specific. Shell vial assay is another method for rapid identification of HPIV. This method yields an average sensitivity of 84% in testing against standard tissue culture-positive HPIV cases.

Isolation of virus

HPIV grows best in primary monkey kidney (PMK) cells (from rhesus, cynomolgus, and African green monkeys). LLC-MK2 cells are also excellent for continued passage and are almost as good as PMK cells for primary isolation. HPIV-2 induces 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 cells, especially osteoclasts. [24]

To recover all HPIV serotypes, trypsin (2-3 mg/mL) must be added to the maintenance medium of LLC-MK2 cells. 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 are rarely demonstrated during primary isolation of HPIV in tissue culture, except in the case of HPIV-2, which shows syncytial formation when cultured. All HPIVs demonstrate greater cytopathic effects upon adaptation to a particular cell line; HPIV-3 is the most aggressive, destroying more than 50% of tissue culture monolayer by day 3.

Viral growth is detected with hemadsorption inhibition using guinea pig erythrocytes within 3-10 days of incubation. However, the prolonged incubation time required severely restricts the usefulness of virus isolation in short-term management. The neuraminidase of HPIV-4 appears to be temperature sensitive because this virus hemadsorbs better at room temperature or 37°C, while all of the other serotypes react well at 4°C.

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 samples. Hemagglutination inhibition, neutralization, complement fixation, ELISA, radioimmunoassay, and Western blotting are frequently used antibody-based serologic tests for diagnosis of HPIV infections.

Genomic detection

HPIV RNA can be detected directly by means of Northern hybridization or a dot blot analysis using virus-specific DNA probes. PCR assay is sensitive and specific in detecting HPIV. In one study, PCR assay yielded twice the sensitivity of cultures and 4 times the sensitivity of immunofluorescent antibody staining. [25] A multiplex reverse transcriptase PCR (RT-PCR) assay for detecting HPIV-1, HPIV-2, and HPIV-3 has been developed.

An RT-PCR enzyme hybridization assay (RT-PCR-EHA) is available for detecting HPIV types 1-4, respiratory syncytial virus (RSV) types A and B, and influenza A and B virus, with a reported sensitivity of 95-100% and specificity of 97-100% in comparison with the results of tissue culture. The RT-PCR-EHA yields results in approximately 7 hours.

A multiplex RT-PCR assay kit using a dual priming oligonucleotide (DPO) system is now available; this kit is capable of detecting 12 common viruses that cause respiratory tract infections in children. [26] A multiplex PCR assay capable of detecting 15 common viruses that cause respiratory infections has been reported. [27]

Currently, multiple multiplex real-time PCRs based on Taqman chemistry are commercially available, which can be used to detect HPIV, along with other respiratory pathogens. [28, 29]

Luminex xTAG assays have enabled multiplexing of multiple targets in a single assay. The commercially available Luminex xTAG Respiratory Viral panel is capable of detecting 19 different pathogens, including all four types of HPIV. Initial comparative studies of Luminex respiratory panel with conventional or multiplex real-time PCR assays have revealed a higher diagnostic yield for most of the respiratory pathogens, including all four types of HPIVs.

In a study by Ko et al, [30] Luminex panel results correlated with real-time assay results for most respiratory pathogens (94.1%-100% of cases), but the correlation percentage was relatively low (94.1%-97.6%) for HPIV-3. The overall yield was higher for the Luminex panel, with 4 additional HPIV-3 detections. Although the technology is high throughput, the high cost of the assay is the limiting factor for wide-scale use.

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Neck and Chest Radiography

Radiographs of the neck or chest are important if epiglottitis, croup, or pneumonia is a possibility.

In patients with croup, anteroposterior views of the neck may demonstrate the classic steeple sign (ie, subglottic swelling with narrowing of the air shadow of the trachea.

Lateral soft tissue films of the neck are normal in most cases. However, such films can be useful if the diagnosis is unclear, especially if conditions such as foreign body aspiration, retropharyngeal abscess, or epiglottitis are in the differential diagnosis. In patients with epiglottitis, lateral views may demonstrate enlargement of the epiglottis and ballooning of the hypopharynx.

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