Molecular quantification of respiratory syncytial virus in respiratory samples: reliable detection during the initial phase of infection.
ABSTRACT Quantitative real-time PCR for the detection of respiratory syncytial virus (RSV) RNA is increasingly used to study the causal role of RSV in lower airway disease. The objective of our study was to evaluate variations in RSV RNA loads at different steps in the RNA quantification process: (i) variation in RSV RNA load within one sample (step 1), (ii) variation in the load in samples from patients who were sampled twice on the same day (step 2), and (iii) variation in the load between simultaneously taken nasopharyngeal aspirate (NPA) samples and tracheal aspirate (TA) samples (step 3). Thirty-two infants with RSV infection at the pediatric intensive care unit (PICU) were included. NPA and TA samples were taken three times a week during ventilation and were not diluted. Intrasample variation (step 1) was shown to be minimal (<0.5 log(10) particles/ml). Intraday variation (step 2) was the lowest for samples with high viral loads (95% limits of agreement, -1.3 to +0.9 log(10)), whereas it increased for samples with relatively lower viral loads (viral load, <6.0 log(10) particles/ml; n = 138 sample pairs from 20 patients). RSV loads in NPA and TA samples (step 3) were found to be the most comparable during the early phase of infection (95% limits of agreement, -1.5 to +1.4 log(10)). The variation increased during the late phase of infection (i.e., in follow-up samples), with the loads in NPA samples remaining significantly higher than the loads in TA samples (n = 138 sample pairs from 31 patients). In conclusion, quantitative detection of RSV RNA in undiluted mucus is a reliable method to quantify viral loads. Nasopharyngeal aspirate samples collected in the initial phase of infection can be used to predict RSV RNA loads in the lower airways.
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ABSTRACT: In 5-40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3'-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3-7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.PLoS ONE 01/2011; 6(1):e16118. · 4.09 Impact Factor