Sampling variability between two mid-turbinate swabs of the same patient has implications for influenza viral load monitoring
© Van Wesenbeeck et al.; licensee BioMed Central. 2014
Received: 30 July 2014
Accepted: 19 December 2014
Published: 24 December 2014
With the clinical development of several antiviral intervention strategies for influenza, it becomes crucial to explore viral load shedding in the nasal cavity as a biomarker for treatment success, but also to explore sampling strategies for sensible and reliable virus collection.
In this study, 244 patients suffering from Influenza like Illness and/or acute respiratory tract infection were enrolled. Sampling was done using mid-turbinate flocked swabs and two swabs per patient were collected (one swab per nostril). The influenza A viral loads of two mid-turbinate flocked swabs (one for each nostril) per patient were compared and we have also assessed whether normalization for human cellular DNA in the swabs could be useful. The Influenza mid-turbinate nasal swab testing resulted in considerable sampling variability that could not be normalized against co-isolated human cellular DNA.
Influenza viral load monitoring in nasal swabs could be very valuable as virological endpoints in clinical trials to monitor treatment efficacy, in analogy to HIV, HBV & HCV viral load monitoring. However, the differences between left and right nostrils, as observed in this study, highlight the importance of proper sampling and the need for standardized sampling procedures.
KeywordsInfluenza viral load Nasal swabs Sampling
Respiratory tract infections (RTI) have an enormous social economic impact, with high incidence of hospitalization and high costs ,. Because of similar clinical symptoms and simultaneous circulation of several different viruses, the etiology is often unknown. Timely detection and discrimination of the infecting pathogens is crucial to optimize treatment and care, to prevent unnecessary antibiotic use, and to prevent secondary spread of infection. Adequate specimen collection is the first crucial step for the correct diagnosis of influenza and other respiratory infections. Correction for the dilution in nasopharyngeal aspirates might improve the detection of respiratory infections ,. Many studies have described that mid-turbinate flocked swabs are less invasive than other specimen collection types (nasopharyngeal swabs, aspirates & washes), have a good sensitivity to detect respiratory viruses and are therefore a good alternative for specimen collection -. Moreover, these mid-turbinate swabs have the possibility for self-collection (either adult patients or parents of children at home) -. To our knowledge, these studies have compared the different specimen types and/or specimen collection by a health care worker and self-collection, but none of them have compared the variability of respiratory virus detection between the two nostrils of the same patient at the same time point. In HIV, HCV, and HBV, viral load monitoring is a key diagnostic parameter to measure treatment success -. With the clinical development of several antiviral intervention strategies for influenza, it becomes crucial to explore viral load shedding in the nasal cavity as a biomarker for treatment success, but also to explore sampling strategies for sensible and reliable virus collection. In this study, the influenza A viral loads of two mid-turbinate flocked swabs (one for each nostril) per patient were compared. We have also assessed whether normalization for human cellular DNA in the swabs could be useful.
A total of 244 patients suffering from Influenza Like Illness (ILI) and/or acute respiratory tract infection (RTI), presenting for medical support at a primary care physicians’ office in Belgium, were enrolled between February-March 2012 and January-March 2013, as previously described . The study was approved by the Committee on Medical Ethics of the University Hospital of Leuven in Belgium and written consent from all participants was obtained. Only patients symptomatic for fewer than 3 days were included in the study. Sampling was done using mid-turbinate flocked swabs (Copan, Italy) and two swabs per patient were collected (one swab per nostril; info on the order of the swab collections (left/right nostril) is not available). One swab was re-suspended in 1 ml Universal transport medium (UTM) (swab A) and the other swab in 3 ml UTM (swab B) to define if the swab collection in a smaller volume results in a higher viral load. Swabs were kept refrigerated (4°C) until transport to central lab where the samples were stored at -80°C. Viral RNA was isolated using the Easymag system (Biomerieux) and Influenza viral load was determined in each swab using qRT-PCR, which was performed according to our in-house protocol for Influenza A (InfA) (targeting the Matrix gene) (based on the CDC protocol ) with a panel of oligonucleotide primers and dual-labeled hydrolysis (TaqMan®) probes, as previously described . Each sample was tested in duplicate. The InfA qRT-PCR is characterized by a low inter- and intra-variability (average %CV < 2.5%) (data not shown). All Ct values were corrected for the loss of RNA during extraction by use of the internal extraction control (IEC) . A standard RNA dilution series (8 dilutions; External quantification control (EQC)) was tested in duplicate in each qRT-PCR experiment . The viral loads of the processed samples were calculated as log10 copies/ml through back calculation on the EQC standard curve, which has the following characteristics y = -3.03x + 40.17 with r2 = 0.99 and a linear range from 4.3 to 10.3 log10 copies/ml (lower and upper limit of quantification (LLOQ and ULOQ)). All samples with an InfA viral load below 4.3 log10 copies/ml were defined as ‘below LLOQ’. To detect the amount of human cells in the mid-turbinate swabs, qPCR (on DNA level) was performed with primers/probe located on the human RNaseP gene, as proposed by CDC . Each sample was tested in duplicate. Amplification and detection were performed on Lightcycler® 480 instrument (Roche Applied Science). The 25 μl reaction mixture of the qPCR was comprised of 5 μl of nucleic acid, 12.5 μl of 2x PCR master mix, 0.5 μl of Platinum® Taq Mix (Life Technologies), 0.5 μl of forward and reverse primers (40 μM) and 0.5 μl of labeled probe (10 μM), and 5.5 μl water. The thermal cycling conditions were as follows: 95°C for 2 min, followed by 45 cycles of PCR amplification (95°C for 15 s and 55°C for 30 s). A 1/10 dilution series of human control genomic DNA (Applied Biosystems) was tested in duplicate on each real-time PCR plate and has the following characteristics: y = -3.33x + 40.04 with r2 = 0.99.
Flu surveillance studies focus on identifying the circulating influenza viruses and their main characteristics, independent of the viral quantity. Influenza viral load monitoring (on different timepoints) in nasal swabs could be very valuable as virological endpoints in clinical trials to monitor treatment efficacy, in analogy to HIV, HBV & HCV viral load monitoring. The differences in InfA viral load between the left and right nostrils, as observed in this study, could be related to variability of the qRT-PCR, biological differences between the nostrils, sampling bias or any combination of these. The qRT-PCR is characterized by a low intra- and inter variability (CV < 2.5%). A right- or left-handed person may obtain the swab differently from the right/left nostril or the order of taking the swabs could also introduce variability. Unfortunately, that info was not collected in this study but this should be included in further studies. The biological differences could include anatomic nose/septum abnormalities, variability in virus shedding or clogging of the nostrils. The differences in Inf A viral load between left and right nostrils, as observed in this study, highlight the importance of proper sampling and the need for standardized sampling procedures. Several options for sampling can be proposed: one swab per patient (which is now the case for most studies), two swabs per patient (one for each nostril), two swabs per patient (one for each nostril) with collection in the same vial, the use of one swab for sampling of both nostrils. Further studies are definitely needed to further explore the use of mid-turbinate swabs, the variability between the nostrils of a patient and to propose one standard procedure for specimen collection which would improve the adequate detection of influenza/respiratory viruses and in the end will contribute to adequately monitor treatment efficacy in clinical trials.
We thank the physicians and all of the volunteers that participated in this study. We also thank Janssen Biobank and the Lab Operations and Medical Affairs department from Janssen Diagnostics for their logistic support and Ole Lagatie, Kristiane Schmidt and Peter Van den Eede for scientific discussions.
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