Refrigeration does not compromise recovery of Neisseria gonorrhoeae from charcoal transport swabs
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《性传输感染》
Health Protection Agency London, Medical Microbiology, King’s College Hospital, Denmark Hill, London SE5 9RS, UK
Keywords: charcoal transport swabs; Neisseria gonorrhoeae; refrigeration
Despite emergent molecular diagnostics, culture recovery of Neisseria gonorrhoeae (NG) remains important for the diagnosis of gonorrhoea, as well as for susceptibility and epidemiological study. Although inoculation of bacteriological media in clinic is optimal, it can prove impractical, or impossible, in some healthcare settings. Further, any healthcare strategy that distances patient testing from diagnostic laboratories reinforces the need for transport media.
Many users assume that commercial transport systems offer comparable performance characteristics, so cost alone may influence choice. However, a proposed NCCLS standard for transport media (M40)1 is likely to confirm significant variations in performance, both between and within different manufacturers’ products. Similarly, little attention has been given to the storage temperature for swabs after use; textbooks offer conflicting recommendations. Overgrowth and killing of NG in transport media by contaminating bacteria may be inhibited by refrigeration, but it is unclear whether refrigeration is detrimental to recovery of NG. To address this we compared the survival of 30 distinguishable clinical strains of NG in charcoal transport swabs held at ambient temperature (AT: 20–22°C) and at 4°C.
Swabs (Transwab; Medical Wire & Equipment Co) were inoculated with a suspension of NG in phosphate buffered saline (PBS). For each strain, four swabs were inoculated, to allow comparison of storage at AT or 4°C, for 24 or 48 hours. At times 24 hours and 48 hours, NG organisms were recovered from swabs by vortexing the tips in 1 ml PBS. Triplicate counts were performed on the 0 hour inocula and the washings on chocolate agar (Oxoid, Basingstoke, UK) using a spiral plater (Don Whitley, Shipley, UK). The median value for each triplicate was taken, and counts compared using the Wilcoxon rank sum test.
At 24 hours there was no significant difference between AT and 4°C counts, with median (interquartile range, IQR) recoverable log10cfu of 4.57 (3.78–4.84) and 4.72 (4.32–4.87), respectively (fig 1). At 24 hours one strain held at AT was not recovered (see fig 1). At 48 hours, six strains held at AT and three at 4°C were not recovered; median counts (IQR) were 3.09 (1.3–3.55) and 3.855 (3.19–4.53) for AT and 4°C, respectively (p = 0.004).
Sng et al in a semiquantitative study tested five strains in Amies medium at four temperatures (4, 18, 26, and 32°C) and found better survival at lower temperatures.2 Arbique et al studied six isolates and found refrigeration improved recovery, though optimum temperature varied with system.3 Perry et al4 using 11 isolates considered that 4°C prolonged survival. Studies using laboratory control strains of NG have usually shown better recovery at 4°C.5,6
It is impossible to reproduce in vitro the NG inoculum and other conditions in clinical swabs. To demonstrate a difference in survival at two temperatures we used a standardised inoculum higher than that likely to be present in clinical samples. Nevertheless, our results add to a growing body of evidence that, compared to AT, refrigeration does not compromise the recovery of NG. Storage at 4°C offers the potential benefit of reducing overgrowth and elimination of NG by contaminating normal flora.
References
www.nccls.org.
Sng E, Rajan VS, Yeo K, et al. The recovery of Neisseria gonorrhoeae from clinical specimens: effects of different temperatures, transport times, and media. Sex Transm Dis 1982;9:74–8.
Arbique JC, Forward KR, LeBlanc J. Evaluation of four commercial transport media for the survival of Neisseria gonorrhoeae. Diagn Microbiol Infect Dis 2000;36:163–8.
Perry JL. Effects of temperature on fastidious organism viability during swab transport. Orlando, FL, USA: Proceedings of the 101st American Society for Microbiology General Meeting, May, 2001:20–4.
Robinson A, Gruver ML. Comparison of bacterial survival in two transport systems stored at room and refrigerator temperatures. Salt Lake City, UT, USA: Proceedings of the 102nd American Society for Microbiology General Meeting, May, 2002:19–23.
Schieven BC, Farrell D. Evaluation of two transport systems for recovery of selective facultative bacteria. Orlando, FL, USA: Proceedings of the 101st American Society for Microbiology General Meeting, May, 2001:20–4.(J J Wade and M A Graver)
Keywords: charcoal transport swabs; Neisseria gonorrhoeae; refrigeration
Despite emergent molecular diagnostics, culture recovery of Neisseria gonorrhoeae (NG) remains important for the diagnosis of gonorrhoea, as well as for susceptibility and epidemiological study. Although inoculation of bacteriological media in clinic is optimal, it can prove impractical, or impossible, in some healthcare settings. Further, any healthcare strategy that distances patient testing from diagnostic laboratories reinforces the need for transport media.
Many users assume that commercial transport systems offer comparable performance characteristics, so cost alone may influence choice. However, a proposed NCCLS standard for transport media (M40)1 is likely to confirm significant variations in performance, both between and within different manufacturers’ products. Similarly, little attention has been given to the storage temperature for swabs after use; textbooks offer conflicting recommendations. Overgrowth and killing of NG in transport media by contaminating bacteria may be inhibited by refrigeration, but it is unclear whether refrigeration is detrimental to recovery of NG. To address this we compared the survival of 30 distinguishable clinical strains of NG in charcoal transport swabs held at ambient temperature (AT: 20–22°C) and at 4°C.
Swabs (Transwab; Medical Wire & Equipment Co) were inoculated with a suspension of NG in phosphate buffered saline (PBS). For each strain, four swabs were inoculated, to allow comparison of storage at AT or 4°C, for 24 or 48 hours. At times 24 hours and 48 hours, NG organisms were recovered from swabs by vortexing the tips in 1 ml PBS. Triplicate counts were performed on the 0 hour inocula and the washings on chocolate agar (Oxoid, Basingstoke, UK) using a spiral plater (Don Whitley, Shipley, UK). The median value for each triplicate was taken, and counts compared using the Wilcoxon rank sum test.
At 24 hours there was no significant difference between AT and 4°C counts, with median (interquartile range, IQR) recoverable log10cfu of 4.57 (3.78–4.84) and 4.72 (4.32–4.87), respectively (fig 1). At 24 hours one strain held at AT was not recovered (see fig 1). At 48 hours, six strains held at AT and three at 4°C were not recovered; median counts (IQR) were 3.09 (1.3–3.55) and 3.855 (3.19–4.53) for AT and 4°C, respectively (p = 0.004).
Sng et al in a semiquantitative study tested five strains in Amies medium at four temperatures (4, 18, 26, and 32°C) and found better survival at lower temperatures.2 Arbique et al studied six isolates and found refrigeration improved recovery, though optimum temperature varied with system.3 Perry et al4 using 11 isolates considered that 4°C prolonged survival. Studies using laboratory control strains of NG have usually shown better recovery at 4°C.5,6
It is impossible to reproduce in vitro the NG inoculum and other conditions in clinical swabs. To demonstrate a difference in survival at two temperatures we used a standardised inoculum higher than that likely to be present in clinical samples. Nevertheless, our results add to a growing body of evidence that, compared to AT, refrigeration does not compromise the recovery of NG. Storage at 4°C offers the potential benefit of reducing overgrowth and elimination of NG by contaminating normal flora.
References
www.nccls.org.
Sng E, Rajan VS, Yeo K, et al. The recovery of Neisseria gonorrhoeae from clinical specimens: effects of different temperatures, transport times, and media. Sex Transm Dis 1982;9:74–8.
Arbique JC, Forward KR, LeBlanc J. Evaluation of four commercial transport media for the survival of Neisseria gonorrhoeae. Diagn Microbiol Infect Dis 2000;36:163–8.
Perry JL. Effects of temperature on fastidious organism viability during swab transport. Orlando, FL, USA: Proceedings of the 101st American Society for Microbiology General Meeting, May, 2001:20–4.
Robinson A, Gruver ML. Comparison of bacterial survival in two transport systems stored at room and refrigerator temperatures. Salt Lake City, UT, USA: Proceedings of the 102nd American Society for Microbiology General Meeting, May, 2002:19–23.
Schieven BC, Farrell D. Evaluation of two transport systems for recovery of selective facultative bacteria. Orlando, FL, USA: Proceedings of the 101st American Society for Microbiology General Meeting, May, 2001:20–4.(J J Wade and M A Graver)