Dry cotton or flocked respiratory swabs as a simple collection technique for the molecular detection of respiratory viruses using real-time NASBA

https://doi.org/10.1016/j.jviromet.2008.08.001Get rights and content

Abstract

This paper describes the molecular detection of influenza A, influenza B, respiratory syncytial virus and human metapneumovirus using real-time nucleic acid sequence based amplification (NASBA) from respiratory samples collected on simple dry cotton swabs, non-invasively and in the absence of transport medium. Viral RNA was detectable on dry cotton and flocked swabs for at least 2 weeks at room temperature and was readily extracted using magnetic silica extraction methods. Dry cotton respiratory swabs were matched with traditionally collected respiratory samples from the same patient, and results of traditional laboratory techniques and real-time NASBA were compared for all four viral targets. The results not only showed a significant increase in the detection rate of the viral targets over traditional laboratory methods of 46%, but also that dry swabs did not compromise their recovery. Over two subsequent winter seasons, 736 dry cotton respiratory swabs were collected from symptomatic patients and tested using real-time NASBA giving an overall detection rate for these respiratory virus targets of 38%. The simplicity of the method together with the increased detection rate observed in the study proves that transporting a dry respiratory swab to the laboratory for respiratory virus diagnosis using molecular methods is a suitable and robust alternative to traditional sample types.

Introduction

Respiratory viruses contribute to significant morbidity and mortality in healthy and vulnerable individuals. The introduction of improved antiviral treatments for respiratory viral infection in recent years has meant that rapid diagnosis of respiratory viral infection is vital to ensure patients are treated and managed appropriately (Englund et al., 1996, Boivin et al., 2004, Templeton et al., 2004, Moore et al., 2004, Deiman et al., 2007).

Achieving a rapid result that is both sensitive and specific is challenging. The timing of sample collection in relation to the onset of symptoms together with the quality of the sample is crucial. For example, although direct immunofluorescence allows results to be available within 1 h of sample receipt, compared to collecting a respiratory swab, more invasive sampling is required to collect a nasopharyngeal aspirate or broncho-alveolar lavage. Also, these samples in adult patients can give poor results due to reduced viral shedding and reduced amounts of cellular material when compared to infants (Hall et al., 1976, Englund et al., 1996, Falsey, 2007). More rapid point of care testing for certain targets like influenza and RSV have their place, but these tests are generally less sensitive than traditional laboratory tests and so it is important that negative results from these assays are later confirmed by a more sensitive test (Moore et al., 2006, Dwyer and Sintchenko, 2007).

For the laboratory diagnosis of respiratory viral infection, cell culture has been historically the gold standard. But for successful isolation it is important that the clinical specimen is collected from the patient close to initial symptom onset and be transported under appropriate conditions to the laboratory. For swabs taken from the respiratory tract, this requires the use of virus transport medium. Using traditional cell culture techniques, a positive result may take several days to become positive depending on the quality of the sample and the viral load, but a negative result may not be available for up to 2 weeks. However, recent advances in cell culture techniques, in particular the commercial shell vial techniques, such as ready-cells (R mix) have reduced the time to result to just 48 h (LaSala et al., 2007).

Molecular tests for respiratory virus detection are being used increasingly in routine diagnostic laboratories (Hibbitts and Fox, 2002). Numerous studies have shown that molecular techniques based on conventional PCR or nucleic acid sequence based amplification (NASBA) vastly improves the detection rate for respiratory viruses over traditional laboratory techniques. Using real-time detection of the amplified product, same day results are now a reality (Englund et al., 1996, Boivin et al., 2004, Templeton et al., 2004, Moore et al., 2004, Moore et al., 2006, Deiman et al., 2007). However, there has been very little work reported on evaluating the most appropriate way of collecting and transporting respiratory samples to the laboratory for molecular testing. The evaluation of new molecular assays is often based on samples received for virus isolation allowing for testing by more than one method and thus a direct comparison of results. By using this approach routinely it means that the sample received might be contaminated with the target virus if processed in a room where cell culture is performed. In an ideal situation, a dedicated respiratory sample for molecular testing would be collected non-invasively, the transportation method would be simple and inexpensive; with viral nucleic acid stability guaranteed allowing for accurate detection of the target virus for several days. Dedicated samples for molecular testing would reduce the risk of contamination in laboratories also performing traditional methods for virus detection and the ease of sample collection would facilitate clinical teams in confirming a diagnosis of respiratory infection. Removing the need for virus transport medium would reduce its associated cost, storage requirements and risk of leakage during transportation.

Other simple collection and transportation methods have been described. One of the most effective methods is a dried blood spot collected onto filter paper (Karapanagiotidis et al., 2005). This method was used in the late 1990s for HIV-1 RNA detection (Cassol et al., 1997, Mwaba et al., 2003, Alvarez-Munoz et al., 2005, Li et al., 2005) genotyping (Plantier et al., 2005) and measles surveillance schemes in Africa (El Mubarak et al., 2004). These showed that the integrity of RNA can be maintained for long periods and at varying temperatures (Abe and Konomi, 1998). A more complex, but commonly used approach is to use FTA® filter paper, which is impregnated with lyophilised chemicals which lyses both viruses and bacteria rendering them non infectious (Moscoso et al., 2005). Filter paper strips have also been used to collect faecal material which is then fixed prior to sending for molecular testing and sequencing studies (Vilcek et al., 2001, Woollants et al., 2004). Recently, a method has been described for collecting respiratory samples for surveillance using swabs fixed in ethanol (Krafft et al., 2005). Similarly, in an outbreak of parainfluenza 3 on a haematology ward at the University Hospital of Wales where nasal samples were collected directly into guanidine thiocyanate lysis buffer for transportation and nucleic acid extraction (Hibbitts et al., 2003).

In the winter season of 2003–2004, a real-time nucleic acid sequence based amplification assay for the detection of influenza A was developed. During the clinical evaluation studies a small number of dry respiratory swabs were received for testing transported in the outer sterile protective cover alone. Rather than discard the swabs as unsuitable for testing, they were broken into lysis buffer on receipt in the laboratory, extracted and tested for influenza as for other samples. In more than one instance influenza A was detected (Moore et al., 2004).

This paper includes three constituent studies that together demonstrate the validity of dry swabs for the detection of respiratory viruses. The first describes a simple RNA stability study comparing the widely available cotton tipped wooden swab and the recently introduced flocked swab designed to increase the surface area of the swab allowing for improved sample collection and yield. The second describes a pilot study comparing dry cotton tipped respiratory swabs with matching nasopharyngeal aspirates or respiratory swabs transported in viral transport medium collected at the same time from the same patient using both real-time NASBA and traditional laboratory techniques and the final part demonstrates how dry cotton respiratory swabs can be used as a simple effective collection technique for routine molecular testing.

Section snippets

Study to determine viral stability

To demonstrate viral stability, dry sterile cotton tipped, wooden swabs and flocked swabs (Bibby Sterilin, Copan, Italy) were compared using serially diluted viral isolate. RSV was selected for this work as it was considered to be most environmentally labile virus being targeted in the study. RSV positive virus culture supernatant was obtained from the Welsh Specialist Virology Centre and its TCID50 was calculated to be 1 × 105 using the method of Reed and Meunch (1938). The virus culture fluid

Virus stability results

RSV RNA could successfully be extracted and detected from all of the swabs. The amount of RNA detected on the cotton tipped swabs decreased towards day 15 with a difference in ‘time to positive’ at day 15 of 8 min in favour of the flocked swab. The consistency in the time to positive results was significantly improved using the flocked swab, which varied very little over the 15 days resulting in a more linear graph over time when compared to the cotton swab (Fig. 1).

Dry respiratory swabs and matching clinical respiratory specimen study

In total 164 dry respiratory

Discussion

As more routine laboratories introduce molecular testing for the detection of respiratory viruses the most appropriate way of collecting respiratory samples to ensure a rapid and sensitive delivery of results also needs to be assessed.

The stability study in this paper demonstrates that nucleic acid from RSV is stable over a long period of time on a dry swab and that the nucleic acid is readily released once the swab is vortexed into the guanidinium based lysis buffer. This means that dry swabs

Conclusion

Collecting a nasal or throat swab and transporting it dry to the laboratory as a dedicated molecular specimen is a simple and robust method for detecting a wide range of respiratory viral targets.

Acknowledgements

The authors would like to thank Dr Sam Hibbitts for her work in developing the assays used in the routine diagnostic service, Dr Diana Westmoreland for her hard work in setting up the respiratory diagnostic service for Wales. The staff of the Welsh Specialist Virology Centre, the Molecular Diagnostics Unit and the R&D staff from bioMérieux in France and the Netherlands for providing kits and technical support. The Welsh Assembly Government provided funding support for the routine surveillance

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