Investigation into the Effect of Temperature on the

Transcription

1 Investigation into the Effect of Temperature on the Viability of Fastidious and Non-Fastidious Bacteria in Two Commercial Amies Gel Transport Systems: BD CultureSwab Max V(+) and Remel BactiSwab. C. BIGGS, THE CHESTER COUNTY HOSPITAL Abstract: At The Chester County Hospital we use basic insulated cooler boxes with ice packs for transport of bacteriology swabs from outreach clinics and physicians offices. During July 2005 we monitored the internal temperature of these coolers using digital temperature recorders. Average daily temperatures ranged between º C with peaks as high as 24.3º C. We decided to evaluate viability of fastidious bacteria in two Amies Gel swab transports; Becton Dickinson CultureSwab Max V(+) (BD) and Remel BactiSwab (RE) held at room temperature (RT) 20 25º C and refrigerator temperature (RF) 4 8º C to understand if upgrading our coolers to units with electrical refrigeration might improve recovery of organisms from swabs. Using the Swab Elution Method described in CLSI M40-A swabs were inoculated with approx CFU of each of the following organisms Neisseria gonorrhoeae (NG) ATCC 43069, Haemophilus influenzae (HI) ATCC 10211, Pseudomonas aeruginosa (PA) ATCC and a clinical isolate of Streptococcus pneumoniae (SP). Each organism/device combination was tested in triplicate at RT and RF for incubation times of 24 & 48h. Zero time baseline counts and viability at each time point was quantified by making vortex suspensions and ten fold dilutions from each swab then culturing aliquots to perform replicate plate counts. At RF all organisms remained viable in both swabs at 24 & 48h but percent recovery was higher in all cases with BD. At 24h percent recovery for NG was 37.5% with BD, 32% RE; HI 89% BD, 13.7% RE; SP 17.6% BD, 4.8% RE; PA 67.3% BD and 64.7% RE. At 48h NG recovery was 2.8% with BD, 1.7% RE; HI 48.5% BD, 9.5% RE; SP 2.4% BD, 1.3% RE; PA 72.2% BD and 68% RE. At RT all organisms remained viable in both swabs at 24h but at 48h RE failed to maintain NG, HI and SP strains. Percent recovery at 24h were NG 42% with BD, 3.4% RE, HI >100% BD, 3.4% RE; SP >100% BD, 3.6% RE. PA demonstrated overgrowth in both swabs at RT and was too numerous to count at 24 & 48h time points. Our study demonstrated that at RF fastidious bacteria are recoverable from BD & RE swab transports for up to 48h. Refrigerated cooler boxes could minimize the overgrowth of PA and improve the recovery of NG, HI or SP from certain swabs. Introduction: Specimen collection and transport are processes often overlooked and undervalued but are a critical part of the total microbiology testing process to deliver quality laboratory information. Bacteriology swab specimen transport and organism viability is one area of particular interest to us as we believe there is room for improvement that could lead to better quality laboratory results. The Network of The Chester County Hospital (CCH) is like many small community hospitals in this country; the core of CCH comprises a 238 bed hospital with satellite locations in West Chester, The Chester County Hospital West Chester, Pennsylvania Downingtown, Exton, Kennett Square, and Lionville, Pennsylvania that include various out-patient labs and drawing stations that collect throat cultures and urine samples. In addition to this we collect samples from various physicians offices. The microbiology lab utilizes a contract courier service that is shared with other hospital departments which manages the collection of patient samples throughout the day within the network. Transit times for microbiology specimens can vary from 15 minutes to several hours depending on timing and courier routing. Before the summer of 2005 we had followed guidelines that appear in microbiology reference manuals which advocate the shipment of microbiology swab samples at room temperature. However, after reviewing recent published studies on organism viability in swab transport systems which demonstrated performance at refrigerator temperature was superior to room temperature we decided to instigate improvements in our specimen management with the goal of lowering the holding temperature during transit to the laboratory down to as near as possible refrigerator temperature (4 8 C) 1-7. Initially we implemented the use of basic insulated cooler boxes filled with ice packs taken from our -20 C freezer and we studied the effectiveness of this combination to provide cold transport temperatures during July 2005 using digital temperature recorders. The results using this combination were not as good as anticipated so we tested insulated cooler boxes incorporating a built-in electrical refrigeration system together with ice packs from our -80 C freezer in the hope that this combination would be more effective at achieving the target temperature of range of 4 8 C. To assess the impact of temperature on organism viability in our patient swab specimens and to provide supporting data to help implement a procedural change for our cooler box courier service at CCH, we conducted a comparative viability study. In this study we tested two brands of Amies Agar Gel; Becton Dickinson CultureSwab Max V(+) (BD) and Remel BactiSwab (RE). Swabs were tested using a selection of aerobic and fastidious bacteria at both room temperature (RT) 20 25º C and refrigerator temperature (RF) 4 8º C. Using data from our cooler box study and viability studies our aim was to draw conclusions that would endorse a new specimen handling procedure for our courier service that would optimize the survival of bacteria and improve laboratory test results.

2 Materials: Cooler Box Temperature Study Rubbermaid 11 x 15 inches - basic insulated cooler box (BCB) with three ice packs from -20 C freezer. Power Travel Cooler 12 x 17 inches (Mfg. by Power On Board) - insulated cooler box with integrated refrigeration unit - refrigerate cooler box (RCB) with two ice packs -80 C. Generic re-usable cooler ice packs Escort Junior Temperature Recorder (EJTR) Model # EJ-IN-D-16-L digital temperature recorder manufactured by Escort Data Loggers, Redmond, WA. Microbiology Lab Microbiology Lab Process Diagram 1. Programming 5. Downloading Loggers ETM Escort Temperature Management System Loggers 2. Programmed Loggers sent inside specimen coolers 3. Transportation of specimens 4. Loggers retrieved from cooler boxes at the end of the day Methods: 1. On 11 days during the month of July and 8 days during the month of August 2005 the temperature inside BCB and RCB coolers respectively, used for the collection and transportation of specimens were digitally recorded at regular time intervals. 2. At the start of each day an EJTR digital recorder was programmed using a laboratory PC and software provided by the manufacture. The recorder was programmed to take temperature readings every 10 minutes between approximately 10:30 am and 4:00 or 4:30 pm. This time frame corresponded to the normal hours of operation of courier specimen transport service to the lab. 3. On the morning of each day of the study at least 30 minutes prior to the first temperature reading the cooler boxes were loaded with ice packs. For the BCB cooler study they were loaded with ice packs prepared overnight in the -20 C freezer. Initially, the RCB coolers were loaded with ice-packs from the -20 C freezer, but the temperature was still too high so the RCB cooler was subsequently loaded with ice packs prepared overnight in the -80 C. The EJTR recorder was then placed in the center of the cooler box and the lid was secured. 4. The cooler boxes were collected by the courier at approximately 10:30 am each day. They were used throughout the day for loading, transportation and delivery of samples to and from the lab and returnedbackto the lab with the final delivery of specimens at 4:00 or 4:30 pm. 5. At the end of each day the EJTR was removed from the cooler and all the temperature measurements from the device for that day were downloaded via an interface to a PC. 6. The procedure was repeated for each day of the study. Comparative Viability Study Materials: Commercial Amies agar gel without charcoal transport swabs: BD - CultureSwab Max V(+), Becton Dickinson, Baltimore, MD (Mfg. by Copan, Brescia, Italy) RE - BactiSwab, Remel, Lenexa, KS (Mfg. by Starplex Scientific, Ontario, Canada) Species Abbreviation Strain Neisseria gonorrhoeae NG ATCC Haemophilus influenzae HI ATCC Streptococcus pneumoniae SP clinical strain Pseudomonas aeruginosa PA ATCC Method(Quantitative Swab Elution Method1) Preparation of inoculum 1. Inocula of test organisms was prepared in 0.85% physiological saline (ph ) to a concentration of approximately 1.5 x 10 8 CFU/mL (equivalent to 0.5 McFarland standard) from an 18 to 24 hour plate culture of each organism. 2. Each inoculum was diluted as described below in 0.85% physiological saline (ph ) solution to provide a concentration of approximately 1.5 x 10 7 CFU/mL. 3. The inoculum of each test organisms was prepared just prior to transferring the organism suspension to microtiter plates where the swab absorption was performed. The whole procedure did not exceed 20 minutes in order to reduce loss of organism viability in inoculum prior to incubation of inoculated swabs at the holding temperature

3 Inoculation procedure 4. Each microorganism/device combination required 9 swab inoculations for both the room temperature (RT) 20 25º C and refrigerator temperature (RF) 4 8º C viability study. The follow steps were carried out for each temperature study. 3 swabs for zero-time inoculum determination 3 swabs for 24 hrs time point viability test 3 swabs for 48 hrs time point viability test µl of inocula was pipetted into each of 9 wells of a microtiter plate for each microorganism to be tested. 6. Each of the 9 swabs for a particular microorganism/device combination was placed into a microtiter well until it touched the bottom and was allowed to absorb the organism inoculum for a minimum of ten seconds. 7. Sampling swabs were then placed directly into their transport device tube or container. 8. All devices were processed in the same manner. Approximately 20 minutes was taken from the preparation of organism suspension to placement of sampling devices in the appropriate transport system. 9. Swab transport devices were held at RT or RF for 0, 24 and 48 hrs and then processed. See summary Table 1. Procedure for colony counting and determining organism viability 1. At the appropriate time point swabs were removed from their transport tube or container and placed into a tube containing 1 ml of 0.85% physiological saline (ph ). This is the primary tube vortex suspension. 2. Each swab was mixed vigorously using a vortex mixer for a minimum of 15 seconds. 3. The primary tube vortex suspension was then thoroughly mixed and 100 ml of this suspension was pipetted onto appropriate plated media. A repeat pipetting step was performed to provide a second plate for duplicate determination. 4. This procedure was repeated for each of the other dilutions 10-1, 10-2 thru All spread plates for colony counts were incubated at ºC in appropriate atmosphere of incubation as described in Table 1. Table 1. Species Inoculum(CFU) Plate Media Incubation T ºC Incubation Atmosphere Testing Time (hours) Neisseria gonorrhoeae 1 x 106 Chocolate agar % CO2 0, 24, 48 Haemophilus influenzae 1 x 106 Chocolate blood agar % CO2 0, 24, 48 Streptococcus penumoniae 1 x 106 5% sheep blood agar % CO2 0, 24, 48 Pseudomonas aeruginosa 1 x 106 5% sheep blood agar % CO2 0, 24, From the original 9 inoculated swabs three of the test swabs for each microorganism/device combination are utilized as zero-time controls. These swabs were removed from their transport containers within 5-15 minutes after they are inoculated. These three swabs serve as zero-time growth controls against which the all other swab holding time points were compared. The remaining test swabs for each organism and temperature/combination were stored for the specified time points and at each time point three swabs were process in an identical manner as described in All spread plate cultures for colony counting were incubated for up to 48 hours before recording the number of Colony Forming Units (CFUs) growth. 8. Counts for each set of 3 swabs held at each time point were combined and averaged. Summary: Organism A - tested against two swab devices Inoculate each swab > Incubate > Vortex in saline > Make three 10-fold dilutions Take 100µl of primary tube vortex suspension and 100µl of each 10-fold dilution and inoculate in duplicate onto culture plates Two swab devices at three time points and tested in triplicate = total of 18 swabs per organism test run.

4 Results Cooler Box Temperature Study The Cooler Box Temperature Study was conducted using two set ups: 1. Basic insulated cooler box (BCB) with three ice packs from -20 Cfreezer. Summary of temp recordings - BCB Basic Insulated Cooler Box with three ice packs from -20 C freezer 7/7/2005 7/8/2005 7/11/2005 7/12/2005 7/13/2005 7/14/2005 7/15/2005 7/18/2005 7/19/2005 7/20/2005 7/21/2005 Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Av Average Average Daily High Insulated cooler box with integrated refrigeration unit - refrigerated cooler box (RCB) with two ice packs -80 C. Summary of temp recordings - RCB - cooler box with electrical refrigeration and -80C ice packs 8/16/2005 8/17/2005 8/18/2005 8/19/2005 8/22/2005 8/23/2005 8/29/2005 8/30/2005 Average Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Time Temp C Temp Average Average Daily High 18.9 only at start

5 Escort Junior Temperature Recorder (EJTR) accurately recorded the temperature at programmed time intervals during the hours of operation 10:30 AM until 4:00 or 4:30 PM. Temperature measurements for this study are summarized in tables 1 and 2. Average daily temperatures for BCB ranged between º C with peaks as high as 24.3º C. During the working day temperatures inside the BCB cooler box varied significantly and this most likely corresponded to erratic open and closure of the box to deposit or remove samples. Only for one hour on one day (7/12/2005) during our study did temperatures inside BCB drop below 9º C. Most often temperatures fluctuated in the mid to low teens Celsius. On the other hand, the RCB set up with -80 C ice packs was very effective at lowering the initial temperature inside the cooler box at the start of each day and the built in refrigeration system was much more effective at maintaining lower temperatures. Average daily temperatures for RCB ranged between º C occasionally temperatures exceeded this range but rarely went above 9º C. Comparative Viability Study The protocol used in this study was based upon the quantitative Swab Elution method as described in the CLSI M40-A standard. This calls for only 0 hrs and 48 hrs endpoint analyses except for Neisseria gonorrhoeae which is measured at 24 hrs endpoint. Acceptable performance is specified as viability at these time points should demonstrate no more than a 3 log 10 (1 x 10 3 ± 10%) decline compare to zero-time CFU counts at both RF (4-8 C) and RT (20-25 C). Tests performed at 4-8 C also include an assessment of bacterial overgrowth as part of the acceptance criteria for organism recovery. Acceptable overgrowth is defined as no more than a 1-log increase in CFU between the zero-time CFU count and the CFU count after incubation for a specified period. For our own interest we recorded viability for all organisms at both 24 and 48 hours. Each swab device/organism combination was tested in triplicate for each time point and colony counts of primary tube vortex suspensions and tenfold dilutions of vortex suspensions were performed in duplicate. Minor test variation was minimized by averaging colony counts from these multiple data points. Colony counts, percent recovery and interpretation of performance are summarized in the following table and graph. Organism / Swab Device 0 hr 0 hr % CFUs Recovery 24 hr CFUs 24 hr % Recovery Interpretation* 48 hr 48 hr % CFUs Recovery Interpretation* Neisseria 4 8 C BD - CultureSwab Max V(+) Pass Remel - BactiSwab Pass Neisseria C BD - CultureSwab Max V(+) Pass Remel - BactiSwab Pass No Growth 0.00 Haemophilus 4 8 C BD - CultureSwab Max V(+) Pass Remel - BactiSwab Pass Haemophilus C BD - CultureSwab Max V(+) > >100 Pass Remel - BactiSwab Fail Streptococcus 4 8 C BD - CultureSwab Max V(+) Pass Remel - BactiSwab Pass Streptococcus C BD - CultureSwab Max V(+) > Pass Remel - BactiSwab Fail Pseudomonas 4 8 C BD - CultureSwab Max V(+) Pass Remel - BactiSwab Pass Pseudomonas C BD - CultureSwab Max V(+) TNTC >100 TNTC >100 Remel - BactiSwab TNTC >100 TNTC >100 Percentage data rounded up to one decimal place for study abstract * Interpretation according to CLSI M40-A for Swab Elution Method. At RF (4-8 C) all organisms remain viable in both swabs at 24 & 48 hrs within the acceptance criteria of M40-A and CFU but percent recovery was higher in all cases with BD CultureSwab Max V(+). In particular Haemophilus influenzae recovery differed significantly; 48.5% with BD and 9.5% with RE at 48 hrs. At RT (20 25 C) only BD was able to recover all four organisms whereas REMEL BactiSwab failed to recover Neisseria gonorrhoeae, Streptococcus pneumoniae and Haemophilus influenzae. CLSI does not recommend overgrowth assessments to be made at room temperature because of the known problems with overgrowth of gram-negative bacilli. In our study no overgrowth was noted with any organisms at RF but Pseudomonas aeruginosa overgrew in both BD and RE swabs at RT and was too numerous to count. In BD at RT the CFU count for HI at 48 hrs was higher than zero-time but was still less than a 1-log increase. Percentage Recovery NG HI SP PA NG HI SP PA 24 Hours 48 Hours 4-8 C Temperature Results Holding Time BD - CultureSwab Max V (+) Remel - BactiSwab C Temperature Results BD - CultureSwab Max V (+) Remel - BactiSwab Conclusions Specimen transport is a critical part of the pre-analytical phase which can have a direct impact on the quality of laboratory test results. Previous studies on transport swabs have indicated that organism survival may be superior at RF compared to RT. We embarked on a project to move from ambient RT specimen transport to cooler temperatures using a combination of insulated coolers and ice packs. Our intention was to present this data along with organism viability data at RT and RF to our laboratory management and contract courier service to instigate a change in specimen transport logistics. 1. A combination of a cooler with a built in refrigeration systems and ice packs taken from our -80 C freezer at the beginning of each day was the most effective way to achieve lower cooler box temperatures throughout the day. 2. From our organism viability study we discovered that holding and transporting samples at elevated temperatures (20 25 C) could result in significance loss in organism viability in certain Amies transport systems (notably Remel with NG, HI and SP). 3. Careful assessment of different Amies Agar Gel swabs is important to demonstrate divergent performance capabilities. BD transport was able to maintain viability of fastidious organisms at both cool and elevated temperatures with notable higher percent recoveries at all time points. 4. At elevated temperatures there is the risk of specimen overgrowth with gram-negative bacilli with all Amies Agar Gel swabs.

6 5. We implemented refrigerator cooler boxes and -80 C ice packs for specimen management. This represents a relatively minimal investment that can lead to improved specimen quality by reducing the risk of loss of organisms and minimizing gram-negative overgrowth. 6. The Escort Junior Temperature Recorder System (one Recorder $133, Interface $48, Key Starter $7.50 and free software) is a low cost, simple means for evaluating and continually auditing the temperature at which samples are transported in cooler boxes. It provides a tool for laboratories to make quality improvements in specimen logistics. References 1.Arbique JC, Forward KR, LeBlanc J. Evaluation of four commercial transport media for the survival of Neisseria gonorrhoeae. Diagnostic Microbiology and Infectious Disease. 2000; 36: Perry JL. Effects of temperature on fastidious organism viability during swab transport. 101st General meeting of the American Society for Microbiology. 2001; Orlando, FL. Abstract C Wilson DA, Tuohy MS, Procop GW, Hall GS. Effects of storage on the recovery of bacteria from three swab transport systems: BD CultureSwab, BD Culturette and Starplex StarSwab II. 101st General meeting of the American Society for Microbiology. 2001; Orlando, FL. Abstract C Arbique J, Campbell S, MacFarlane M, Davidson RJ. Comparison of methodologies described in NCCLS document M40-P Quality Control of Microbiology Transport Devices. 103rd General meeting of the American Society for Microbiology. 2003; Washington, DC. Abstract C Mitchell E, Berman M, Ginocchio CC. Evaluation of two new Liquid Stuart transport systems: Platinum StarSwab II (Starplex Scientific) and BBL CultureSwab (Becton Dickinson). 102nd General meeting of the American Society for Microbiology. 2002; Salt Lake City, UT. Abstract C Perry JL, Matthews JS. Compliance of two popular swab transport systems with performance standards detailed by the new NCCLS Proposed Standard, M40-P. 103rd General meeting of the American Society for Microbiology. 2003; Washington, DC. Abstract C Robinson A, Gruver ML. Comparison of bacterial survival in two transport systems stored at room temperature and refrigerator temperatures. 102nd General meeting of the American Society for Microbiology. 2002; Salt Lake City, UT. Abstract C Clinical Laboratory Standards Institute CLSI (formerly National Committee for Clinical Laboratory Standards NCCLS) Quality Control of Microbiology Transport Systems. M40-A Vol 23 No. 34. Page 6 of 6