Research Article
16 February 2012

Evaluation of Swabs, Transport Media, and Specimen Transport Conditions for Optimal Detection of Viruses by PCR

ABSTRACT

Depletion of swabs and viral transport medium during epidemics may prompt the use of unvalidated alternatives. Swabs collected and transported dry or in saline were compared to commercially available swab/medium combinations for PCR detection of influenza, enterovirus, herpes simplex virus, and adenovirus. Each was detected at an ambient temperature (22°C) and 4°C for 7 days. Detection of influenza on dry or saline swabs is important because of its capacity to cause outbreaks involving large numbers of cases.

TEXT

During the 2009 influenza A H1N1 pandemic, the number of samples referred for influenza virus testing to our laboratory increased substantially (3). Virological swabs and transport medium became in short supply, and alternatives were often used, although systematic data regarding their suitability were lacking.
Molecular detection methods do not require replication competent virus, but preservation of nucleic acid is essential. Previous studies have examined the swab types (1, 6, 9, 10) and transport medium (7) optimal for identification of viruses, but often they do not assess variables such as transit times and holding temperatures between sample collection and testing. While many environmental studies have investigated the effects of temperature on virus survivability (2, 4, 5, 8), less is known about the impact of temperature on nucleic acid detection by PCR.
This study examined the stability of four viruses with different physicochemical properties in several swab and transport medium combinations. The viruses, each of which were isolated in our laboratory, were influenza A/New Caledonia/20/99 (H1N1) and an enterovirus (echovirus type 30), representing enveloped and nonenveloped RNA viruses, respectively; and herpes simplex virus type 2 (HSV-2) and adenovirus type 7, representing enveloped and nonenveloped DNA viruses, respectively. We examined the impact of swab type, viral transport medium, and the duration and temperature of transport.
Swabs were purchased from Copan (Brescia, Italy). The types of swabs were as follows: plain (rayon-tipped) swabs without medium (catalogue no. 8154CIS), virus transport medium swabs in a medium-soaked sponge (VTM) (catalogue no. 147CV), flocked swabs with universal transport medium (UTM) (catalogue 359C), flocked nylon fiber swabs with liquid Amies medium (E swabs) (catalogue 480CE), and swabs with Amies gel containing charcoal (catalogue E114).
Virus stocks were prepared in minimum essential medium (MEM) (Sigma, Manheim, Germany) containing 2% fetal bovine serum (FBS) (Gibco, Auckland, New Zealand) (MEM2%) to give cycle threshold (CT) values of between 22 and 25 (approximately 10,000 to 1,000 nucleic acid copies per ml, respectively). Swabs were immersed in the virus for 2 s, and those with associated transport media were immediately placed in that medium. Amies gel swabs were resheathed in the gel. Plain swabs were either resheathed in their original housings without added medium (dry) or placed in a tube containing 2 ml sterile saline. Duplicates of each swab type exposed to the individual viruses were immediately stored at −70°C as time zero controls. The remaining swabs were divided into 3 groups and held at 4°C, ambient temperatures (20 to 22°C), or 37°C. Duplicate swab/medium combinations from each treatment condition were then collected and stored at −70°C every 24 h for 7 days. All swabs were incubated for a further 7 days at −70°C and then thawed at 4°C overnight. The medium on VTM, UTM, E, and saline swabs was used directly for nucleic acid extraction (the volume in VTM swabs was increased by 2 ml with MEM2%). Plain swabs without transport medium (dry) and the Amies gel swabs were placed in 2 ml MEM2% immediately prior to extraction.
Total nucleic acid in a 200-μl volume of sample was extracted using Qiagen DX extraction kits (Qiagen Sciences, Germantown, MD) and a QIAxtractor robot (Qiagen, Hilden, Germany). The elution volume was 60 μl. To obtain cDNA, 10 μl extract was linearized at 65°C for 10 min, quenched on ice, added to 12 μl reverse transcription (RT) master mix containing random hexamers (Roche Diagnostics, Mannheim, Germany), deoxynucleoside triphosphates (dNTPs) (Amersham Biosciences, Buckinghamshire, United Kingdom), and avian myeloblastosis virus (AMV)-RT enzyme and buffers (Promega, Madison WI), and then incubated for 30 min at 42°C and 10 min at 100°C.
PCR used 4 μl DNA and 16 μl ABI Fast master mix (Applied Biosystems, Foster City, CA) containing primers at 0.9 μM and probes at 0.2 μM (sequences available on request). Cycling conditions were 95°C for 2 min then 45 cycles of 95°C for 2 s and 60°C for 30 s using an ABI 7500 fast real-time system (Applied Biosystems, CA).
CT values obtained for influenza virus under the conditions evaluated are shown (Fig. 1). Use of UTM, VTM, and E was consistently associated with an amplifiable product. Plain swabs in saline also yielded an amplifiable product at each time point and temperature, with CT levels similar to those of the commercial products. Using linear regression, there was a statistically significant difference in CT over time between saline swabs held at either 4°C or an ambient temperature (22°C) and those at 37°C (P = 0.001), but not between 4°C and an ambient temperature (P = 0.11). The results for plain dry swabs showed that an influenza virus-specific product could be reliably detected only following transport at temperatures at or below ambient (Fig. 1). Using linear regression, there was no statistically significant difference in CT over time between dry swabs held at 4°C and those at an ambient temperature, respectively (P = 0.31). However, the difference between dry swabs held at either 4°C or an ambient temperature and those at 37°C was statistically significant (P < 0.001). Nucleic acid extracted from influenza virus collected using Amies gel swabs was consistently more difficult to detect at each temperature and time point.
Fig 1
Fig 1 Detection of influenza virus RNA by real-time PCR analysis from swabs maintained at (a) 4°C, (b) room temperature (RT), and (c) 37°C for up to 7 days. A CT of 45 indicates a result was not detected for both replicates.
Each of the swab/transport medium combinations produced an amplifiable enterovirus, HSV-2, or adenovirus product (results not shown). However, CT values obtained for each of these viruses on dry swabs increased after day 3 when the holding temperature was 37°C, indicating a gradual loss of nucleic acid integrity. Similar to the result obtained for influenza virus, CT values obtained from Amies gels were consistently higher for these 3 viruses. This was not unexpected, since this product has been developed for preservation of anaerobic bacteria, but is sometimes used to collect and transport specimens for viral studies.
This study differed from several previous investigations that compared swab types and specimen collection methods within a clinical setting (1, 6). Our approach was laboratory based, and no patients were involved. Although CT values provide some quantitative information, issues such as the relationship between viral load and disease severity could therefore not be assessed. A study similar to ours has shown that respiratory syncytial virus (RSV) could be detected using PCR on dry cotton swabs after 15 days at room temperature (9). In the same study, dry swabs used for clinical sampling showed utility for several respiratory viruses, although no direct comparison to other transport media was made (9).
We did not evaluate the impact of swabs, transport media, or transport conditions on virus isolation, although it is unlikely that isolation would match the sensitivity of PCR analysis under the conditions chosen. However, reference laboratories need to be mindful that during outbreaks representative virus isolates are sometimes needed for purposes such as strain characterization, supply of candidate vaccine strains, and drug resistance phenotyping.
The utility of swab/medium combinations for the molecular detection of influenza virus is of considerable importance. This virus is historically associated with large epidemics and is more likely than other viruses to cause depletion of commercially available stocks of swabs and other consumables during times of high testing demand. Our observation that collection and transport of influenza virus on dry swabs in saline is appropriate for PCR detection is important for future pandemic planning.

REFERENCES

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Published In

cover image Journal of Clinical Microbiology
Journal of Clinical Microbiology
Volume 50Number 3March 2012
Pages: 1064 - 1065
PubMed: 22205810

History

Received: 30 November 2011
Accepted: 16 December 2011
Published online: 16 February 2012

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Contributors

Authors

Julian Druce
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
Katherine Garcia
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
Thomas Tran
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
Georgina Papadakis
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia
Chris Birch
Victorian Infectious Diseases Reference Laboratory, North Melbourne, Victoria, Australia

Notes

Address correspondence to Julian Druce, [email protected].

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