Cellular DNA quantification in respiratory samples for the normalization of viral load: a real need?

Abstract

Background: Respiratory tract infections have an enormous social economic impact, with high incidence of hospitalization and high costs. Adequate specimen collection is the first crucial step for the correct diagnosis of viral respiratory infections.

Objectives: The present retrospective study aimed: i) to verify the cell yield obtained from sampling the nasal respiratory tract using mid-turbinate flocked swabs; ii) to evaluate the normalization of viral load, based on cell number; and iii) to compare the kinetics of viral infection obtained with normalized vs non-normalized viral load.

Study design: The number of cells were quantified by real-time PCR in residual extract of nasal swabs tested for respiratory viruses detection and stored at -80 °C in a universal transport medium (UTM™).

Results: A total of 513 virus-positive and 226 virus-negative samples were analyzed. Overall, a median of 4.42 log₁₀ β2-microgolubin DNA copy number/ml of UTM^™ (range 1.17-7.26) was detected. A significantly higher number of cells was observed in virus-positive as compared to virus-negative samples (4.75 vs 3.76; p < 0.001). Viral loads expressed as log₁₀ RNA copies/ml of UTM^™ and log₁₀ RNA copies/median number of cells were compared in virus-positive samples and a strict correlation (r = 0.89, p < 0.001) and agreement (R2 = 0.82) were observed. In addition, infection kinetics were compared using the two methods with a follow-up series of eight episodes of viral infection and the mean difference was -0.57 log₁₀ (range -1.99 to 0.40).

Conclusions: The normalization of viral load using cellular load compliments the validation of real-time PCR results in the diagnosis of respiratory viruses but is not strictly needed.

Keywords: Cell number; Flocked mid-turbinate nasal swabs; Quantification; Respiratory viruses; Viral load.

Fig. 1

Comparison of the number of cells measured in mid-turbinate flocked nasal swabs in the overall, virus-positive and virus-negative samples (A). Frequency distribution of the number of cells measured in respiratory virus-positive (white bars) versus virus-negative (grey bars) samples (B). Log-Log linear regression plots comparing the viral load and number of cells expressed as log₁₀ β2-microgolubin DNA copy numbers/ml of UTM™ (C).

Fig. 2

Correlation analysis between viral loads expressed in log₁₀ copies/ml of UTM™ or normalized to log₁₀ copies/ median number of cells (A). Bland-Altman plots of log₁₀ differences in viral RNA loads against the two methods of expressing results (B). The acceptability range (1 to -1 Log₁₀ difference) is shaded in grey.

Fig. 3

Viral load kinetics measured in 8 episodes of respiratory infection expressed as log₁₀ RNA copies/ml of UTM™ (solid line) and as log₁₀ RNA copies/median number of cells (dashed line). HRV, human rhinovirus; FluA, influenza A virus; RSV, respiratory syncytial virus.