Transwab -Transwab -Virocult -VCM Virocult

Transcription

1 Transwab -Transwab -Virocult -VCM Virocult M40-A COMPLIANCE

2 Medical Wire Compliance with CLSI M40-A Since the launch in 1975 of Transwab, the world s first commercially produced all-in-one transport swab, Medical Wire has remained committed to the quality of its products, ensuring reliable handling of the patient s specimen, and dependable performance for all of our laboratory colleagues. Accordingly, Medical Wire was one of the early adopters of CLSI s M40-A standard, implementing the requirements of the then proposed standard (M40- P) in We have always seen it as an essential reference to measure and where possible improve the performance of existing products, and for the development of new transport devices. This document provides details of those of our products for which M40-A is relevant, with details of compliance. Reference CLSI. Quality Control of Microbiological Transport Systems ; Approved Standard M40-A. CLSI (formerly NCCLS) document M40-A [ISBN ]. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania USA, a

3 Purpose of M40-A CLSI s a document M40-A was first published as an approved standard in 2003 b by the then NCCLS c. It set out criteria for the guidance of manufacturers of microbiological transport devices, including urine specimen containers, blood culture bottles, and transport swabs. These same criteria were also intended to assist end users in assessing the relative performance characteristics of available devices. The essential requirement of M40-A is to show that the microbiological quality of a patient s specimen is being maintained in a stable manner during the anticipated transport period and conditions. In the case of transport swabs where live microorganisms are required to remain viable without drastic increase or decrease in numbers, the performance is assessed with a set of ten specified ATCC strains of bacteria in categories of aerobes, anaerobes, and fastidious organisms. Survival is assessed for up to 48 hours at either ambient or refrigeration temperatures. Swabs for virology are assessed using specified viruses. Transport swabs which are intended for a more restricted set of criteria can be assessed, but the limitations of any claims must be clearly stated. Since publication, M40-A has become established as the international benchmark for transport devices, perhaps particularly in relation to transport swabs for bacteriology and virology. Many studies have been presented or published invoking its requirements, although sadly not all such studies are genuine, and may show conclusions which cannot be reconciled with the data presented. Notes a. CLSI : Clinical Laboratory Standards Institute b. M40-A is currently being revised, and will be reissued in the near future as CLSI M40-A2 c. NCCLS: National Committee for Clinical Laboratory Standards Please note CLSI M40-A is a copyrighted document and copies can be purchased from Clinical Laboratory Standards Institute at CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania USA

4 Table 1 M40-A Compliance MW169C MW169P MW170 MW171 MW172C MW172P MW173C MW173P MW175C MW175P MW176S MW176S MW167S MW177S MW910S MW950S MW950 Pseudomonas aeruginosa (ATCC BAA-427) Streptococcus pyogenes (ATCC 19615) Streptococcus pneumoniae (ATCC 6305) Haemophilus influenzae (ATCC 10211) Bacteroides fragilis (ATCC 25285) Peptostreptococcus anaerobius (ATCC 27337) Fusobacterium nucleatum (ATCC 25586) Prevotella melaninogenica (ATCC 25845) Propionibacterium acnes (ATCC 6919) Neisseria gonorrhoeae (ATCC 43069) Adenovirus Influenza A Herpes Simplex Virus Type 2 Respiratory Syncitial Virus Mycoplasma hominis Ureaplasma urealyticum Chlamydia trachomatis

5 Table 2 M40-A Compliance for Transwab Products MW169C MW169P MW170 MW171 MW172C MW172P MW173C MW173P MW175C MW175P Pseudomonas aeruginosa (ATCC BAA-427) Streptococcus pyogenes (ATCC 19615) Streptococcus pneumoniae (ATCC 6305) Haemophilus influenzae (ATCC 10211) Bacteroides fragilis (ATCC 25285) Peptostreptococcus anaerobius (ATCC 27337) Fusobacterium nucleatum (ATCC 25586) Prevotella melaninogenica (ATCC 25845) Propionibacterium acnes (ATCC 6919) Neisseria gonorrhoeae (ATCC 43069)

6 Validation studies for Transwabs with Plain Medium and Transwabs with Charcoal Medium Recovery Studies Various batches of Medical Wire Transwabs with Amies Medium and Transwab with Amies Medium with Charcoal transport swabs were tested in accordance with CLSI Quality Control of Microbiological Transport Systems ; Approved Standard M40-A. Testing was carried out using both the Roll Plate Method, and the Swab Elution Method. Known concentrations of Pseudomonas aeruginosa (ATCC BAA-427), Streptococcus pyogenes (ATCC 19615), Streptococcus pneumoniae (ATCC 6305), Haemophilus influenzae (ATCC 10211), Bacteroides fragilis (ATCC 25285), Peptostreptococcus anaerobius (ATCC 27337), Fusobacterium nucleatum (ATCC 25586), Propionibacterium acnes (ATCC 6919), Prevotella melaninogenica (ATCC 25845) and Neisseria gonorrhoeae (ATCC 43069) were inoculated in triplicate onto swabs and held at 22 O C or 4 O C for up to 48 hours. Recoveries were measured at 0hours, 24 hours or 48 hours according to strain as shown in Tables 1-4. For the Roll Plate Method swabs are inoculated by dipping in tubes or microtitre plate wells containing 100μl of diluted inoculum containing 10 8, 10 7, 10 6, 10 5, or 10 4 cfu /ml. The swabs are then held in their transport medium for the designated period before plating out. For the Swab Elution Method, swabs are inoculated with 100 μl of the 10 7 cfu/ml, and held in their transport medium for the designated period. After the holding period the swab is transferred to a tube containing 1 ml of 0.85% saline. The tube is vortexed, and then 100μl is added to 900μl of saline, and this procedure is repeated to give a series of dilutions from 10 6 cfu/ml to 10 1 cfu/ml. From each tube, 100μl is plated onto appropriate medium. The results showed that Medical Wire Transwabs with Amies Medium and Transwab with Amies Medium with Charcoal transport swabs maintain the viability of these organisms as set out in the standard.

7 Table 3: Results for Transwab with Plain Medium ( Roll Plate Method) Temp Time (hours) Cfu/ml Pseudomonas aeruginosa (ATCC BAA-427) x 10 8 Pseudomonas aeruginosa (ATCC BAA-427) 4C x 10 8 Streptococcus pyogenes (ATCC 19615) x 10 7 Streptococcus pyogenes (ATCC 19615) 4C x 10 7 Streptococcus pyogenes (ATCC 19615) RT x 10 7 Streptococcus pneumoniae (ATCC 6305) x 10 5 Streptococcus pneumoniae (ATCC 6305) 4C x 10 5 Streptococcus pneumoniae (ATCC 6305) RT x 10 4 Haemophilus influenzae (ATCC 10211) x 10 8 Haemophilus influenzae (ATCC 10211) 4C x 10 7 Haemophilus influenzae (ATCC 10211) RT x 10 7 Bacteroides fragilis (ATCC 25285) x 10 8 Bacteroides fragilis (ATCC 25285) 4C x 10 8 Bacteroides fragilis (ATCC 25285) RT x 10 7 Peptostreptococcus anaerobius (ATCC 27337) x 10 5 Peptostreptococcus anaerobius (ATCC 27337) 4C x 10 4 Peptostreptococcus anaerobius (ATCC 27337) RT x 10 4 Propionibacterium acnes (ATCC 6919) x 10 8 Propionibacterium acnes (ATCC 6919) 4C x 10 8 Propionibacterium acnes (ATCC 6919) RT x 10 7 Prevotella melaninogenica (ATCC 25845) x 10 7 Prevotella melaninogenica (ATCC 25845) 4C x 10 4 Neisseria gonorrhoeae (ATCC 43069) x 10 6 Neisseria gonorrhoeae (ATCC 43069) 4C x 10 5 Table 4: Results for Transwab with Plain Medium (Swab Elution Method) Temp Time (hours) SEM Pseudomonas aeruginosa (ATCC BAA-427) x 10 7 Pseudomonas aeruginosa (ATCC BAA-427) 4C x 10 7 Streptococcus pyogenes (ATCC 19615) x 10 7 Streptococcus pyogenes (ATCC 19615) 4C x 10 7 Streptococcus pyogenes (ATCC 19615) RT x 10 6 Streptococcus pneumoniae (ATCC 6305) x 10 6 Streptococcus pneumoniae (ATCC 6305) 4C x 10 5 Streptococcus pneumoniae (ATCC 6305) RT x 10 5

8 Haemophilus influenzae (ATCC 10211) x 10 6 Haemophilus influenzae (ATCC 10211) 4C x 10 5 Haemophilus influenzae (ATCC 10211) RT Bacteroides fragilis (ATCC 25285) x 10 8 Bacteroides fragilis (ATCC 25285) 4C x 10 8 Bacteroides fragilis (ATCC 25285) RT x 10 7 Peptostreptococcus anaerobius (ATCC 27337) x 10 6 Peptostreptococcus anaerobius (ATCC 27337) 4C x 10 6 Peptostreptococcus anaerobius (ATCC 27337) RT x 10 4 Propionibacterium acnes (ATCC 6919) x 10 8 Propionibacterium acnes (ATCC 6919) 4C x 10 8 Propionibacterium acnes (ATCC 6919) RT x 10 8 Prevotella melaninogenica (ATCC 25845) x 10 7 Prevotella melaninogenica (ATCC 25845) 4C x 10 6 Neisseria gonorrhoeae (ATCC 43069) x 10 5 Neisseria gonorrhoeae (ATCC 43069) 4C x 10 5 Table 5: Results for Transwab with Charcoal Medium ( Roll Plate Method) Temp Time (hours) RPM Pseudomonas aeruginosa (ATCC BAA-427) x 10 7 Pseudomonas aeruginosa (ATCC BAA-427) 4C x 10 7 Streptococcus pyogenes (ATCC 19615) x 10 7 Streptococcus pyogenes (ATCC 19615) 4C x 10 6 Streptococcus pyogenes (ATCC 19615) RT x 10 6 Streptococcus pneumoniae (ATCC 6305) x 10 5 Streptococcus pneumoniae (ATCC 6305) 4C x 10 5 Streptococcus pneumoniae (ATCC 6305) RT x 10 5 Haemophilus influenzae (ATCC 10211) x 10 7 Haemophilus influenzae (ATCC 10211) 4C x 10 7 Haemophilus influenzae (ATCC 10211) RT x 10 6 Bacteroides fragilis (ATCC 25285) x 10 7 Bacteroides fragilis (ATCC 25285) 4C x 10 7 Bacteroides fragilis (ATCC 25285) RT x 10 6 Peptostreptococcus anaerobius (ATCC 27337) x 10 7 Peptostreptococcus anaerobius (ATCC 27337) 4C x 10 6 Peptostreptococcus anaerobius (ATCC 27337) RT x 10 4 Fusobacterium nucleatum (ATCC 25586) x 10 7 Fusobacterium nucleatum (ATCC 25586) 4C x 10 7 Fusobacterium nucleatum (ATCC 25586) RT x 10 5 Propionibacterium acnes (ATCC 6919) x 10 8 Propionibacterium acnes (ATCC 6919) 4C x 10 8

9 Propionibacterium acnes (ATCC 6919) RT x 10 8 Prevotella melaninogenica (ATCC 25845) x 10 6 Prevotella melaninogenica (ATCC 25845) 4C x 10 5 Neisseria gonorrhoeae (ATCC 43069) x 10 5 Neisseria gonorrhoeae (ATCC 43069) 4C x 10 5 Neisseria gonorrhoeae (ATCC 43069) RT x 10 4 Table 6: Results for Transwab with Charcoal Medium (Swab Elution Method) Temp Time (hours) Pseudomonas aeruginosa (ATCC BAA-427) x 10 7 Pseudomonas aeruginosa (ATCC BAA-427) 4C x 10 6 Streptococcus pyogenes (ATCC 19615) x 10 5 Streptococcus pyogenes (ATCC 19615) 4C x 10 4 Streptococcus pyogenes (ATCC 19615) RT x 10 3 Streptococcus pneumoniae (ATCC 6305) x 10 5 Streptococcus pneumoniae (ATCC 6305) 4C x 10 5 Streptococcus pneumoniae (ATCC 6305) RT x 10 5 Haemophilus influenzae (ATCC 10211) x 10 5 Haemophilus influenzae (ATCC 10211) 4C x 10 5 Haemophilus influenzae (ATCC 10211) RT x 10 5 Bacteroides fragilis (ATCC 25285) x 10 7 Bacteroides fragilis (ATCC 25285) 4C x 10 7 Bacteroides fragilis (ATCC 25285) RT x 10 6 Fusobacterium nucleatum (ATCC 25586) x 10 7 Fusobacterium nucleatum (ATCC 25586) 4C x 10 4 Fusobacterium nucleatum (ATCC 25586) RT x 10 5 Propionibacterium acnes (ATCC 6919) x 10 7 Propionibacterium acnes (ATCC 6919) 4C x 10 7 Propionibacterium acnes (ATCC 6919) RT x 10 6 Neisseria gonorrhoeae (ATCC 43069) x 10 7 Neisseria gonorrhoeae (ATCC 43069) 4C x 10 6 Neisseria gonorrhoeae (ATCC 43069) RT x 10 5 SEM Conclusion: The results showed that Medical Wire Transwabs with Amies Medium and Transwab with Amies Medium with Charcoal maintain the viability of these organisms as set out in the standard. Recovery study completed by: Alexandra Standke, Corsham, England May 2012

10 Table 7 M40-A Compliance for -Transwab Products MW176S MW167S MW176S3 MW177S Pseudomonas aeruginosa (ATCC BAA- 427) Streptococcus pyogenes (ATCC 19615) Streptococcus pneumoniae (ATCC 6305) Haemophilus influenzae (ATCC 10211) Bacteroides fragilis (ATCC 25285) Peptostreptococcus anaerobius (ATCC 27337) Fusobacterium nucleatum (ATCC 25586) Prevotella melaninogenica (ATCC 25845) Propionibacterium acnes (ATCC 6919) Neisseria gonorrhoeae (ATCC 43069)

11 Validation studies for -Transwab with Liquid Amies Medium Three different batches of -Transwab transport swabs were tested in accordance with CLSI Quality Control of Microbiological Transport Systems 4 ; Approved Standard M40-A. Known concentrations of Pseudomonas aeruginosa (ATCC BAA-427), Streptococcus pyogenes (ATCC 19615), Streptococcus pneumoniae (ATCC 6305), Haemophilus influenzae (ATCC 10211), Bacteroides fragilis (ATCC 25285), Peptostreptococcus anaerobius (ATCC 27337), Fusobacterium nucleatum (ATCC 25586), Propionibacterium acnes (ATCC 6919), Prevotella melaninogenica (ATCC 25845) and Neisseria gonorrhoeae (ATCC 43069) were inoculated in triplicate onto swabs from three different lot numbers and held at 22 O C and 4 O C for up to 48 hours. Recoveries were measured at 0hours, 24hours (Neisseria gonorrhoeae) and/or 48 hours (all other test strains). The results showed that -Transwab transport swabs maintain the viability of these organisms as set out in the standard. METHODS: Testing was performed according to the Swab Elution Method as described in CLSI Quality Control of Microbiological Transport Systems 4 ; Approved Standard M40-A. Inocula were prepared in 0.85% physiological saline to a concentration of approximately 1.5 x These suspensions were diluted 1:5, and 50μl dispensed into microtitre plate wells. The 50μl aliquots were absorbed onto the swab tips for each swab being tested. The swabs were placed in their respective tube of medium and held at 4C or 22C for 0, 24 or 48 hours as shown in the table. After the holding time 100μl aliquots were removed from each tube and plated on appropriate agar, using a spiral plater. Following incubation of the plates, the resultant colonies were counted and used to calculate the concentration of bacteria in the Transwab medium. The original suspensions were also tested in this way.

12 Table 8: Results for Sigma-Transwab (Swab Elution Method) Organism Temp Time (hours) cfu/ml (average) Pseudomonas aeruginosa (ATCC BAA-427) x 10 6 Pseudomonas aeruginosa (ATCC BAA-427) 4C x 10 6 Pseudomonas aeruginosa (ATCC BAA-427) RT x 10 7 Streptococcus pyogenes (ATCC 19615) x 10 5 Streptococcus pyogenes (ATCC 19615) 4C x 10 5 Streptococcus pyogenes (ATCC 19615) RT x 10 5 Streptococcus pneumoniae (ATCC 6305) x 10 6 Streptococcus pneumoniae (ATCC 6305) 4C x 10 5 Streptococcus pneumoniae (ATCC 6305) RT x 10 7 Haemophilus influenzae (ATCC 10211) x 10 6 Haemophilus influenzae (ATCC 10211) 4C x 10 6 Haemophilus influenzae (ATCC 10211) RT x 10 6 Bacteroides fragilis (ATCC 25285) x 10 6 Bacteroides fragilis (ATCC 25285) 4C x 10 6 Bacteroides fragilis (ATCC 25285) RT x 10 6 Peptostreptococcus anaerobius (ATCC 27337) x 10 6 Peptostreptococcus anaerobius (ATCC 27337) 4C x 10 6 Peptostreptococcus anaerobius (ATCC 27337) RT x 10 5 Fusobacterium nucleatum (ATCC 25586) x 10 5 Fusobacterium nucleatum (ATCC 25586) 4C x 10 5 Fusobacterium nucleatum (ATCC 25586) RT x 10 5 Propionibacterium acnes (ATCC 6919) x 10 6 Propionibacterium acnes (ATCC 6919) 4C x 10 6 Propionibacterium acnes (ATCC 6919) RT x 10 5 Prevotella melaninogenica (ATCC 25845) x 10 6 Prevotella melaninogenica (ATCC 25845) 4C x 10 6 Prevotella melaninogenica (ATCC 25845) RT x 10 3 Neisseria gonorrhoeae (ATCC 43069) x 10 5 Neisseria gonorrhoeae (ATCC 43069) 4C x 10 5 Neisseria gonorrhoeae (ATCC 43069) 4C x 10 4 Conclusion: The results showed that -Transwab transport swabs maintain the viability of these organisms as set out in the standard. Recovery study performed by: Monika Stuczen, Manchester Metropolitan University, Manchester, England November 2012 REFERENCES 1. Cumitech - Various American Society for Microbiology, Washington D.C., various dates Garcia, L., (3 ed.), Clinical Microbiology Procedures Handbook. American Society for Microbiology, Washington, D.C., Manual of Clinical Microbiology, 9 th Edition, ASM Press, Washington D.C. 4. CLSI. Quality Control of Microbiological Transport Systems ; Approved Standard M40-A. CLSI (formerly NCCLS) document M40-A [ISBN ]. CLSI, 940 West Valley Road, Suite 1400, Wayne, Pennsylvania USA, 2003.

13 Laboratory Evaluation of Σ-Virocult Transport Swabs Edwin Sharma and Jenni Lindeman Virology Laboratory, Health Waikato Laboratories, Waikato Hospital, Hamilton, New Zealand ABSTRACT Background: Collection and storage of viral specimen is the most important factor in obtaining a reliable and accurate diagnostic result. Swabs are often used to collect specimens for detecting and identifying viral infections Scope of study: In this study Σ-Virocult transport swabs (Medical Wire and Equipment) were evaluated using the CLSI Quality Control of Microbiological Transport Systems Approved Standard M40-A. The standard presents a specified culture protocol to assess a viral culture transport device manufactured to facilitate virus preservation. The study was carried out using two different temperatures. Method: Herpes simplex virus type 2 and Adenovirus type 3 were inoculated into Σ- Virocult swabs at a concentration of 5 x 10 4 TCID 50 and held at 22ºC and 4ºC. The swabs were sampled every day for four days, then at day seven. Results: The results showed that the swabs maintain the viability of virus as set out in the standard and that survival is better at 4ºC. Conclusion: The Σ-Virocult transport swabs may be recommended as a reliable virus transport device. INTRODUCTION Viral particles vary widely in composition, structure, morphology size and stability (2). Viral isolation has been widely accepted as the gold standard for laboratory confirmation of viral infection; however, it requires specimen storage and transport in a viral medium maintained at low temperatures to optimally preserve infectious viral particles (1). Clinical specimens for virus isolation should ideally be inoculated into cell culture with as little delay as possible (2). However, many common viruses are able to withstand both storage at room temperature and transport for extended periods of time when placed in a suitable transport medium (2). A suitable transport medium for swabs must maintain viability of viruses, prevent overgrowth of bacteria and fungi, and prevent drying of the specimen (6). Swabs are generally used to collect cells and fluid from nasal passages, the throat, the rectum, vesicles, eyes, and cervical, genital and skin lesions. The Σ-Virocult is designed for specimen collection, transport and maintenance of virus viability. The device consists of soft polyurethane foam budded swab and a transport tube of liquid virus transport medium which contains antimicrobials to inhibit growth of any bacteria or fungi present in the specimen (5,10). This study evaluates the Σ-Virocult transport swab to determine whether it is able to maintain the viability of viruses at different temperatures over a certain period of time. MATERIALS AND METHODS Materials Viruses: Stock concentrations of wild-type (clinical isolates) Adenovirus type 3 and Herpes simplex virus type 2 were used in the swab evaluation study. Cell lines: HEP2 (human larynx carcinoma) and A549 (human lung carcinoma) cell lines routinely utilised in the laboratory were utilised as the cell lines to support the growth of Adenovirus (HEP2) and Herpes simplex type 2 (A549). Fresh working lots of low passage number (<15) were split into tube cell cultures to have a confluent monolayer after 24 hours. Swabs: Σ-Virocult, Lot No. 09J30, expiry date Sep 2010 were used in the study. Methods The wild-type viruses were inoculated into a flask containing a confluent monolayer of HEP2 for Adenovirus type 3 and A549 for Herpes simplex type 2. Once most of the monolayer showed typical cytopathic effect (CPE), the virus was harvested and stored for use as the stock suspension of the virus. Stock suspensions of Adenovirus type 3 and Herpes simplex

14 type 2 were serially diluted in cell culture maintenance medium using 10-fold dilutions from 10-1 to For each dilution, 5 tubes of fresh HEP2 and A549 cell cultures were inoculated with 100µl of diluted viral suspension. The cultures were incubated at 37ºC and examined daily for CPE over a period of 10 days. TCID 50 (tissue culture infective dose which is the amount of virus which gives a CPE in 50% of inoculated cultures) was calculated as described by Reed and Muench (7). The TCID 50 for the Adenovirus type 3 was calculated to be 4.22 x 10 5 /0.1ml. And the TCID 50 for the HSV type 2 was calculated to be 3.16 x 10 5 /0.1ml. The Σ-Virocult swabs contain 2mls of liquid transport medium and the inoculation to achieve 5 x 10 4 TCID 50 as per the Approved Standard M40-A was calculated to be 230µl of the Adenovirus stock suspension and 300µl for the HSV stock suspension. 40 swabs were inoculated with 230µl of the Adenovirus stock suspension and 40 swabs were inoculated with 300µl of the HSV stock suspension. For each virus, 20 swabs were stored in the fridge at 4ºC and the other 20 were stored at room temperature, 22ºC. The swabs were sampled every day for four days, then at day 7. On each appropriate day, 200µl of the liquid medium from the swabs was inoculated into the appropriate cell culture and incubated at 37ºC. Tubes were removed once CPE was observed. RESULTS Table 9: Adenovirus CPE after being inoculated in swabs TEMP 4ºC 22 ºC Day Tube 1 Tube 2 Tube 3 Tube 4 Tube 1 Tube 2 Tube 3 Tube Table 10: Herpes simplex virus CPE after being inoculated into swabs TEMP 4ºC 22 ºC Day Tube 1 Tube 2 Tube 3 Tube 4 Tube 1 Tube 2 Tube 3 Tube : 25% of cell monolayer showed CPE 2+: 50% of cell monolayer showed CPE 3+: 75% of cell monolayer showed CPE 4+: 100% of cell monolayer showed CPE

15 DISCUSSION The Σ-Virocult swab was able to maintain the viability of the viruses for up to 7 days at two different temperatures. An optimal viral transport system can be defined as that system which preserves the virus in the system, prevents loss of the specimen or test due to microbial contamination, has a long shelf life, is readily available and is inexpensive (4). The results demonstrated that the swabs held at 4ºC provide better viability than those held at room temperature and therefore users should be encouraged to place swabs in the refrigerator if delay in sending the swabs to the laboratory is likely to occur. Typical cytopathic effect was seen in all inoculated tubes which demonstrated the tubes do not inhibit virus viability and that the swab is non-toxic to virus in the specimen. The Σ-Virocult transport swab is compact, enclosed and resistant to breakage or damage during shipping therefore viral specimens can be reliably transported to the laboratory at ambient temperatures and viral infections can be routinely diagnosed by culture (9). In the laboratory, the swab containers fit easily into racks, stand alone and may be centrifuged. These features are very useful when processing swab specimens. In summary, the Σ-Virocult transport swab complied with the Approved Standard M40-A and may be recommended as a reliable virus transport device. References 1. Krafft, A.E., Russell, K.L & Hawksworth, A.W. (2005). Evaluation of PCR Testing of Ethanol-fixed Nasal Swab Specimens as an Augmented Surveillance Strategy for Influenza Virus and Adenovirus Identification. Journal of Clinical Microbiology, 43 (4): Johnson, F.B. (1990). Transport of Viral Specimens. Clinical Microbiology Reviews, 3 (2): Yeary, T.J., & Kapil, S. (2004). A Primer on Diagnostic Virology: Specimen Selection and Serology. Veterinary Learning Systems Inc. Kansas State University, Manhattan. 4. Kimberly, A.S., Bema, K.B &Stoner, E (2010). Comparison of Polyurethane Foam to Nylon Flocked Swabs for Collection of Secretions from the Anterior Nares in Performance of a Rapid Influenza Virus Antigen Test in a Pediatric Emergency Department. Journal Of Clinical Microbiology, 48 (3): Medical Wire and Equipment. (2010). Σ-Virocult 2ml with standard Σ-Swab. Retrieved April 16 from 6. Daley, P., Castriciano, S & Chernesky, M (2006). Comparison of Flocked and Rayon Swabs for Collection of Respiratory Epithelial Cells from Uninfected Volunteers and Symptomatic Patients. Journal of Clinical Microbiology, 44(6): Specter, S & Hodinka, R.L, Young, S.A. (2000). Clinical Virology Manual (3 rd Ed): ASM Press. Washington DC 8. Body, B.A., & Arbique, J.C. (2003). Quality Control of Microbiological Transport Systems; Approved Standard. NCCLS document M40-A (ISBN ). Clinical and Laboratory Standards Institute (CLSI), Pennsylvania, USA. 9. Johnson, F.B., Leavitt, R.W & Richards, D.F. (1984). Evaluation of the Virocult Tube for Isolation of Herpes simplex Virus from Clinical Specimens. Journal of Clinical Microbiology, 20(1): June 2010

16 Study of Σ VCM Culture Transport Devices with Virus This study was performed in accordance with CLSI M40 A. Protocol for virus. Viruses to be included are Adenovirus, Influenza A, HSV 2, RSV, and CMV For Adenovirus, Influenza A, HSV 2, RSV each device being tested is inoculated with 50μl of suspension containing TCID50/50μl of virus. The suspension is prepared and 50μl aliquots are dispensed by micropipette into the wells of a microtitre plate. Each swab to be tested in dipped into a well and twisted around to absorb as much as possible of the medium. The swab is then transferred to its transport tube, the swab snapped off at its breakpoint, and the cap screwed on securely. Device is held for 0h and 96h at 4 O C or Room temp (20 25 O C)*.This was done in triplicate for each time/temperature combination. Device is then processed in normal way, and serial dilutions performed. (10 0, 10 1 and 10 2 ). For CMV each device is inoculated with 50μl of suspension containing TCID50/50μl of virus, and tested as above, with the addition of a 48 hour holding time, and without further dilutions because of the much lower inoculum. 0.2ml aliquots from each dilution are inoculated onto appropriate cell lines, and the culture dishes,flasks or tubes are incubated for up to 10 days and monitored daily for the appearance of CPE (cytopathic effect). The days to positivity are recorded for each device/time/temp/dilution combination. Acceptable performance is when viable virus is still detectable after 96 hours holding time. Tests were initially performed on the standard product (MW910S) using all 4 viruses. The tests were repeated for Influenza A (H1N1) with a batch of the Minitip version of the swab. Results Table 11: Results for regular Sigma-VCM (eg MW910S) VCM Test Lot No. Virus Dilution Time & Temperature Virus Adenovirus Influenza A (H1N1) Herpes Simplex Type 2 Respiratory Syncytial Virus 10J20 N 0h N 96h RT N 96h 4C J20-1 0h h RT h 4C

17 10J20-2 0h h RT h 4C /- 1+: 25% of cell monolayer showed CPE 2+: 50% of cell monolayer showed CPE 3+: 75% of cell monolayer showed CPE 4+: 100% of cell monolayer showed CPE Table 12: Results for VCM with Minitip Swab VCM Test Lot No. Virus Dilution Time & Temperature Influenza A (H1N1) 10B08 N 0h 4+ N 96h RT 4+ N 96h 4C 4+ 10B08-1 0h h RT +/ h 4C +/- 10B08-2 0h h RT +/ h 4C +/- Biobest Laboratories Ltd, 6 Charles Darwin House, The Edinburgh Technopole, Milton Bridge, Nr Penicuik EH26 0PY January 2011

18 Study of Sigma VCM Culture Transport Devices with Chlamydia Trachomatis Objective To confirm the stability of Chlamydia Trachomatis stored in the Sigma-VCM transport system swab over a period of 48 hours at -80 C, +4 C or room temperature. Materials Sigma-VCM swabs as supplied by Medical Wire: Batch 11D13. Chlamydia: Chlamydia Trachomatis (ATCC-VR-1477) Cell lines: Hep-2 Culture of chlamydia stocks Chlamydia stocks were cultured using standard procedures and cultured in the Cell Line named below. Chlamydia Chlamydia Trachomatis Cell Line Hep-2 Study Protocol 1. On the day prior to inoculation the required numbers of cell culture plates were prepared in order to perform the titration of the time zero viral transport media. Plates were 70% 90% confluent at the time of infection. 2. The growth media was gently removed from the cell culture plates using a multichanel and 100 µl of 10% EMEM containing filtered DEAE Dextran (45µg/ml) was added to each well of the cell culture plate and incubated at 37 C to pre-treat the cells before infection. The dextran media was then removed and 50µl of Chlamydia was added to the required number of wells in a sterile tissue culture plate. The now inoculated cell culture plate was then centrifuged for 60 minutes (750g, 21 C). Whilst the plates were in the centrifuge, 20 µl of cycloheximide was added to1 litre of 10% EMEM and 100 µl of this media was added to each well of the cell culture plates before they are incubated for 5-7 days. 3 swabs were used per incubation condition. 3. Swabs were loaded with Chlamydia by inserting the tip into the well. The swab was moved around the well until the full volume had been absorbed. The swab was placed into transport media, mixed and was then incubated at the desired temperature of -80 C, + 4 C or room temperature (20 C-25 C) for 48 hours. A time zero swab swab was also inoculated for each strain of Chlamydia, 250µl of VTM was

19 removed from this swab immediately after inoculation for titration. The remaining media from the time zero sample was then stored at -80 C hours after inoculation of the swabs the required numbers of cell culture plates were prepared in order to perform the titration of the viral transport media. Plates were 70% 90% confluent at the time of infection hours post inoculation the transport media from each tube will be titrated in a cell culture appropriate to the Chlamydia being isolated. A positive and negative control will also be titrated on the same plate. Any unused transport media will be stored at -80 C for later testing if required. In the event of cell culture plates not being suitable for infection, swabs will be transferred to -80 C for subsequent testing. 6. Plates will be fixed, stained and read for specific fluorescence under a U.V microscope 5-7 days after infection. The results are recorded using the following criteria: + = positive for specific fluorescence 0 = negative for specific fluorescence 7. Results are reported as Chlamydia recovered or no Chlamydia recovered. Table 13: Results Temperature 0 hrs 48 hrs Room Fridge (+4C) Temperature Freezer (-80C) Positive Control Swab Swab Swab = Chlamydia recovered Sigma-VCM swabs showed acceptable recovery of Chlamydia when incubated at room temperature, + 4 C and -80 C when used from a neat starting dilution. Recovery was also recorded using swabs that were stored at room temperature and -80 C that were infected with Chlamydia at Biobest Laboratories Ltd, 6 Charles Darwin House, The Edinburgh Technopole, Milton Bridge, Nr Penicuik EH26 0PY Feb 2012

20 Σ VCM Validation with Mycoplasma Mycoplasma The following was a study done in 2009 at a French Mycoplasma laboratory using mycoplasma culture techniques. TA is room temperature. Recoveries seemed to be acceptable to the investigators. A7 Plates : Strain T0 T0+24H T0+48H T0+72H M.hominis 10 3 TA : <10 3 TA : <10 3 TA : < C : C : C : 10 3 M.hominis PG21 U.urealyticum S8 U.urealyticum TA : 10 4 TA : 10 4 TA : C : C : C : TA : 10 5 TA : 10 5 TA : C : C : C : TA : 10 4 TA : 10 4 TA : C : C : C : 10 3 colonies number = X X 1 = < X < 5 = < X < 15 =10 5 X > 15 = 10 6 T0 = Time Zero AT= Room Temperature Where <10 3 is shown, it means recovery was as for 10 2

21 Validation Studies for Virocult virus transport swabs In the present study Virocult swabs (Medical Wire) were evaluated in accordance with CLSI M40-A. The panel of viruses was increased to include Adenovirus 1, Influenza A, Parainfluenza 3, Rhinovirus, and Herpes Simplex Types 1 and 2. Survival was studied after holding times of up to 7 days, and at holding temperatures of 4 O C (refrigeration) and 22 O C (representing ambient temperatures). Virus strains were all obtained from ATCC, with the exception of Influenza A for which a clinical isolate strain was used. Titres were established so that an inoculum close to 5 x 10 4 TCID 50 could be used. Appropriate cell lines were used for each virus, for initial growth, and for the detection of live virus following simulated transport. In one case (Adenovirus 1) live virus following transport was detected using fluorescent antigen. Virocult was shown to successfully recover all 6 viruses after holding for up to 7 days, and whether held at 4 O C or ambient temperature. Method Freeze dried virus reconstituted with H 2 O Confirm identity of virus and titrate Dilute virus in Eagles MEM (Biowhittaker) (0.3ml inoculum +2.7ml Eagles MEM) Immerse Virocult swabs in each dilution (4 swabs per dilution) for 10 seconds Insert each swab into its own Virocult tube with medium Squeeze tubes Incubate at 2-8 O C or at Room Temperature for holding period (see table) After holding period add 4ml liquid medium (Eagles MEM) to tube, vortex for 10 seconds. 100μl of medium inoculated onto cell culture (X2) Cell culture monitored daily for appearance of CPE The results shown are for an inoculum of 3 x 10 4 TCID 50

22 Table 15: Results Virus Adenovirus 1 ATCC VR-1 HSV Type 2 ATCC VR-734 HSV Type 1 ATCC VR-539 Parainfluenza 3 ATCC VR-93 Rhinovirus ATCC VR-1118 Time (days) Temperature Inoculum (TCID50) Cell line CPE detected or detection by FA 4 4C 3 x 10 4 Hep-2 YES (FA) n/a Days to CPE 4 RT 3 x 10 4 Hep-2 YES (FA) n/a 7 4C 3 x 10 4 Hep-2 YES 2 7 RT 3 x 10 4 Hep-2 YES 2 3 4C 3 x 10 4 MRC-5 YES 1 3 RT 3 x 10 4 MRC-5 YES 1 7 4C 3 x 10 4 MRC-5 YES 2 7 RT 3 x 10 4 MRC-5 YES 3 3 4C 3 x 10 4 MRC-5 YES 2 3 RT 3 x 10 4 MRC-5 YES 2 7 4C 3 x 10 4 MRC-5 YES 1 7 RT 3 x 10 4 MRC-5 YES 2 3 4C 3 x 10 4 PLC YES 4 3 RT 3 x 10 4 PLC YES 7 7 4C 3 x 10 4 PLC YES 6 7 RT 3 x 10 4 PLC YES C 3 x 10 4 MRC-5 YES 2 3 RT 3 x 10 4 MRC-5 YES 3 7 4C 3 x 10 4 MRC-5 YES 3 7 RT 3 x 10 4 MRC-5 YES 5 Influenza A 3 4C 3 x 10 4 PLC YES 4 3 RT 3 x 10 4 PLC YES 5 8 4C 3 x 10 4 PLC YES 5 8 RT 3 x 10 4 PLC YES 8 Virocult was shown to successfully recover all 6 viruses after holding for at least 7 days, and whether held at 4 O C or ambient temperature. M40-A requires the transport device to maintain live virus at detectable levels starting with a titre of 5 x 10 4 for up to 96 hours. In the study Virocult was shown to meet and exceed the requirements of M40-A, by maintaining a selection of viruses in a viable condition when held at 4OC, or at ambient/room temperatures for 96 hours, and beyond until at least 7 or 8 days. The device should be suitable for respiratory or skin lesion specimens that require to be transported from remote sites, even if they cannot be frozen or transported with dry ice.