Comparison of quantitative reverse transcription-PCR to viral culture for assessment of respiratory syncytial virus shedding

Abstract

Respiratory syncytial virus (RSV) has recently been recognized as a serious pathogen in elderly and immunocompromised adults. Diagnosis of acute infection in adults is often difficult due to the insensitivity of viral culture, and reverse transcription-PCR (RT-PCR) is a more sensitive alternative. The relationship of quantitative RT-PCR to viable virus has never been studied for RSV. Therefore, we compared a quantitative real-time RT-PCR with viral culture to assess viral load in adult volunteers challenged with the RSV A2 strain. Twelve of 13 volunteers were infected, and there was a high correlation (r = 0.84) between quantitative RT-PCR and viral titer by cell culture. However, RT-PCR was more sensitive, with 73 of 169 (43%) samples positive compared to 58 (34%) samples positive by culture. The correlation between the two tests was highest early in the course of viral shedding (r = 0.91, days 0 to 6), whereas during days 7 to 13, there was more variability (r = 0.70). All subjects were culture negative by day 11, whereas one subject remained RT-PCR positive on day 12. All subjects were RT-PCR negative at day 28 postinfection. Quantitative RT-PCR has an excellent correlation with viral titers, as measured by culture, and should be a useful tool for future studies addressing viral load and disease pathogenesis.

FIG. 1.

Quantitative RT-PCR. (A) The PCR uses the Taqman principle with RFU monitored on an iCycler thermocycler. RFU were plotted, and the Ct for each reaction was determined. Six 10-fold dilutions of stock virus (10⁶ PFU/ml) were used to generate a standard curve. Shown from left to right are the curves of decreasing concentrations of stock virus from dilutions of 10⁻¹ to 10⁻⁶. The Ct is defined as the point at which the curve crosses the horizontal threshold line at 60 RFU. The limit of detection for the assay was defined as the 10⁻⁵ dilution (10 PFU/ml), because the 10⁻⁶ dilution was not reliably positive. (B) The log₁₀ titer of virus in a specimen was plotted against the Ct value, and a best fit line was constructed. The linear range of the assay is from 1 to 10⁵ PFU/ml, with a correlation coefficient of 0.99. RSV quantity in unknown clinical samples, expressed as log₁₀ PFU-per-milliliter equivalents, is derived by plotting the Ct of an unknown on the standard curve.

FIG. 2.

Graphic representation of the relationship of RSV viral load in nasal specimens (left y axis) and nasal IgA titer (right y axis and shaded line) in 12 infected volunteers challenged with the RSV A2 strain. The virus titer is reported as TCID₅₀ per milliliter for culture (solid line) and as PFU-per-milliliter equivalents for RT-PCR (dotted line) as described in Materials and Methods and the legend to Fig. 1. Each graph represents 1 of the 12 subjects infected with RSV.

FIG. 2.

Graphic representation of the relationship of RSV viral load in nasal specimens (left y axis) and nasal IgA titer (right y axis and shaded line) in 12 infected volunteers challenged with the RSV A2 strain. The virus titer is reported as TCID₅₀ per milliliter for culture (solid line) and as PFU-per-milliliter equivalents for RT-PCR (dotted line) as described in Materials and Methods and the legend to Fig. 1. Each graph represents 1 of the 12 subjects infected with RSV.