Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Comparison of sampling methods for the detection of human rhinovirus RNA.

Author(s): Waris M, Österback R, Lahti E, Vuorinen T, Ruuskanen O, Peltola V

Publication: J Clin Virol, 2013, Vol. 58, Page 200-4

PubMed ID: 23810645 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of room temperature storage duration on human rhinovirus RNA levels in nasopharyngeal aspirate specimens measured by real-time qRT-PCR. Specimens were collected from 39 children with symptoms of viral respiratory infection (nasal discharge and stuffiness, cough, sore throat, or fever). Nasopharyngeal aspirates were collected using a disposable mucus extractor by inserting the catheter through the nostril until the resistance of the nasopharynx was felt, and pulling it back while applying suction from an electronic device. Six flocked, cotton swabs were dipped into the aspirate, placed in dry tubes, and RNA was extracted immediately with or without frozen storage at −70°C and after room temperature storage for 0, 1, 2, 3, or 4 days. RNA was extracted using an E.Z.N.A. Viral RNA Isolation Kit and viral RNA copy numbers were determined by real-time qRT-PCR targeting the human rhinovirus 5’ non-coding region. Specimens from eight rhinovirus positive individuals were included in the study.

   Summary of Findings:

   The viral RNA CT values were not affected by storage of nasopharyngeal aspirate swabs at room temperature for up to 4 days and there were no significant differences between the calculated mean amounts of rhinovirus RNA in specimens for any of the time points (P=1.000).

   Biospecimens

   • Cell - Nasopharynx

   Preservative Types

   • Frozen
   • None (Fresh)

   Diagnoses:

   • Pneumonia/Respiratory Infection

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Time at room temperature	0 days 1 day 2 days 3 days 4 days
   Real-time qRT-PCR Specific	Targeted nucleic acid	human rhinovirus 5’ non-coding region

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2. Study Purpose

   This study compared human rhinovirus RNA levels measured by real-time qRT-PCR in nasal swab types and among different specimen types (nasal swab specimens, oropharyngeal swab specimens, induced sputum, and nasopharyngeal aspirates). The effect of mailing nasopharyngeal specimens on viral loads was also investigated. Specimens were collected from 39 children with symptoms of viral respiratory infection (nasal discharge and stuffiness, cough, sore throat, or fever), and from their accompanying parents if they also had acute respiratory symptoms. Nasal specimens were collected using a wood-shafted cotton swab or a nylon flocked swab, and oropharyngeal (throat) specimens were collected using a wood-shafted cotton swab. Swabs were placed in dry, sterile tubes and frozen immediately at -70°C. Nasopharyngeal specimens were collected using a disposable mucus extractor by inserting the catheter through the nostril until the resistance of the nasopharynx was felt and pulling it back while applying suction from an electronic device. Two cotton swabs were dipped in the nasopharyngeal aspirate; placed in dry, sterile tubes; and one was frozen immediately at -70°C and the other was sent to the laboratory using standard mail. Paired induced sputum and nasopharyngeal specimens collected by unspecified methods for pneumonia diagnosis were also compared for viral loads. RNA was extracted with the Nuclisense easy-Mag automated nucleic acid extractor and viral RNA copy numbers were determined by real-time qRT-PCR targeting the human rhinovirus 5’ non-coding region and overall cellular content was measured by real-time qRT-PCR of β-actin mRNA.

   Summary of Findings:

   Compared to freshly frozen nasopharyngeal aspirates, nasal nylon flocked swab specimens had comparable average rhinovirus loads (mean log copies/sample ± SD) (7.3 ± 1.0 versus 7.3 ± 1.1, P=1.000) and nasopharyngeal specimens collected on cotton swabs had slightly, but not significantly lower loads (6.9 ± 1.2 versus 7.3 ± 1.1, P=0.313); however, there was a 26-fold lower average yield in the matched oropharyngeal swabs (5.9 ± 1.3, P<0.0001).  There was a 2.6-fold decrease observed when the nasopharyngeal swab specimen was mailed (6.9 ± 1.3, P=0.009). There was a modest positive correlation of rhinovirus and β-actin copy numbers between induced sputum and nasopharyngeal specimens (R2= 0.586 and R2=0.647, respectively) but copy numbers tended to be higher in induced sputum (5.3 ± 1.9 vs. 5.0 ± 1.7, P=0.079), while no differences were observed for β-actin mRNA (4.3 ± 1.7 vs. 4.5 ± 1.6, P=0.369). The overall correlation between rhinovirus RNA and β-actin mRNA copy numbers was poor (R2=0.181). When the ratio of rhinovirus RNA/β-actin mRNA was compared, the relative virus loads in sputum and nasopharyngeal specimens were similar (P=0.184).

   Biospecimens

   • Cell - Nasal cavity
   • Cell - Sputum
   • Cell - Oral Cavity
   • Cell - Nasopharynx

   Preservative Types

   • Frozen

   Diagnoses:

   • Pneumonia/Respiratory Infection

   Platform:

   Analyte	Technology Platform
   RNA	Real-time qRT-PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Method of cell acquisition	nylon flocked swab cotton swab
   Biospecimen Acquisition	Biospecimen location	nasopharynx oropharynx nasal cavity induced sputum
   Real-time qRT-PCR Specific	Targeted nucleic acid	human rhinovirus 5’ non-coding region β-actin mRNA
   Storage	Between site transportation method	Mailed Not transported

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