Comparison of the Copan ESwab System with Two Amies Agar Swab Transport Systems for Maintenance of Microorganism Viability

Swab transport systems are used for a variety of specimen typesand must maintain organism viability throughout the transportprocess. The Copan ESwab is a new nylon-flocked swab designedto optimize specimen collection and to minimize entrapment ofthe specimen. We used the quantitative elution method with recommendedstrains, as described in CLSI document M40-A, to evaluate theESwab for maintenance of viability of aerobic and anaerobicmicroorganisms for 0, 6, 24, and 48 h during room temperatureand refrigerated temperature storage. The Becton Dickinson CultureSwabMaxV swab and the Remel BactiSwab were used as comparators.The ESwab met CLSI acceptance criteria for all aerobic isolatesstored at both temperatures and for all anaerobic isolates storedat refrigerated temperature. The ESwab also met CLSI criteriafor four of five anaerobic strains at room temperature. Prevotellamelaninogenica was not recovered after 24 or 48 h of room temperaturestorage with any of the three swab transport systems tested.Overall, the ESwab was equivalent to the Becton Dickinson CultureSwabMaxV swab in organism recovery but recovered more isolates thanRemel BactiSwab.

INTRODUCTION

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INTRODUCTION

Appropriate specimen collection and transport are essentialfor accurate laboratory diagnosis of bacterial infections. Becauseof their convenience, swab systems with transport media areoften used to collect and transport specimens of various types.These systems must maintain organism viability during transitto a laboratory. Given the increasing frequency of transportdelays due to cost containment measures, consolidations, andservices being shifted to centralized or reference laboratories,robust transport systems are becoming increasingly relevant.The CLSI M40-A method (2) was used recently to evaluate severalswab collection and transport devices for maintenance of bacteriaviability (3, 4, 6, 7, 8, 9).

Swab tips, which are typically rayon or Dacron, should be preparedwith material that collects sufficient specimen material, isnontoxic to microorganisms, maintains viability in conjunctionwith the transport medium, and releases specimen material efficientlyonto agar media. A new nylon-tipped swab (ESwab; Copan Diagnostics,Inc., Corona, CA) prepared by spray-on flocked fiber technologyhas been developed for transport of bacteria and viruses. Thistechnology provides stronger capillary action and strong hydraulicuptake of liquids, which should result in better specimen collection.This design should also provide more efficient release of specimenmaterial and, therefore, less entrapment of specimen than occurswith typical rayon or Dacron fiber-tipped swabs. The ESwab shaftis scored for ease and consistency of tip breakage into themodified liquid Amies transport medium. A swab capture mechanismin the cap locks the broken swab shaft into the cap when itis fully closed.

In this study, we evaluated the new ESwab with liquid Amiestransport medium for maintenance of viability of aerobic andanaerobic bacteria according to the CLSI M40-A quantitativeelution method (2). The Becton Dickinson CultureSwab MaxV swaband Remel BactiSwab (RBS; Remel, Lenexa, KS), both with Amiesagar gel, were tested as comparators.

(This work was presented in part at the 106th General Meetingof the American Society for Microbiology, Orlando, FL [9a].)

MATERIALS AND METHODS

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MATERIALS AND METHODS

Organisms. The 10 strains tested were those recommended by CLSI documentM40-A (2), as follows: Haemophilus influenzae ATCC 10211, Neisseriagonorrhoeae ATCC 43069, Pseudomonas aeruginosa ATCC BAA-427,Streptococcus pneumoniae ATCC 6305, Streptococcus pyogenes ATCC19615, Bacteroides fragilis ATCC 25285, Fusobacterium nucleatumATCC 25586, Prevotella melaninogenica ATCC 25845, Peptostreptococcusanaerobius ATCC 27337, and Propionibacterium acnes ATCC 6919.All strains were freshly subcultured to appropriate media atleast twice prior to being tested. Media included tryptic soyagar with 5% sheep blood, for P. aeruginosa, S. pneumoniae,and S. pyogenes; chocolate agar, for H. influenzae and N. gonorrhoeae;and anaerobic Brucella agar with 5% sheep blood, for all fiveanaerobes (media were obtained from Becton Dickinson, Sparks,MD). All isolates were incubated at 37°C with an atmosphereappropriate for each organism, i.e., non-CO₂ for P. aeruginosaand S. pyogenes, 5% CO₂ for H. influenzae, N. gonorrhoeae, andS. pneumoniae, and anaerobic (EZ anaerobe container system;Becton Dickinson) for the anaerobic isolates.

Quantitative elution. Tests were performed by the CLSI M40-A quantitative elutionmethod (2). An organism suspension from a freshly grown isolateof each strain was prepared in sterile saline to a turbidityequivalent to that of a 0.5 McFarland standard. The suspensionwas further diluted 1:10 in sterile saline to achieve an inoculumconcentration of approximately 1.5 x 10⁷ CFU/ml. For growthcontrols, serial 10-fold dilutions were prepared from the suspensionand plated on duplicate plates of the appropriate medium. Theplates were then incubated at 37°C in the appropriate atmosphere,and colony counts were obtained to confirm that the inoculumconcentration was acceptable (10⁷ to 10⁹ CFU/ml).

For swab testing, 12 100-µl aliquots of the 10⁷-CFU/mlinoculum of each organism suspension were placed onto the surfaceof a sterile Petri plate. Each of 12 swabs from one manufacturerwas rolled into 1 of the 12 100-µl inoculum spots for15 s to ensure complete absorption. Triplicate swabs of eachtype were inoculated for each storage time period (0, 6, 24,or 48 h), for a total of 12 swabs of each manufacturer's swabtype inoculated for each storage temperature to be tested. Allswabs were immediately placed into their respective transportsystems and stored at a controlled 23 ± 1°C for roomtemperature storage or 4°C for refrigerated storage foreach time period prior to plate inoculation.

After the appropriate storage time, including 0 h (tested within15 min of inoculation), each of the Amies agar gel swabs (MaxVand RBS) was removed from the transport medium, placed into0.9 ml sterile saline, and mixed by vortexing for 15 s. TheESwab was mixed in the transport tube by being vortexed for15 s, and 100 µl of the liquid Amies medium was transferredto 0.9 ml of sterile saline. Serial 10-fold dilutions of eachswab system suspension were prepared to obtain suspensions theoreticallyequivalent to 10⁶ to 10¹ CFU/ml. The ESwab transport tube wasconsidered the 10⁶-CFU/ml tube because of the initial 1:10 dilutioninto the liquid Amies medium. A 100-µl aliquot of eachdilution was removed, placed onto the surface of duplicate agarmedia appropriate for the organism tested, and inoculated overthe entire agar surface with a sterile spreader. All plateswere incubated at 37°C under appropriate atmospheric conditionsfor the organism tested. Incubation of the plates was done forup to 48 h for aerobic organisms and up to 96 h for anaerobicorganisms. After incubation, two readers counted colonies onplates with 30 to 300 countable colonies, and the average colonycount was used. The average CFU at storage times of 6, 24, and48 h were compared to the average CFU at 0 h. CLSI M40-A (2)criteria were used for evaluation, as follows: a swab systemwas considered acceptable for the organism tested if the changein CFU from the 0-h value declined no more than 3 log₁₀ (1 x10³ CFU ± 10%) for storage at room temperatures and,for storage at refrigerated temperature, no more than a 1-logincrease or no more than a 3-log₁₀ (1 x 10³ CFU ± 10%)decline compared to the 0-h CFU. CLSI has established a storageevaluation time of 24 h for N. gonorrhoeae and 48 h for allother organisms. There is no CLSI M40-A interpretation for N.gonorrhoeae beyond a 24-h storage time. Similarly, no CLSI standardexists for evaluation of overgrowth in swab transport systemsstored at room temperature. Comparison of the swab transportsystems was based on average CFU and percent recovery of viableorganisms at each incubation time and at each storage temperature.

RESULTS

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RESULTS

Recovery of aerobic isolates. Recovery of the aerobic isolates after room temperature storageis summarized in Table 1. The ESwab and MaxV systems yieldedacceptable recovery of all five isolates according to CLSI standards;the RBS yielded acceptable recovery results for four isolates,failing to recover H. influenzae after 24 h and 48 h of storage.All three swabs yielded acceptable recovery of N. gonorrhoeaeafter 24 h of storage. Heavy overgrowth was observed for P.aeruginosa with all three swab systems at 24 and 48 h.

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TABLE 1. Recovery of aerobic microorganisms from three swab systems stored at room temperature

Recovery of the aerobic isolates after refrigerated storageis summarized in Table 2. All three swab systems yielded acceptablerecovery of the five isolates. There was no observed overgrowth,and all three swab systems also recovered N. gonorrhoeae after48 h of storage.

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TABLE 2. Recovery of aerobic microorganisms from three swab systems stored at refrigerated temperature

Recovery of anaerobic isolates. Recovery of the anaerobic isolates after room temperature storageis summarized in Table 3. The ESwab and MaxV systems yieldedacceptable recovery of four of the five isolates, while theRBS recovered only three of the five isolates at 24 and 48 h.No swab system recovered the P. melaninogenica isolate after24 h or 48 h of room temperature storage. The RBS also failedto recover the P. anaerobius isolate after 24 and 48 h of incubation.No distinct overgrowth was observed for the anaerobes at roomtemperature, but the B. fragilis isolate showed some apparentgrowth in the ESwab at 24 h, with decreased counts at 48 h.

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TABLE 3. Recovery of anaerobic microorganisms from three swab systems stored at room temperature

Recovery of anaerobic isolates after refrigerated storage issummarized in Table 4. The ESwab and MaxV systems yielded acceptablerecovery for all five anaerobic isolates. The RBS yielded acceptablerecovery for three isolates, failing to recover P. anaerobiusand yielding unacceptable recovery of P. melaninogenica after48 h of storage. No overgrowth was observed with the anaerobesstored at refrigerated temperature.

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TABLE 4. Recovery of anaerobic microorganisms from three swab systems stored at refrigerated temperature

DISCUSSION

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DISCUSSION

CLSI document M40-A (2) provides standardized methods, for bothroll plates and elution, to aid manufacturers and laboratoriesin determining the performance characteristics of swab transportdevices. The roll plate method more closely resembles routinelaboratory practices of mechanical swabbing actions that influenceorganism release onto the agar plate but provides only semiquantitativeapproximation of recovery. Although the elution method doesnot reflect typical laboratory swab practices, we performedthe assays by the elution method to provide quantitative comparisonsthat could more effectively detect performance characteristicdifferences between the swab transport systems. To our knowledge,this is the first report of an evaluation of the ESwab systemutilizing all CLSI-recommended bacterial strains tested at bothstorage temperatures.

The ESwab met CLSI acceptance criteria at both storage temperaturesfor all isolates tested except for P. melaninogenica. In fact,none of the three swab transport systems maintained P. melaninogenicaviability after 24 h or 48 h of room temperature storage, possiblybecause inoculation preparation procedures were performed underaerobic conditions. However, this explanation does not fullyaccount for the unacceptable results in that the same preparationprocedures were used for the refrigerated storage studies, whichrecovered P. melaninogenica isolates with all three swab transportsystems (the RBS gave an unacceptable CFU/ml). Others have alsoreported difficulty in recovering P. melaninogenica from swabtransport systems stored at room temperature (5, 6). We arenot aware of any published swab studies of P. melaninogenicatested at refrigerated storage temperatures.

Overgrowth of an organism in a swab transport system is problematicand would misrepresent the true relative proportions of a mixedinfection, therefore yielding misleading culture results. However,there are no CLSI standards for organism overgrowth at roomtemperature incubation (2). We observed heavy overgrowth (>10⁵%increase) of P. aeruginosa after room temperature storage forthe ESwab and the MaxV systems, and the RBS also showed lesserovergrowth. Overgrowth of P. aeruginosa has been reported forother swab systems after storage at room temperature (6, 7,8, 9) and for enterics (1, 8). The ESwab allowed less, but distinct,growth of S. pyogenes during room temperature storage (>10-foldrecovery at 24 and 48 h). Growth of S. pyogenes in swab systemshas been observed previously (7, 8). In the present study, theMaxV swab also yielded S. pyogenes growth of >100-fold. However,this amount of growth would still be within acceptable CLSIlimits (2) if refrigerated storage limits were applied. Theincrease in numbers of the B. fragilis isolate with the ESwabsystem after 24 h of room temperature storage remains unexplained,but this could have been due to initial growth in the ESwabsystem followed by a decline due to loss of viable cells at48 h. No swab showed overgrowth at refrigerated temperatures,consistent with previous studies of other swab systems (3, 6,7).

Overall, the ESwab performed as well as the MaxV and RBS systemsfor maintenance of aerobic organism viability at refrigeratedtemperature storage. At room temperature, the ESwab maintainedorganism viability as well as the MaxV swab and better thanthe RBS, which failed to recover H. influenzae. The ESwab maintainedanaerobic organism viability equivalent to that with the MaxVswab at both room and refrigerated temperatures and was consideredbetter than the RBS, which failed to recover P. anaerobius atboth storage temperatures. No swab system recovered P. melaninogenicaafter 24 h of room temperature storage. The RBS also yieldedunacceptable recovery of P. melaninogenica after 48 h of refrigeratedtemperature storage. Only one lot of each swab type was used,and this may have accounted for the failures with the RBS. Furtherstudies that test multiple lots of each swab type might needto be performed to help determine if lot-to-lot variations existwith regard to this type of evaluation.

In conclusion, the ESwab system is an acceptable swab transportsystem for both aerobes and anaerobes. This system met CLSIacceptance criteria for all aerobic and anaerobic isolates whenit was tested under refrigerated storage conditions and failedto allow growth of only P. melaninogenica when it was testedat room temperature (as did both comparator swabs). For thelaboratory, caution might be advised in using swab transportdevices that are not specifically designed for transport ofspecimens for anaerobic culture, since our study indicates thatvery fastidious anaerobes might be missed after longer storageand transport (>6 h), especially if they are maintained underroom temperature conditions.

ACKNOWLEDGMENTS

Funding and materials for this project were supplied by CopanDiagnostics, Inc., Corona, CA.

We gratefully acknowledge the assistance of Jose Matud (FocusDiagnostics, Inc.) and Jeff Radcliff (Quest Diagnostics NicholsInstitute, San Juan Capistrano, CA) with preparation of themanuscript.

FOOTNOTES

* Corresponding author. Mailing address: Focus Diagnostics, Inc., 5785 Corporate Ave., Cypress, CA 90630. Phone: (714) 503-1392. Fax: (714) 484-1296. E-mail: kvanhorn{at}focusdx.com

Published ahead of print on 19 March 2008.

Present address: Agensys, 1545 17th Street, Santa Monica, CA90404.

REFERENCES

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