Abstract
The efficacy of swab transport systems in maintaining viability of Neisseria gonorrhoeae and Streptococcus pneumoniae is crucial both for establishing definitive diagnosis and for monitoring emerging resistance. We tested the efficacy of a newly modified Amies charcoal swab transport system, the StarSwab SP131X (Starplex Scientific, Inc., Etobicoke, Ontario, Canada), by using a combined total of 31 clinical and American Type Culture Collection stock reference strains of N. gonorrhoeae and S. pneumoniae in 46 suspensions of concentrations ranging from 105 to 108 CFU/ml. Triplicate swabs per strain held at room temperature for 0, 24, and 48 h were plated without prior vortexing, and their growths were graded. All 31 strains were viable at 0 and 24 h. Gonococcal viability at 48 h varied considerably, even among strains with comparable inoculum sizes, suggesting that viability might be strain dependent and confirming the different structural and growth profiles of gonococcal strains. S. pneumoniae strains showed consistent viability, with all strains recovered at all holding periods. This study demonstrates that the StarSwab SP131X is capable of maintaining the viability of N. gonorrhoeae and S. pneumoniae for at least 24 and 48 h, respectively, and reinforces the need for adequate sampling and for timely processing of specimens to maintain optimum performance.
One of the crucial steps for effective laboratory diagnosis of infection is adequate collection and transport of specimens for culture. For years, swabs have been used to sample and transport clinical material obtained from infected sites for microbiological examination. While swab systems are considered less optimal than direct plating for culturing purposes, they have become increasingly important in view of the delay of specimen transport necessitated by recent strategies of cost containment and consolidation of laboratory services. As a result, there have been continuous efforts to improve and assess the efficacies of swab systems and to develop industry-wide standards to monitor their performances (1, 6, 7, 12, 13, 16, 18; C. Hetchler, C. Brown, and J. C. Galbraith, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-152, p. 164, 2000).
Among the key factors impacting the efficacy of a swab system is its ability to maintain viability of fastidious organisms for sufficient duration. In the present study, we tested the efficacy of the newly modified StarSwab SP131X (Starplex Scientific, Inc., Etobicoke, Ontario, Canada) with two fastidious organisms, Neisseria gonorrhoeae and Streptococcus pneumoniae. Both organisms are significant pathogens, commonly encountered in clinical specimens and known for their fragile viability. We opted to examine the Amies charcoal-enriched rather than charcoal-free StarSwab, because of the former's common usage as a multipurpose swab in many clinical facilities, owing to the ability of the added charcoal to neutralize toxic materials (7, 17). During the initial part of this study, it was noted that while all of the pneumococcal strains exhibited consistent viability profiles, the five gonococcal strains in the study exhibited significant differences in colonial morphology and growth yield at various intervals. As a result, we extended the gonococcal portion of the study with an additional 20 clinical strains, with the purpose of obtaining more objective and reliable data to assess the efficacy of this swab transport system.
(Results of this work were previously presented in part at the 100th General Meeting of the American Society for Microbiology, Los Angeles, Calif., 21 to 25 May 2000 [S. E. Farhat, M. Thibault, and S. J. Finn, Abstr. 100th Gen. Meet. Am. Soc. Microbiol., abstr. C-156, p. 164, 2000].)
A total of 31 clinical and reference isolates were used in this study, including 25 N. gonorrhoeae and 6 S. pneumoniae strains, as listed in Tables 2 and 3, respectively. The clinical strains were isolated from patient specimens and were identified by standard methods (8, 14). The stock reference strains belonged to the American Type Culture Collection (ATCC) and were obtained from lyophilized pellets (Kwik-Stik; MicroBiologics, Inc., St. Cloud, Minn.) or freeze-dried cultures (Cryocults; Quality Technologies, Newbury Park, Calif.). Each strain was subcultured three times prior to testing to ensure its purity and stability. Suspensions of test organisms were initially prepared to a concentration equivalent to a McFarland no. 1 standard, which was diluted with Trypticase soy broth (TSB; PML Microbiologicals, Mississauga, Ontario, Canada) to yield final concentrations of 108, 107, 106, or 105 CFU/ml, as assigned for each strain. Briefly, 1 ml of the 108-CFU/ml suspension (at a McFarland no. 1 standard, diluted 1:3) was added to 9 ml of TSB to yield a 107-CFU/ml concentration. Serial 10-fold dilutions were prepared to yield 106- and 105-CFU/ml concentrations. A portion of the 105-CFU/ml suspension was further diluted 1:100 to obtain a 103-CFU/ml concentration. Reference plates were prepared in duplicate by plating 0.1 ml of the 103-CFU/ml suspension onto chocolate agar plates (PML Microbiologicals) for the N. gonorrhoeae strains and 5% Columbia sheep blood agar plates (PML Microbiologicals) for the S. pneumoniae strains, by using the spread plate technique to obtain colony counts and determine inoculum size (18). The inoculum size was calculated by multiplying the average number of colonies recovered on the reference plates by the dilution factor used. The swabs were inoculated with the assigned strains at the predetermined concentration. Each swab was removed from its device and dipped into 0.1 ml of the suspension for 3 to 4 s to allow adsorption and then returned to its transport device. Triplicate swabs per strain, held at room temperature for 0, 24, and 48 h, were plated without prior vortexing, to simulate the regular laboratory practice of swab processing. Although vortexing of swabs has been advocated on the basis that it enhances the recovery of entrapped bacteria (11, 19), most clinical laboratories do not vortex swabs prior to inoculation of culture media, nor is swab vortexing indicated in the procedure according to the manufacturers' instructions. The S. pneumoniae and N. gonorrhoeae plates were streaked and incubated in 5% CO2 at 36°C and were read at 36 and 48 h, respectively. Growth obtained from each of the cultured swabs was graded according to criteria adapted from Thompson and French (18). Briefly, a score of between 0 and 5 was assigned to each plate based on the amount and distribution of colonies within the plate's four quadrants (Table 1). The average yield score for each triplicate set (average growth score [AGS]) was calculated as a measure of the organism viability at the test interval.
TABLE 2.
Recovery of Neisseria gonorrhoeae strains in various concentrations at different holding periods
| Prepared suspension concn (CFU/ml) | Strain | Inoculum size (CFU/ml) | AGS ata:
|
||
|---|---|---|---|---|---|
| 0 h | 24 h | 48 h | |||
| 108 | ATCC 43069 | 1.3 × 108 | 5.0 | 3.7 | 2.3 |
| ATCC 31426 | 0.8 × 108 | 4.3 | 4.6 | 4.0 | |
| ATCC 19424b | 1.4 × 108 | 5.0 | 5.0 | 4.3 | |
| ATCC 19424b | 1.7 × 108 | 4.8 | 3.9 | 1.0 | |
| ATCC 49226 | 0.9 × 108 | 5.0 | 5.0 | 3.3 | |
| Clinical no. 1 | 0.6 × 108 | 5.0 | 4.0 | 2.7 | |
| Clinical no. 4 | 0.9 × 108 | 5.0 | 5.0 | 3.7 | |
| Clinical no. 9 | 1.1 × 108 | 5.0 | 4.7 | 2.0 | |
| Clinical no. 13 | 1.7 × 108 | 5.0 | 5.0 | 4.7 | |
| Clinical no. 18 | 2.5 × 108 | 5.0 | 3.3 | 2.0 | |
| Clinical no. 21 | 1.4 × 108 | 5.0 | 5.0 | 4.7 | |
| 107 | ATCC 31426 | 0.9 × 107 | 5.0 | 3.0 | 2.3 |
| Clinical no. 3 | 0.4 × 107 | 4.0 | 1.3 | 0.7 | |
| Clinical no. 7 | 0.8 × 107 | 5.0 | 2.0 | 0.7 | |
| Clinical no. 11 | 0.5 × 107 | 5.0 | 2.3 | 1.0 | |
| Clinical no. 15 | 0.5 × 107 | 4.7 | 1.7 | 0.7 | |
| Clinical no. 20 | 2.1 × 107 | 4.0 | 2.0 | 1.0 | |
| 106 | ATCC 19424 | 1.4 × 106 | 3.0 | 2.3 | 0.0 |
| ATCC 49226 | 0.9 × 106 | 3.3 | 3.0 | 1.7 | |
| Clinical no. 2 | 1.7 × 106 | 5.0 | 4.7 | 3.0 | |
| Clinical no. 6 | 2.7 × 106 | 5.0 | 1.7 | 0.7 | |
| Clinical no. 10 | 2.1 × 106 | 5.0 | 3.3 | 2.0 | |
| Clinical no. 14 | 0.4 × 106 | 3.7 | 1.7 | 0.0 | |
| Clinical no. 17 | 1.8 × 106 | 5.0 | 3.7 | 1.3 | |
| Clinical no. 21 | 1.4 × 106 | 4.0 | 2.7 | 2.3 | |
| 105 | ATCC 43069 | 1.3 × 105 | 3.0 | 1.7 | 0.0 |
| ATCC 31426b | 0.9 × 105 | 2.3 | 1.0 | 0.7 | |
| ATCC 31426b | 0.8 × 105 | 2.0 | 1.6 | 0.0 | |
| ATCC 19424 | 1.7 × 105 | 3.0 | 1.0 | 0.0 | |
| Clinical no. 5 | 2.8 × 105 | 5.0 | 3.0 | 0.0 | |
| Clinical no. 8 | 0.7 × 105 | 2.3 | 0.7 | 0.0 | |
| Clinical no. 12 | 0.6 × 105 | 2.3 | 2.0 | 1.0 | |
| Clinical no. 16 | 0.7 × 105 | 3.7 | 2.0 | 0.0 | |
| Clinical no. 19 | 0.9 × 105 | 3.3 | 0.7 | 0.0 | |
The AGS of a triplicate set was calculated by dividing the sum of individual scores by the number of plates at each holding period (for grading scores, consult Table 1).
ATCC strains 19424 and 31426, prepared at 108 and 105 CFU/ml, respectively, were tested in two separate trials.
TABLE 3.
Recovery of Streptococcus pneumoniae strains in two concentrations at different holding periods
| Prepared suspension concn (CFU/ml) | Strain | Inoculum size (CFU/ml) | AGS ata:
|
||
|---|---|---|---|---|---|
| 0 h | 24 h | 48 h | |||
| 108 | ATCC 6303 | 0.3 × 108 | 4.9 | 4.0 | 4.0 |
| ATCC 41619 | 0.4 × 108 | 5.0 | 5.0 | 5.0 | |
| Clinical no. 1 | 0.7 × 108 | 5.0 | 5.0 | 5.0 | |
| Clinical no. 2 | 1.2 × 108 | 5.0 | 5.0 | 5.0 | |
| Clinical no. 3 | 0.4 × 108 | 5.0 | 5.0 | 5.0 | |
| Clinical no. 4 | 0.5 × 108 | 5.0 | 5.0 | 5.0 | |
| 105 | ATCC 6303 | 0.3 × 105 | 2.6 | 1.0 | 1.0 |
| ATCC 41619 | 0.4 × 105 | 4.0 | 3.0 | 4.0 | |
| Clinical no. 1 | 0.7 × 105 | 4.0 | 3.0 | 4.0 | |
| Clinical no. 2 | 1.2 × 105 | 4.0 | 4.0 | 3.0 | |
| Clinical no. 3 | 0.4 × 105 | 4.0 | 3.0 | 4.0 | |
| Clinical no. 4 | 0.5 × 105 | 4.0 | 4.0 | 4.0 | |
The AGS of a triplicate set was calculated by dividing the sum of individual scores by the number of plates at each holding period (for grading scores, consult Table 1).
TABLE 1.
Criteria for evaluation of growth obtained from cultured swabs
| Description of growth | Grade |
|---|---|
| No growth | 0 |
| 1–50 colonies in 1st quadrant (primary inoculum) | 1 |
| >50 colonies in 1st quadrant | 2 |
| Growth in 1st and 2nd quadrants | 3 |
| Growth in 1st, 2nd, and 3rd quadrants | 4 |
| Growth in 1st, 2nd, 3rd, and 4th quadrants | 5 |
We compared bacterial yields of reference versus clinical strains by using AGSs of suspensions prepared at the same concentrations to determine whether the type of strain influenced viability in the StarSwab. The mean AGS was calculated for both reference and clinical types (data not shown). We found that in general both strain types had equivalent viability. The effects of duration of swab storage and of inoculum size on organism viability were also studied (Tables 2 and 3). Although all S. pneumoniae strains were recovered at all holding periods regardless of inoculum size, prolonged storage at ambient temperature reduced the yield of N. gonorrhoeae, a phenomenon universally observed (1, 18). As early as 1954, Stuart and his colleagues had remarked that “Over 24 hours, the viability of gonococci deteriorates progressively.” (17). Later studies also showed that prolonged transport adversely affected gonococcal recovery (9). In our hands, gonococcal viability was obtained in all strains at 24 h, confirming the adequacy of swab performance for at least 24 h from the time of sampling. Viability was still recoverable at 48 h in strains with inoculum sizes greater than 1.4 × 106 CFU/ml, but as can be seen from the results in Table 2, inoculum size was not the sole factor impacting gonococcal recovery. There were strains of equivalent inoculum sizes that displayed different yields. For example, two different strains of N. gonorrhoeae, one clinical (no. 21, Table 2) and the other a reference strain (ATCC 19424, Table 2), both with an inoculum size of 1.4 × 106 CFU/ml, were both recovered at 24 h, but only one remained viable at 48 h. In strains with lower inocula, 48-h viability was obtained with some strains with an inoculum size as low as 0.6 × 105 CFU/ml, but not with others at even higher inocula. There was no consistency in yield within this group, suggesting that viability in some strains was strain dependent and not only contingent on the number of colonies on the swab, an observation noted by other investigators (1). While a few recent investigations of swab transport systems had tested a minimal number of gonococcal strains, often with a single strain (usually an ATCC strain), to study efficacy (6, 7, 12, 13; Hetchler et al., Abstr. 100th Gen. Meet. Am. Soc. Microbiol., 2000), the use of multiple clinical strains has been recommended as a more accurate marker of swab performance (18). What induces gonococcal heterogeneity is not clear, but several recent studies have suggested that strains of N. gonorrhoeae possess diverse structural and growth profiles, which may explain their antigenic and physiologic diversity. It is known that the surfaces of N. gonorrhoeae strains display a highly variable antigenic structure, such as that of pilin expression (2, 15). In addition, growth patterns within certain serogroups as well as structural differences among typed strains, based on sequenced genes encoding protein products responsible for serovar specificity, have been identified (5, 10). Characterization of gonococcal strains also demonstrated strain differences based on auxotyping, serotyping, plasmid profiling, and DNA amplification fingerprinting (3).
Recent studies of the Amies StarSwab have shown variable recovery of N. gonorrhoeae at different holding periods, with questionable performance at 24 and 48 h when compared to other products (1, 6, 18; Hetchler et al., Abstr. 100th Gen. Meet. Am. Soc. Microbiol., 2000). The results we have presented here refer to the improved Amies charcoal StarSwab product. While the swab rayon tips are made of nonbacteriostatic fibers to allow adequate absorption of organisms from the sample, the ample fibrous material may have contributed to organism entanglement and reduced release. Reduction or loss of recovery has been previously attributed to organism entrapment, particularly if the number of entrapped organisms was too low to be adequately expressed once the swab was applied to the culture medium (4, 16).
In summary, we have conducted an efficacy study to test the ability of a commercially available swab transport system to maintain viability of N. gonorrhoeae and S. pneumoniae. While viability of some N. gonorrhoeae isolates may be strain dependent, this study has demonstrated that the newly modified Amies charcoal StarSwab is capable of maintaining viability of N. gonorrhoeae and S. pneumoniae for at least 24 and 48 h, respectively, and reinforces the need for adequate sampling and for timely processing of specimens to maintain optimum performance.
Acknowledgments
This work was supported in part by Starplex Scientific, Inc.
We thank the Public Health Laboratories of the Ontario Ministry of Health for kindly providing four of the gonococcal clinical strains used in this study.
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