Nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control

Abstract

BackgroundCarefully conducted, community-based, longitudinal studies are required to gain further understanding of the nature and timing of respiratory viruses causing infections in the population. However, such studies pose unique challenges for field specimen collection, including as we have observed the appearance of mould in some nasal swab specimens. We therefore investigated the impact of sample collection quality and the presence of visible mould in samples upon respiratory virus detection by real-time polymerase chain reaction (PCR) assays.MethodsAnterior nasal swab samples were collected from infants participating in an ongoing community-based, longitudinal, dynamic birth cohort study. The samples were first collected from each infant shortly after birth and weekly thereafter. They were then mailed to the laboratory where they were catalogued, stored at -80°C and later screened by PCR for 17 respiratory viruses. The quality of specimen collection was assessed by screening for human deoxyribonucleic acid (DNA) using endogenous retrovirus 3 (ERV3). The impact of ERV3 load upon respiratory virus detection and the impact of visible mould observed in a subset of swabs reaching the laboratory upon both ERV3 loads and respiratory virus detection was determined.ResultsIn total, 4933 nasal swabs were received in the laboratory. ERV3 load in nasal swabs was associated with respiratory virus detection. Reduced respiratory virus detection (odds ratio 0.35; 95% confidence interval 0.27-0.44) was observed in samples where the ERV3 could not be identified. Mould was associated with increased time of samples reaching the laboratory and reduced ERV3 loads and respiratory virus detection.ConclusionSuboptimal sample collection and high levels of visible mould can impact negatively upon sample quality. Quality control measures, including monitoring human DNA loads using ERV3 as a marker for epithelial cell components in samples should be undertaken to optimize the validity of real-time PCR results for respiratory virus investigations in community-based studies.

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

“…In these studies, molecular cell quantification using real-time PCR was performed and therefore the data obtained could be compared with those presented here. In addition, our results were consistent with the findings observed in these two studies, in which samples positive for single or multiple viruses had a greater number of cells as compared to virus-negative nasal swabs [14,15]. In these studies, several reasons such as increased epithelial desquamation or a cytopathic effect induced in vivo have previously been hypothesized to explain this observation [14,15].…”

“…The median number of cells measured in mid-turbinate flocked nasal swab samples was similar to that observed in other recent studies [14,15]. In these studies, molecular cell quantification using real-time PCR was performed and therefore the data obtained could be compared with those presented here.…”

Nasal swab bacteriology by PCR during the first 24‐months of life: A prospective birth cohort study

“…Routine laboratory quality assurance included assaying samples for the human DNA marker, ERV3, to ensure human cells were present. While important for viral detection, it seems also relevant for bacteria as we observed considerably higher bacterial detection rates in samples containing ERV‐3. Consequently, only high‐quality nasal swabs were used to avoid compromising sensitivity in the analysis.…”

“…Any extract having a >3 cycle threshold (Ct) difference to that of the expected value by EHV real‐time PCR assay was considered to have failed quality control and the sample was re‐extracted. Nasal swab specimen quality was determined by screening for human DNA using an endogenous retrovirus‐3 (ERV3) assay as described previously . Swabs with ERV3 Ct values >38 were deemed lower‐quality and they, and associated person‐time, were excluded from analyses.…”

Influenza vaccine efficacy in young children attending childcare: A randomised controlled trial

“…Qualitative real‐time PCR was performed for Influenza A and B and other 13 respiratory viruses . This method is well established and short delays between swab collection and processing in the laboratory do not impact on the likelihood of identifying any/total respiratory virus(es) from nasal swabs …”