INTRODUCTION MATERIALS & METHODS

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1 Evaluation of Three Bacterial Transport Systems, The New Copan M40 Transystem, Remel Bactiswab And Medical Wire & Equipment Transwab, for Maintenance of Aerobic Fastidious and Non-Fastidious Organisms Nabeel Rishmawi, Issa Shomali, Musa Hindiyeh - Caritas Baby Hospital, Bethlehem, Palestine ABSTRACT It is well established that aspirates of fluids and exudates from suspected infected sites are superior to samples collected on swabs. However, because of the ease of using swabs, clinical microbiology laboratories continue to receive clinical samples on swabs. The aim of this study was to compare the performance of the new Copan M40 Transystem [C-M40) (Copan Innovation Inc.) to the Remel BactiSwab [R-BS] (Remel) and Medical Wire & Equipment Transwab [MW-TS] (Medical Wire & Equipment Co. Ltd.). The survival of the following isolates in the three transport systems was evaluated: S. pneumoniae (ATCC 49619), H. influenzae (ATCC 49766), N. gonorrhoeae (ATCC 49226), N. meningitidis (ATCC 13090); L. monocytogenes (ATCC 19115), S. aureus (ATCC 25923); S. pyogenes (ATCC 19615); S. agalactiae (Clinical isolate); P. aeruginosa (ATCC 27853), M. catarrhalis (ATCC 25239), and E. coli (ATCC 25922). The procedure outlined by NCCLS guideline M40-P was followed to evaluate the three swabs. Briefly, 0.5 MacFarland standard of each organism freshly grown at 37 C for 18-24hrs was prepared. A 1:10 dilution of each organism was made and 100µl was used to inoculate each of the swabs in triplicate. The survival of the organisms at C was determined at 0, 6, 24, 48, and 72 h. In triplicate, 100µl samples were used to quantify viable organisms on 5% SBA or Chocolate agar. With regard to the fastidious organisms, the C-M40 out performed the other two swabs with maintaining the viability of N. gonorrhoeae, and H. influenzae. Unlike R-BS and MW-TS swabs, which maintained the viability of N. gonorrhoeae for 6 hrs, CM-40 maintained the viability of N. gonorrhoeae for up to 24 hours. Furthermore, H. influenzae survived for up to 72 hrs in CM-40 while in the R-BS and MW-TS swabs H. influenzae survived for 72 and 24hrs, respectively. The survival rate of S. pneumoniae in CM-40, R-BS and MW-TS swabs was (6, 72, 72hrs, respectively). All three swabs maintained an increasing or stable number of non-fastidious organisms with time. In conclusion, C-M40 outperformed the other two swabs evaluated by maintaining the viability of two of three fastidious organisms for at least 24 hours. This time period should be sufficient for transport of specimens to the clinical microbiology laboratory without compromising organism recovery. INTRODUCTION Diagnosis of bacterial pathogens in patients samples largely depends on sample collection and transport to the laboratory. Failure to collect an appropriate patients sample can lead to patient misdiagnosis and inappropriate treatment. It is well established that aspirates of fluids, and exudates from suspected infected sites are superior to samples collected on swabs (1). However, because of the ease of using swabs, clinical microbiology laboratories continue to receive clinical samples on swabs. In addition, the gold standard for collecting some clinical samples (e.g.: throat) is swabs. With the advancements in technology, swab transport systems utilizing semi-solid medium have been developed for transporting patient samples. Moreover, swabs have been shown to protect both anaerobic and fastidious aerobic organisms (2, 3, 4). In this study we compared the performance of the new Copan M40 Transystem [C-M40) (Copan Innovation Inc.) to the Remel BactiSwab [R- BS] (Remel) and Medical Wire & Equipment Transwab [MW-TS] (Medical Wire & Equipment Co. Ltd.) in maintaining the viability of fastidious and non-fastidious aerobic microorganisms. MATERIALS & METHODS Bacterial Stains: The following bacterial strains were used: Haemophilus influenzae (ATCC 49766) Neisseria gonorrhoeae (ATCC 49226) Neisseria meningitidis (ATCC 13090) Moraxella catarrhalis (ATCC 25238) Listeria monocytogenes (ATCC 19115) Staphylococcus aureus (ATCC 25923) Streptococcus pneumoniae (ATCC 49619) Streptococcus pyogenes (ATCC 19615) Streptococcus agalactiae (Clinical isolate) Pseudomonas aeruginosa (ATCC 27853) Escherichia coli (ATCC 25922) page 1 of 6

2 Transport Swab Systems: M40 Transystem [C-M40) (Copan Innovation Inc.) Remel BactiSwab [R-BS] (Remel) Medical Wire & Equipment Transwab [MW-TS] (Medical Wire & Equipment Co. Ltd.). Bacterial identification: The identification of all the bacterial strains used in the study was confirmed by the standard identification protocols as described by the Manual of Clinical Microbiology [5] (Murray el al. 7th edition). Agar Plates: 5% sheep blood agar plates (Cat # Hy-PD 049) and Chocolate Agar (Cat # PD-013) were purchased from Hy.Labs (Park Tamara, Rehovot, Israel). Bacterial strain cultures: All bacterial strains were cultured on the appropriate media (5% Sheep blood Agar or Chocolate Agar) for hours at 37 C. Well isolated colonies were used for sample preparation. Some fastidious organisms required 48 hours incubation before sample preparation. Experimental Design: The NCCLS procedure M40-A was followed to evaluate the different transport swabs. Briefly, a 0.5 MacFarland (3 x 10 8 organisms/ml) standard of each organism freshly grown at 37 C for hrs was prepared using BioMerieux Vitek Inc. colorimeter in 0.85% saline. A 1:10 dilution (3 x 10 7 organisms/ml) of each organism was made and 100µl (3 x 10 6 organisms/ml) was used to inoculate each of the swabs in triplicate. The survival of the organisms at C was determined at 0, 6, 24, 48, and 72 h. At each of the time points, the viable organisms on the swabs were recovered in 1ml saline after vortexing of the swab for 30s, and 1:10, 1:100, and 1:1000 serial dilutions were made in sterile saline. In triplicate, 100-ul samples were used to quantify the organisms in each of the dilutions on 5% SBA or Chocolate agar. The organisms were spread over the agar surface with a plate spreader, and the plates were incubated at 37 C in 5% CO2 incubator. Data Analysis: Bacterial recovery was determined by counting the colonies recovered in each of the dilutions. The number of the organisms recovered is expressed as an average for triplicate samples evaluated and as a percentage of the baseline counts (counts at time zero). RESULTS - (Following Pages) page 2 of 6

3 RESULTS TABLE 1 RECOVERY OF FASTIDIOUS BACTERIA Haemophilus influenzae (ATCC 49766) C-M40 6.E E E E R-BS 4.E E E E E+02 0 MW -TS 5.E E E E+00 0 Neisseria gonorrhoeae (ATCC 49226) C-M40 6.E E E+00 0 R-BS 7.E E E E E+00 0 MW-TS 4.E E E E E+00 0 Streptococcus pneumoniae (ATCC 49619) C-M40 8.E E E E E+00 0 R-BS E E E E+01 0 MW-TS E E E E+02 0 Neisseria meningitidis (ATCC13090) C-M40 6.E E E R-BS 9.E E E E E+00 0 MW-TS E E E E (Log count olony C Figure 1: Recovery of Haemophilus influenzae (ATCC 49766) 0 1 (Log count olony C Figure 2: Recovery of Neisseria gonorrhoeae (ATCC 49226) Figure 3: Recovery of Streptococcus pneumoniae (ATCC 49619) olony Count (Log 1 cfu/ml ) C 0 Figure 4: Recovery of Neisseria meningitidis (ATCC 13090) page 3 of 6

4 TABLE 2 RECOVERY OF GRAM-POSITIVE BACTERIA Staphylococcus aureus (ATCC 25923) C-M40 2.E E E E R-BS 3.E E E E E MW-TS 5.E E E E Streptococcus pyogenes (ATCC 19615) C-M40 3.E E E E E R-BS 3.E E E E+04 8 MW-TS 6.E E E E E Streptococcus agalactiae (Clinical isolate) C-M40 2.E E E E R-BS 4.E E E E MW-TS 6.E E E E+04 9 Listeria monocytogenes (ATCC 19115) C-M40 4.E E E E E R-BS 6.E E E E MW-TS E E Figure 5: Recovery of Staphylococcus aureus (ATCC 25923) (Log10 Colony Count Figure 6: Recovery of Streptococcus pyogenes (ATCC 19615) C olony 1 0 (Log count Figure 7: Recovery of Streptococcus agalacticae (Clinical isolate) Figure 8: Recovery of Listeria monocytogenes (ATCC 19115) page 4 of 6

5 TABLE 3 RECOVERY OF GRAM-NEGATIVE BACTERIA Moraxella catarrhalis (ATCC 25239) C-M E E E R-BS 2.E E E E MW-TS 2.E E E E+03 2 Pseudomonas aeruginosa (ATCC 27853) C-M E E R-BS E E E MW-TS E E Escherichia coli (ATCC 25922) C-M40 2.E E E E E R-BS 2.E E E E MW-TS 3.E E E E E Figure 9: Recovery of Morexella catarrhalis (ATCC 25239) Figure 10: Recovery of Pseudomonas aeruginosa (ATCC 27853) (Log10 Colony count Figure 11: Recovery of E. coli (ATCC 25922) page 5 of 6

6 DISCUSSION In this study new Copan M40 Transystem outperformed the other two swabs systems (Tables 1-3). The new Copan M40 Transystem Amies Gel transport swab is flushed with Nitrogen gas and packed in this modified atmosphere. The Copan M40 Transystem maintained the viability of the fastidious organism H. influenzae for 72 hours (Fig. 1). Indeed the number of viable H. influenzae was maintained through 72 hours. On the other hand, the Remel BactiSwab maintained the number of viable organisms for 24 hours, and drops by 1 & 3 logs was observed at at 48 and 72 hours, respectively (Fig. 1). Medical Wire & Equipment Transwab maintained the number of viable H. influenzae for 6 hours; this was followed by 0.5 log reduction at 24 hours, and no viable organisms were maintained after 24 hours (Fig. 1). Copan M40 Transystem also out performed the other two swabs in maintaining the viability of N. gonorrhoaeae (Fig. 2). Copan M40 maintained viable organisms for 48 hours; however, there was a 3 log reduction at 48 hours. The other two swabs maintained viable organisms for 6 hours; however, there was 2 and 3 log reduction in the number of viable organisms (Fig. 2). No viable organisms were maintained at 24 and 48 hours. The Copan M40 Transystem also out performed the other two swabs in maintaining the viability of N. meningitidis (Fig. 3). Copan M40 maintained constant number of viable organisms for 72 hours, while the other two swabs maintained a viable number of organisms for 24 hours (Fig. 3). There was one log reduction in the number of viable organisms in the Remel BactiSwab at 24 hours, while there was a 3 log reduction in the number of viable organisms in the Medical Wire & Equipment Transwab. Copan M40 Transystem did not perform as well in maintaining the viability of S. pneumoniae (Fig. 4). Copan M40 maintained viable organism for 6 hours with 2 log reduction in the number of viable organisms. No viable organisms were detected after 6 hours. On the other hand the two other swabs maintained viable S. pneumoniae for 72 hours (Fig. 4). About three log reduction was observed after 72 hours incubation. Upon repeat testing of the three swabs with two clinical isolates of S. pneumoniae, similar results were obtained. All three swabs performed similarly with regard to maintaining the viability of gram-positive organisms, except for the S. pyogenes (Table 2). The Copan M40 increased the number of viable organisms by 2 logs at 72 hours while there was one log reduction in the number of viable organisms in the RemelBactiSwab (Fig 6). Similar results were also obtained for the three swabs for maintaining the viability of non-fastidious gram negative organisms (Table 3), except for M. catarrhalis, where one log reduction in the number of viable organisms was observed after 72 hours in the Medical Wire & Equipment Transwab swabs (Fig 9). Other factors should be considered in the evaluation of specimen transport swabs. Ease of use, cost effectiveness, durability and specimen absorption. All three swabs were easy to use, inexpensive, and did not break when dropped. Sample absorption by the Copan M40 Transystem was much better than the other two swabs. The Medical Wire & Equipment Transwab had a problem with maintaining the semi-gel in the bottom of the tube, while the other two swabs did not have this problem. CONCLUSION Copan M40 Transystem swabs outperformed the other swabs evaluated for maintaining the viability three of the four fastidious organisms evaluated. H. influenzae N. gonorrhoeae N. meningitidis Remel Bactiswab & Medical Wire & Equipment Transwab outperformed Copan M40 Transystem in maintaining viability of S. pneumoniae All three swabs maintained the viability of non-fastidious gram-positive and gram-negative organisms up to 72 hours Flushing with nitrogen and packaging in this modified atmosphere appears to maintain the viability of the fastidious organisms We rank Copan M40 Transystem as the best swab followed by Remel Bactiswab, and lastly, the Medical Wire & Equipment Transwab REFERENCES 1. Perry, J. L. (1989) Factors influencing transfer of bacteria from collection / transport systems to culture media. Clin Lab Sci. 1989; Perry, J. L. (1997) Assessment of swabs transport systems for aerobic and anaerobic organisms recovery. J. Clin. Microbiol. 25, Hindiyeh, M., V. Acevedo, and K. C. Carroll. (2001). Comparison of three transport systems (Starplex StarSwab II, the new Copan Vi-Pak Amies agar gel collection and transport swabs, and the BBL port-a-cul) for maintenance of anaerobic and fastidious aerobic organisms. J. Clin. Microbiol. 39: Farhat, S. E., M. Thibault, and R. Devlin. (2001). Efficacy of a swab transport system in maintaining viability of Neisseria gonorrhoeae and Streptococcus pneumoniae. J. Clin. Microbiol. 39, Murray, P.R., E. J. Baron, M. A. Pfaller, F. C. Tenover, R. H. Yolken. (1999). Manual of Clinical Microbiology 7th edition. page 6 of 6