Influenza A viral loads in respiratory samples collected from patients infected with pandemic H1N1, seasonal H1N1 and H3N2 viruses

Abstract

BackgroundNasopharyngeal aspirate (NPA), nasal swab (NS), and throat swab (TS) are common specimens used for diagnosis of respiratory virus infections based on the detection of viral genomes, viral antigens and viral isolation. However, there is no documented data regarding the type of specimen that yields the best result of viral detection. In this study, quantitative real time RT-PCR specific for M gene was used to determine influenza A viral loads present in NS, NPA and TS samples collected from patients infected with the 2009 pandemic H1N1, seasonal H1N1 and H3N2 viruses. Various copy numbers of RNA transcripts derived from recombinant plasmids containing complete M gene insert of each virus strain were assayed by RT-PCR. A standard curve for viral RNA quantification was constructed by plotting each Ct value against the log quantity of each standard RNA copy number.ResultsCopy numbers of M gene were obtained through the extrapolation of Ct values of the test samples against the corresponding standard curve. Among a total of 29 patients with severe influenza enrolled in this study (12 cases of the 2009 pandemic influenza, 5 cases of seasonal H1N1 and 12 cases of seasonal H3N2 virus), NPA was found to contain significantly highest amount of viral loads and followed in order by NS and TS specimen. Viral loads among patients infected with those viruses were comparable regarding type of specimen analyzed.ConclusionBased on M gene copy numbers, we conclude that NPA is the best specimen for detection of influenza A viruses, and followed in order by NS and TS.

Virtual quantification of influenza A virus load by real-time RT-PCR

“…The intra-laboratory variability (CV) was lower than 5% in all dilutions and the inter-laboratory variability ranged from 0.92% to 2.70%. The Ct values and the standard curve equation were in agreement with data presented by Ngaosuwankul et al 2 A total of 120 respiratory samples (30 from each center) were quantified by comparison of Ct values with usual locally generated standard curves and using the VQT. The median copy number according to the locally generated standard curve was 2.5 × 10 5 RNA copies/ml sample (range 1.9 × 10 2 to 5.4 × 10 8 RNA copies/ml sample) while according to VQT was 3.1 × 10 5 RNA copies/ml sample (range 1.7 × 10 2 to 6.3 × 10 8 RNA copies/ml sample).…”

Virus shedding and environmental deposition of novel A (H1N1) pandemic influenza virus: interim findings

“…• Nasal swab Although a nasopharyngeal aspirate (NPA) is considered to be the best specimen for detecting influenza A viruses, [53][54][55] this procedure causes more discomfort and is more difficult to perform, particularly in children. Indeed, studies attempting to collect daily NPA samples from subjects have reported problems with subjects' tolerance and compliance with the procedure.…”

Nucleic acid amplification-based diagnosis of respiratory virus infections

“…A sensitive and specific TaqMan assay targeting a single nucleotide polymorphism within the NP gene has been described that can differentiate pandemic H1N1 from other swine virus [45]. Recently, a quantitative RT-PCR targeting the M gene was used to compare the pandemic H1N1 viral loads in NP, NPA and throat swab samples from 12 patients [46]. NPA was found to contain the highest amount of virus followed by nasal swab and throat swab.…”