Ʃ-Virocult. Virus specimen transport TECHNICAL FILE. For Molecular and Culture Techniques. Part of the new generation of preanalyticals

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1 Ʃ-Virocut Virus specimen transport For Moecuar and Cuture Techniques TECHNICAL FILE Part of the new generation of preanayticas

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3 Virus Specimen Transport for Moecuar and Cuture Techniques Virocut Medium Respiratory viruses STD s Skin esions Enteric viruses Compatibe with moecuar techniques Compatibe with RT-PCR Compatibe with cuture F LOT C31 09 F LOT wire mwe 0088 Ref. MWE9505 Open-ceed foam bud Optimum absorption and reease Optimum performance with moecuar test systems medica M Date Ward Hosp N0. Time Spec Name D. O. B. M Date MWE9505 Ward Ref. Time 0088 Hosp N0. wire mwe medica Spec Name D. O. B. F LOT M Date MWE9505 Ward Ref. Time 0088 Hosp N0. wire mwe medica Spec Name D. O. B. C31 C31 -Swab - Virocut features a unique open ce structure. Part of the new generation of preanayticas mwe.co.uk www. mwe.co.uk www. - Virocut

4 - Virocut Virus specimen transport For Moecuar and Cuture Techniques -Virocut combines Medica Wire s open ce bud Sigma-Swab with Virocut medium, the eading transport medium for virus specimens. -Virocut just for viruses Virocut has ong been recognised as one of the best transport devices for viruses, demonstrating surviva of many types of virus at ambient temperatures, incuding Herpes Simpex Virus, Varicea-Zoster Virus, Infuenza Type A (incuding Nove H1N1, H5N1, and H3N2), Infuenza Type B, respiratory syncytia virus, mumps virus, adenovirus, rhinovirus, and various enterovirus. Virocut medium stabiises virus partices aowing ong surviva, and contains antimicrobias to prevent the growth of any bacteria and fungi present in the specimen. -Virocut Sigma-Virocut is suppied with SigmaSwab, for optimum uptake and reease of target microorganisms, and compete fowthrough of reagents, C31 09 C31 09 F LOT Etringham, G.J.A., A. Rudsdae & C. Hi, Detection of Infuenza A (Pandemic H1N1v), RSV, Rhinovirus and other respiratory viruses in different popuations using Sigma-Virocut Poster P10,European Society for Cinica Viroogy, Winter Meeting, London, January Etringham, G.J.A., A. Rudsdae & C. Hi, Prevaence of respiratory viruses in different popuations using Sigma-Virocut IBMS, Biomedica Science Congress, Birmingham September, Sharma, E. & J. Lindeman, 2010, Laboratory Evauation of Σ -Virocut Transport Swabs with Herpes simpex virus type 2 and Adenovirus type 3. Σ -Virocut Technica Fie 5 Rudsdae. A. & D. Shedden, Investigation of the suitabiity of the Virocut swab transport device for infuenza A specimens which are to be anaysed by ce-cuture or moecuar techniques. Poster M42. 25th Cinica Viroogy Symposium Daytona Beach. 6 Rudsdae. A Evauation of a viroogy specimen transport device with six viruses using CLSI Standard M40-A. Poster C-053 ASM 109th Genera Meeting, Phiadephia. 7 Lina. B., et a Surveiance of community-acquired vira infections due to respiratory viruses in Rhone-Aps (France) during winter 1994 to J Cin. Microbio. 34: CLSI, 2003 Quaity Contro of Microbioogica Transport Systems ; Approved Standard M40-A. CLSI (formery NCCLS) document M40-A [ISBN ]. CLSI, 940 West Vaey Road, Suite 1400, Wayne, Pennsyvania USA, Johnson F. B., 1990,Transport of Vira Specimens,. Cinica Microbioogy Reviews, 3: Code Via Swab configuration Fi Pack MW951S Sma 1 Standard Sigma Swab with breakpoint 1.m 125 MW951SENT Sma 1 Mini tip Sigma Swab with breakpoint 1.m 125 MW951S2 Sma 2 Standard Sigma Swab with breakpoint 1.m 125 MW951T Sma Tube of medium ony 1.m MW9501S Large 1 Standard Sigma Swab with breakpoint 50 2.m 125 MW950SENT Large 1 Mini tip Sigma Swab with breakpoint 2.m 125 MW950S2 Large 2 Standard Sigma Swab with breakpoint 2.m 125 MW950SE2 Large 1 Standard, 1 Mini tip Sigma Swab with breakpoint 2.m 125 MW950T Large Tube of medium ony 2.m 125 -Virocut is CE-marked Sigma-Virocut conforms to the requirements of the European Medica Devices Directive and In Vitro Medica Devices Directives -Virocut vaidated to M40-A Virocut & Sigma-Virocut are vaidated according to CLSI s M40-A standard for vira cuture transport devices, which requires surviva of reference strains for at east 96 hours at ambient or refrigerated temperatures. 09 F C31 09 LOT Date MWE9505 Ward Ref. Time 0088 Hosp N0. wire mwe medica Spec D. O. B. M Date LOT F Ward MWE9505 M Ref. Time 0088 Hosp N0. wire mwe medica Spec Name D. O. B. M MWE9505 Ward Ref. Time 0088 Hosp N0. wire mwe medica Spec Corsham, Witshire, SN13 9RT, U.K. Teephone: Fax: E-mai: info@mwe.co.uk Date LOT F 09 C31 C31 Optimum recovery of target organisms Optimum compatibiity with moecuar test systems Antibiotics inhibit bacteria and fungi Recovers wide range of respiratory, genita and enteric viruses Transport specimens at ambient temperatures Choice of fi voume Order Information Name Open-ceed foam bud Optimum absorption and reease Optimum performance with moecuar test systems Standard shaft or ENT/urethra Virocut medium 2 Name M Date Ward MWE9505 wire medica Spec Name D. O. B. Sigma-Swab Vaette, M., M. Bouscambert-Duchamp, R. Fanget, S. Lambert & B. Lina, 2010, Comparison Of Virocut Swab, Σ -Swab And Σ -Virocut For Infuenza A Viabiity For Ce Cuture And Moecuar Detection, Poster S84, Cinica Viroogy Symposium 2010, Daytona Beach Ref. Time 0088 is avaiabe in a range of formats mwe Hosp N0. -Virocut Standard Sigma-Swab is suitabe for genera swab appications such as skin esions, nose and throat. Mini Tip SigmaSwab is suitabe for nasopharyngea and urethra samping. It is stored at room temperature, with a shef ife of 1 year. Specimens, once coected, can be transported under ambient or refrigerator temperature conditions. 1 D. O. B. F LOT M Date MWE9505 Ward Ref. Time 0088 Hosp N0. wire mwe medica Spec Name D. O. B. best for coection References:

5 Σ- VIROCULT MW950S, MW950S2, MW950SENT, MW950SE2, MW950T, MW951S, MW951SENT, MW951T PRODUCT INFORMATION Σ-VIROCULT MW950S, MW950S2, MW950SENT, MW950SE2, MW950T, MW951S, MW951SENT, MW951T Vira Specimen Transport Device Intended Use Medica Wire & Equipment Σ-Virocut Virus Coection and Transport System is intended to preserve the viabiity and infectivity of vira specimens for vira cuture after their coection and during transport from the coection site to the testing aboratory. Σ-Virocut specimens are processed using standard cinica aboratory operating procedures for vira and ce cuture. Principe and Summary One of the routine procedures in the diagnosis of infections caused by viruses invoves the coection and transportation of a cinica swab specimen from the patient to the aboratory. Specimens containing ive viruses may be submitted to a aboratory for diagnosis or confirmation of the patient s iness. Σ-Virocut tubes contain a iquid medium to keep the specimen moist, and to maintain any viruses in a viabe condition unti they can be investigated at the aboratory by vira cuture. The iquid medium consists of a baanced sat soution for maintaining osmotic pressure within physioogica imits and phosphate buffers to stabiize the ph of the medium. For specific recommendations about the coection of specimens for viruses and primary isoation techniques, consut the foowing ASM pubications: Cumitech 15A 1, Cinica Microbioogy Procedures Handbook 2, Manua of Cinica Microbioogy 3, Cinica Viroogy Manua 4, and Johnson F. B. 5 Precautions FOR VIROLOGY SPECIMENS ONLY FOR IN VITRO DIAGNOSTIC USE DO NOT FREEZE Observe aseptic techniques and estabished precautions against microbioogica hazards throughout a procedures. Prior to discarding, swabs and other contaminated materias must be steriized by autocaving. Once a swab sampe is coected it shoud be paced immediatey into the transport tube where it comes into contact with transport medium. Swab specimens for virus isoation and/or detection shoud be submitted to the aboratory as quicky as possibe after coection. Do not use if package sea is broken. Reagent Formua Virocut medium is a baanced sat soution, buffered with disodium hydrogen orthophosphate, and aso contains actabumin hydroysate as a stabiiser, and antibiotics to inhibit the growth of any bacteria contaminants in the specimen. Active ingredients: Choramphenico Amphotericin g per iter 0.003g per iter Storage and Stabity Σ-Virocut shoud be stored in a dry pace at temperatures between +5ºC to 25ºC. Expiration Date 12 months from date of manufacture. Expiration date stated on tube abe, pee pouch, and box abe. Materias provided Swab for coection of specimen.(mw950t & MW951T are provided without swabs) Transport tube with Virocut medium Materias required but not provided Eage s Minimum Essentia Medium (buffered) or Hanks Baanced Sat Soution (buffered). Pipette to withdraw 0.2m fuid from Virocut tube. Ce cuture faciities and ce ines appropriate for target viruses. Directions For Use CODE DESCRIPTION INTENDED USE MW950S Σ-Virocut Singe standard Sigma Swab, pastic shaft green coour coded cap, Mouth, nose, throat, skin

6 Σ- VIROCULT MW950S, MW950S2, MW950SENT, MW950SE2, MW950T, MW951S, MW951SENT, MW951T PRODUCT INFORMATION 2.0m medium MW950SENT MW950S2 MW950SE2 MW951S MW951SENT MW950T MW951T Σ-Virocut Singe mini-tip Sigma Swab, pastic shaft green coour coded cap, 2.0m medium Σ-Virocut Two standard Sigma Swabs, pastic shaft green coour coded cap, 2.0m medium Σ-Virocut One standard Sigma Swab, and one minitip Sigma Swab, pastic shaft green coour coded cap, 2.0m medium Σ-Virocut Singe standard Sigma Swab, pastic shaft green coour coded cap, 1.0m medium Σ-Virocut Singe mini-tip Sigma Swab, pastic shaft green coour coded cap, 1.0m medium Via ony, 2.0m Virocut medium Via ony, 1.0m Virocut medium Nasopharyngea, pediatric, urogenita Mouth, nose, throat, skin Mutipe sites Mouth, nose, throat, skin Nasopharyngea, pediatric, urogenita Instructions for taking specimen Pee back pouch, remove via and pace on a fat surface. Loosen cap by partiay unscrewing. Withdraw swab and use to take specimen. Remove cap from via, insert swab into via and snap off the non-bud end so that the remaining shaft fits within the via. Repace cap, and tighten unti secure. Transport to aboratory immediatey. Processing Specimens (Cuture method) Add approximatey 2.0m of Eage s Minimum Essentia Medium or Hanks soution to the transport tube with the swab in situ. Mix thoroughy using a vortex mixer. Using a pipette withdraw the iquid and add approximatey 0.2m of the suspension to each tissue cuture we, dish or tube. In compiance with CLSI M40-A, inocuation of specimens onto ce cutures shoud be performed within 96 hours of specimen coection. Virus is detected by the appearance of cytopathic effect in the ce cuture. Processing Specimens (Moecuar methods) Refer to the test system manufacturer s instructions. Any use with non-cuture methods must be vaidated by the user. Quaity contro With reference to CLSI M40-A it is recommended that Herpes Simpex Type 2 ATCC VR-734 be used as a contro strain. The swab is inocuated from a suspension containing 5 x 10 4 TCID per m, and paced into the transport tube. The tube is hed at the desired transport temperature (4 O C or room temperature) for up to 96 hours. The transport tube is processed as described above ( Processing Specimens ) and 0.2m of suspension is inocuated onto a suitabe tissue cuture monoayer. Any recovery of virus is acceptabe performance. Limitations 1.This device is NOT SUITABLE FOR THE TRANSPORT OF BACTERIA OR FUNGI because antibiotics are used in the medium. 2.Traces of antivira reagents at site of specimen prior to samping may affect recovery of virus 3.Σ-Virocut has not been vaidated for use with moecuar techniques such as PCR, so any such use must be vaidated by the user. Performance Characteristics* Recovery Studies Virocut virus transport swabs were tested in accordance with CLSI (NCCLS) Quaity Contro of Microbioogica Transport Systems 6 ; Approved Standard M40-A. Known concentrations of Herpes simpex virus type 2 (HSV 2) and Adenovirus type 3 were inocuated, in tripicate, into the Virocut swabs of different ot numbers and expiration dates and hed at 22 C and 4 C. The swabs were samped every day for four days (24 to 96 hours as per the standard). The resuts showed the Virocut swabs maintain the viabiity of virus as set out in the standard. Hep-2 and A549 ce ines used routiney in the aboratory were utiised as the ce ines to support the growth of Adenovirus and HSV. Fresh working ots of ow passage number (<15) were spit into tube cutures to have a confuent monoayer after 24 hours. Adenovirus type 3, ATCC VR-3 and HSV 2, ATCC VR-734 were obtained from the American Type Cuture Coection and used to assess the performance of the swabs.

7 Σ- VIROCULT MW950S, MW950S2, MW950SENT, MW950SE2, MW950T, MW951S, MW951SENT, MW951T PRODUCT INFORMATION The study was performed in June METHODS: The viruses were inocuated into a fask containing a confuent monoayer of Hep-2 for Adenovirus 3 and A549 for HSV 2. Once the most of the monoayer showed CPE, the virus was harvested and stored for use as the stock suspension of virus. Stock suspensions of Adenovirus 3 and HSV type 2 were seriay diuted using 10-fod diutions and, in tripicate, inocuated into fresh Hep-2 and A549 tube cutures. Once CPE was obtained, the inocuum for 50,000TCID/m was cacuated as originay described by Reed and Muench 7. For both viruses this was cacuated to be 0.3m of stock suspension. The swabs were inocuated, in tripicate, with 0.3m of stock suspension of virus, mixed thoroughy with the medium in the device and hed at either 4 C or 22 C. On each appropriate day, 0.2m was withdrawn, inocuated into the appropriate tube cuture and observed for CPE. The tubes were removed once ++++ CPE (100%) was observed. To compare using a much ower virus titre, the procedure was aso carried out using an inocuum of stock virus into the swabs of 10000TCID/m. Concusion: The studies showed that Virocut aowed the recovery of Adenovirus Type 3 and HSV 2 viruses for at east 96 hours after inocuation at both 4 O C and 22 O C. Adenovirus Type 3 and HSV 2 had vira titres of 50,000 and 10,000 TCID/m respectivey. *Performance testing with Medica Wire Virocut was conducted using pure aboratory virus strains spiked onto a swab and not using human specimens. Recovery study performed by: Jenni Lindemann, Viroogy Laboratory, Heath Waikato Laboratories. Waikato Hospita, Hamiton New Zeaand June 2006 Suppementary studies The above studies have been repeated for Σ-Virocut and confirmed that Σ-Virocut aso aowed the recovery of Adenovirus Type 3 and HSV 2 viruses for at east 96 hours after inocuation at both 4 O C and 22 O C. June 2010 REFERENCES 1. Geaves C. A., R. L. Hodinka, S. L. G. Johnston and E. M. Swierkosz, Cumitech 15A. Laboratory Diagnosis of Vira Infections, p. 7. American Society for Microbioogy, Washington D.C., Mier, M. J., and A.L. Warford. Preparation of specimens for inocuation of ce cutures, p In H.D. Isenberg (ed.), Cinica Microbioogy Procedures Handbook. American Society for Microbioogy, Washington, D.C., Chapin, K.C., & F.W. Westenfed, 2003, Reagents, Stains, Media, and Ce Lines: Viroogy, p.1250 in Murray P.R., E.J. Baron, J.H. Jorgensen, M.A. Pfaer, & R.H. Yoken, 2003, Manua of Cinica Microbioogy, 8 th Edition, ASM Press, Washington D.C. 4. Specter, S., R.L. Hodinka, and S.A. Young, 2000, Cinica Viroogy Manua, 3 rd Edition, ASM Press, Washington D.C. 5. Johnson F. B., Transport of Vira Specimens, p Cinica Microbioogy Reviews, Vo. 3, No. 2, Apri CLSI. Quaity Contro of Microbioogica Transport Systems ; Approved Standard M40-A. CLSI (formery NCCLS) document M40-A [ISBN ]. CLSI, 940 West Vaey Road, Suite 1400, Wayne, Pennsyvania USA, Reed, L. J., and H. Muench A simpe method of estimating fifty percent endpoints. Am. J. Hyg. 27: Medica Wire & Equipment Co (Bath), Corsham, Witshire, Engand, SN13 9RT Te: Fax: Emai: info@mwe.co.uk Website: 26/08/10

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9 Infuenza A Virus COMPARISON OF VIROCULT SWAB, Σ-SWAB AND Σ-VIROCULT FOR INFLUENZA A VIABILITY FOR CELL CULTURE AND MOLECULAR DETECTION Martine Vaette 1, Maude Bouscambert-Duchamp 1, Rémi Fanget 1, Simone Lambert 1 and Bruno Lina 1 1 Hospices Civis de Lyon, Nationa Infuenza Centre (South of France), Laboratory of Viroogy - Bât A3, 59 Bouevard Pine, F Bron Cedex, France POSTER S84, Cinica Viroogy Symposium 2010, Daytona Beach

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13 Laboratory Evauation of Σ-Virocut Transport Swabs Edwin Sharma and Jenni Lindeman Viroogy Laboratory, Heath Waikato Laboratories, Waikato Hospita, Hamiton, New Zeaand ABSTRACT Background: Coection and storage of vira specimen is the most important factor in obtaining a reiabe and accurate diagnostic resut. Swabs are often used to coect specimens for detecting and identifying vira infections Scope of study: In this study Σ-Virocut transport swabs (Medica Wire and Equipment) were evauated using the CLSI Quaity Contro of Microbioogica Transport Systems Approved Standard M40-A. The standard presents a specified cuture protoco to assess a vira cuture transport device manufactured to faciitate virus preservation. The study was carried out using two different temperatures. Method: Herpes simpex virus type 2 and Adenovirus type 3 were inocuated into Σ- Virocut swabs at a concentration of 5 x 10 4 TCID 50 and hed at 22ºC and 4ºC. The swabs were samped every day for four days, then at day seven. Resuts: The resuts showed that the swabs maintain the viabiity of virus as set out in the standard and that surviva is better at 4ºC. Concusion: The Σ-Virocut transport swabs may be recommended as a reiabe virus transport device. INTRODUCTION Vira partices vary widey in composition, structure, morphoogy size and stabiity (2). Vira isoation has been widey accepted as the god standard for aboratory confirmation of vira infection; however, it requires specimen storage and transport in a vira medium maintained at ow temperatures to optimay preserve infectious vira partices (1). Cinica specimens for virus isoation shoud ideay be inocuated into ce cuture with as itte deay as possibe (2). However, many common viruses are abe to withstand both storage at room temperature and transport for extended periods of time when paced in a suitabe transport medium (2). A suitabe transport medium for swabs must maintain viabiity of viruses, prevent overgrowth of bacteria and fungi, and prevent drying of the specimen (6). Swabs are generay used to coect ces and fuid from nasa passages, the throat, the rectum, vesices, eyes, and cervica, genita and skin esions. The Σ-Virocut is designed for specimen coection, transport and maintenance of virus viabiity. The device consists of soft poyurethane foam budded swab and a transport tube of iquid virus transport medium which contains antimicrobias to inhibit growth of any bacteria or fungi present in the specimen (5,10). This study evauates the Σ-Virocut transport swab to determine whether it is abe to maintain the viabiity of viruses at different temperatures over a certain period of time. MATERIALS AND METHODS Materias Viruses: Stock concentrations of wid-type (cinica isoates) Adenovirus type 3 and Herpes simpex virus type 2 were used in the swab evauation study. Ce ines: HEP2 (human arynx carcinoma) and A549 (human ung carcinoma) ce ines routiney utiised in the aboratory were utiised as the ce ines to support the growth of Adenovirus (HEP2) and Herpes simpex type 2 (A549). Fresh working ots of ow passage number (<15) were spit into tube ce cutures to have a confuent monoayer after 24 hours. Swabs: Σ-Virocut, Lot No. 09J30, expiry date Sep 2010 were used in the study. Methods The wid-type viruses were inocuated into a fask containing a confuent monoayer of HEP2 for Adenovirus type 3 and A549 for Herpes simpex type 2. Once most of the monoayer showed typica 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 simpex

14 type 2 were seriay diuted in ce cuture maintenance medium using 10-fod diutions from 10-1 to For each diution, 5 tubes of fresh HEP2 and A549 ce cutures were inocuated with 100µ of diuted vira suspension. The cutures were incubated at 37ºC and examined daiy for CPE over a period of 10 days. TCID 50 (tissue cuture infective dose which is the amount of virus which gives a CPE in 50% of inocuated cutures) was cacuated as described by Reed and Muench (7). The TCID 50 for the Adenovirus type 3 was cacuated to be 4.22 x 10 5 /0.1m. And the TCID 50 for the HSV type 2 was cacuated to be 3.16 x 10 5 /0.1m. The Σ-Virocut swabs contain 2ms of iquid transport medium and the inocuation to achieve 5 x 10 4 TCID 50 as per the Approved Standard M40-A was cacuated to be 230µ of the Adenovirus stock suspension and 300µ for the HSV stock suspension. 40 swabs were inocuated with 230µ of the Adenovirus stock suspension and 40 swabs were inocuated with 300µ 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 samped every day for four days, then at day 7. On each appropriate day, 200µ of the iquid medium from the swabs was inocuated into the appropriate ce cuture and incubated at 37ºC. Tubes were removed once CPE was observed. RESULTS Adenovirus CPE after being inocuated in swabs TEMP 4ºC 22 ºC Day Tube 1 Tube 2 Tube 3 Tube 4 Tube 1 Tube 2 Tube 3 Tube Herpes simpex virus CPE after being inocuated into swabs TEMP 4ºC 22 ºC Day Tube 1 Tube 2 Tube 3 Tube 4 Tube 1 Tube 2 Tube 3 Tube : 25% of ce monoayer showed CPE 2+: 50% of ce monoayer showed CPE 3+: 75% of ce monoayer showed CPE 4+: 100% of ce monoayer showed CPE

15 DISCUSSION The Σ-Virocut swab was abe to maintain the viabiity of the viruses for up to 7 days at two different temperatures. An optima vira transport system can be defined as that system which preserves the virus in the system, prevents oss of the specimen or test due to microbia contamination, has a ong shef ife, is readiy avaiabe and is inexpensive (4). The resuts demonstrated that the swabs hed at 4ºC provide better viabiity than those hed at room temperature and therefore users shoud be encouraged to pace swabs in the refrigerator if deay in sending the swabs to the aboratory is ikey to occur. Typica cytopathic effect was seen in a inocuated tubes which demonstrated the tubes do not inhibit virus viabiity and that the swab is non-toxic to virus in the specimen. The Σ-Virocut transport swab is compact, encosed and resistant to breakage or damage during shipping therefore vira specimens can be reiaby transported to the aboratory at ambient temperatures and vira infections can be routiney diagnosed by cuture (9). In the aboratory, the swab containers fit easiy into racks, stand aone and may be centrifuged. These features are very usefu when processing swab specimens. In summary, the Σ-Virocut transport swab compied with the Approved Standard M40-A and may be recommended as a reiabe virus transport device. References 1. Krafft, A.E., Russe, K.L & Hawksworth, A.W. (2005). Evauation of PCR Testing of Ethano-fixed Nasa Swab Specimens as an Augmented Surveiance Strategy for Infuenza Virus and Adenovirus Identification. Journa of Cinica Microbioogy, 43 (4): Johnson, F.B. (1990). Transport of Vira Specimens. Cinica Microbioogy Reviews, 3 (2): Yeary, T.J., & Kapi, S. (2004). A Primer on Diagnostic Viroogy: Specimen Seection and Seroogy. Veterinary Learning Systems Inc. Kansas State University, Manhattan. 4. Kimbery, A.S., Bema, K.B &Stoner, E (2010). Comparison of Poyurethane Foam to Nyon Focked Swabs for Coection of Secretions from the Anterior Nares in Performance of a Rapid Infuenza Virus Antigen Test in a Pediatric Emergency Department. Journa Of Cinica Microbioogy, 48 (3): Medica Wire and Equipment. (2010). Σ-Virocut 2m with standard Σ-Swab. Retrieved Apri 16 from 6. Daey, P., Castriciano, S & Chernesky, M (2006). Comparison of Focked and Rayon Swabs for Coection of Respiratory Epitheia Ces from Uninfected Vounteers and Symptomatic Patients. Journa of Cinica Microbioogy, 44(6): Specter, S & Hodinka, R.L, Young, S.A. (2000). Cinica Viroogy Manua (3 rd Ed): ASM Press. Washington DC 8. Body, B.A., & Arbique, J.C. (2003). Quaity Contro of Microbioogica Transport Systems; Approved Standard. NCCLS document M40-A (ISBN ). Cinica and Laboratory Standards Institute (CLSI), Pennsyvania, USA. 9. Johnson, F.B., Leavitt, R.W & Richards, D.F. (1984). Evauation of the Virocut Tube for Isoation of Herpes simpex Virus from Cinica Specimens. Journa of Cinica Microbioogy, 20(1): June 2010

16 POSTER C-053 ASM 109 TH GENERAL MEETING, PHILADELPHIA, 2009 EVALUATION OF A VIROLOGY SPECIMEN TRANSPORT DEVICE WITH SIX VIRUSES USING CLSI STANDARD M40-A Andrew Rudsdae Heath Protection Agency, Newcaste Upon Tyne, United Kingdom Abstract Methods Resuts Athough PCR is becoming estabished as the method of choice for the diagnosis of vira infections, to recover ive virus for fu characterisation and future reference sti requires the traditiona methods of ce cuture. With ce cuture faciities being increasingy restricted to a few reference aboratories, it is even more essentia that transport devices for vira specimens are reiabe for transportation over onger distances. CLSI s Quaity Contro of Microbioogica Transport Systems ; Approved Standard M40-A 1 provides a method for determining whether particuar devices are suitabe for the transport of vira specimens over extended distances. In the present study Virocut swabs (Medica Wire) were evauated in accordance with M40-A. The pane of viruses was increased to incude Adenovirus 1, Infuenza A, Parainfuenza 3, Rhinovirus, and Herpes Simpex Types 1 and 2. Surviva was studied after hoding times of up to 7 days, and at hoding temperatures of 4 O C (refrigeration) and 22 O C (representing ambient temperatures). Virus strains were a obtained from ATCC,with the exception of infuenza A for which a cinica isoate strain was used. Titres were estabished so that an inocuum cose to 5 x 10 4 TCID50 coud be used. Appropriate ce ines were used for each virus, for initia growth, and for the detection of ive virus foowing simuated transport. In one case (Adenovirus 1) ive virus foowing transport was detected using fuorescent antigen. Virocut was shown to successfuy recover a 6 viruses after hoding for up to 7 days, and whether hed at 4 O C or ambient temperature. Introduction The current emergency with a pandemic strain of swine fu 2,3 has highighted the continuing need for virus cuture faciities to be avaiabe for the typing, fu characterisation and monitoring of previousy unknown strains. Whie PCR and other rapid methods provide convenient and highy effective toos for the diagnosis and monitoring of previousy known strains, they are as yet unabe to precisey identify new strains which may share those highy conserved target DNA or RNA sequences with their ess viruent antecedents, have nove features which aow them to be agressivey infectious. Athough moecuar techniques such as PCR have become estabished as the methods of choice for the diagnosis of vira infections, to recover ive virus for fu characterisation and future reference sti requires the traditiona methods of ce cuture 4. With ce cuture faciities being increasingy restricted to a few reference aboratories, it is even more essentia that transport devices for vira specimens are reiabe for transportation over onger distances. CLSI s Quaity Contro of Microbioogica Transport Systems ; Approved Standard M40-A provides a method for determining whether particuar devices are suitabe for the transport of vira specimens over extended distances. In the present study Virocut swabs (Medica Wire) were evauated in accordance with M40-A. The pane of viruses was increased to incude Adenovirus 1, Infuenza A, Parainfuenza 3, Rhinovirus, and Herpes Simpex Types 1 and 2, to refect the typica range of organisms expected to be present in submitted specimens. Surviva was studied after hoding times of up to 7 days, and at hoding temperatures of 4 O C (refrigeration) and 22 O C (representing ambient temperatures).m40-a ony requires surviva for up to 96 hours, but the test period was extended to refect situations where specimens might require to be forwarded to a reference faciity after initia rapid testing. Virus strains were a obtained from ATCC,with the exception of infuenza A for which a cinica isoate strain was used. Titres were estabished so that an inocuum cose to 5 x 10 4 TCID 50 coud be used. Appropriate ce ines were used for each virus, for initia growth, and for the detection of ive virus foowing simuated transport. In one case (Adenovirus 1) ive virus foowing transport was detected using fuorescent antigen. Freeze dried virus reconstituted with H 2 O Confirm identity of virus and titrate (see pane beow) Diute virus in Eages MEM (Biowhittaker) (0.3m inocuum +2.7m Eages MEM) Immerse Virocut swabs in each diution (4 swabs per diution) for 10 seconds Insert each swab into its own Virocut tube with medium Squeeze tubes Incubate at 2-8 O C or at Room Temperature for hoding period (see tabe) After hoding period add 4m iquid medium (Eages MEM) to tube, vortex for 10 seconds. 100μ of medium inocuated onto ce cuture (X2) Ce cuture monitored daiy for appearance of CPE The resuts shown are for an inocuum of 3 x 10 4 TCID 50 Identification and Titration of HSV-2 Freeze dried virus reconstituted with H 2 O Confirmation of identity of stock virus Reconstituted virus 200μ into 3 tubes of MRC-5 ces 3 days od in EMEM + 1% foeta bovine serum) Reconstituted virus 100μ into 1 tube of MRC-5 ces 3 days od in EMEM + 1% foeta bovine serum) Incubated stationary at 37 O C for 1 day. Examined by ight microscopy: tttt Cytopathic Effect in a 4 tubes Stained with Chemicon IF stain HSV 2 or IF stain HSV 1 Examined by IF HSV-2 tttt HSV-1 - Seria diutions (100μ inocuum to 1m with diuent) in EMEM (Biowhittaker) + 1% foeta bovine serum (x2) Incubate stationary at 37C Observe for CPE VIRUS Hoding Time (days) Hoding Temp Inocuum (TCID 50 ) Ce ine CPE detected or virus detected by FA Days to CPE Adenovirus O C 3 x 10 4 Hep-2 YES n/a ATCC VR-1 4 RT Hep-2 YES n/a 7 4 O C Hep-2 YES 2 7 RT Hep-2 YES 2 HSV Type O C 3 x 10 4 MRC-5 YES 1 ATCC VR RT MRC-5 YES O C MRC-5 YES 2 7 RT MRC-5 YES 3 HSV Type O C 3 x 10 4 MRC-5 YES 2 ATCC VR RT MRC-5 YES O C MRC-5 YES 1 7 RT MRC-5 YES 2 Parainfuenza O C 3 x 10 4 PLC YES 4 ATCC VR-93 3 RT PLC YES O C PLC YES 6 7 RT PLC YES 15 Rhinovirus 3 4 O C 3 x 10 4 MRC-5 YES 2 ATCC VR RT MRC-5 YES O C MRC-5 YES 3 7 RT MRC-5 YES 5 Infuenza A 3 4 O C 3 x 10 4 PLC YES 4 Cinica isoate 3 RT PLC YES O C PLC YES 5 8 RT PLC YES 8 Virocut was shown to successfuy recover a 6 viruses after hoding for up to 7 days, and whether hed at 4 O C or ambient temperature. CLSI s document M40-A provides a usefu gauge for the assessment of transport devices. For bacterioogy where there is a quick turn round of cutures, it is a practica method of judging the suitabiity of a device for a particuar appication. However, for viroogy this is not the case, and the work is extremey time consuming, and expensive in materias, and not practica for a routine aboratory. This may expain why there have been so few reported viroogy based M40 studies, whie there is a pethora of bacterioogica studies. The Virocut swab has been around for many years, and there have been many studies in the past showing it to be effective for most of the common respiratory and skin associated viruses. The present study has provided the opportunity to assess the Virocut swab in a new era, using current materias and ce ines. References 1. NCCLS, 2003, Quaity Contro of Microbioogica Transport Systems: Approved Standard NCCLS Document M40-A 2. WHO - Pubic Heath Emergency of Internationa Concern 3. Statement by WHO director-genera, Dr Margaret Chan [25 Apr 2009] Swine infuenza CDC Heath Advisory Distributed via Heath Aert Network Apri 25, 2009, 3:00 EST (03:00 PM EDT) CDCHAN ALT-N Investigation and Interim Recommendations: Swine Infuenza (H1N1) 4. Rudsdae, A.. & D. Shedden, 20th Apri, 2009, Investigation of the Suitabiity of the Virocut Swab Transport Device for Infuenza A Specimens Which Are to be Anayzed by Ce Cuture or Moecuar Techniques, POSTER M42, Cinica Viroogy Symposium 2009, Daytona Beach Virocut swabs, and ATCC virus cutures for this study were provided by Medica Wire Incubation period 24 hours 48 hours 72 hours Diution Tube 1 Tube 2 Tube 1 Tube 2 Tube 1 Tube Ce Contro TCID 50 = hours These procedures were carried out for each virus using appropriate ce ines, IF stains, etc M40-A requires the transport device to maintain ive virus at detectabe eves starting with a titre of 5 x 10 4 for up to 96 hours. The resuts shown here are for an inocuum of 3 x 10 4, as being cosest to that standard. Other diutions were aso used, and recovery demonstrated, but these are not shown here. In this study the test period was extended to 7 days to refect situations where specimens might require to be forwarded to a reference faciity after initia rapid testing. In the case of infuenza Type A, the reading had to be taken at 8 days because of a hoiday situation, but surviva was sti demonstrated. In the study Virocut was shown to meet and exceed the requirements of M40-A, by maintaining a seection of viruses in a viabe condition when hed at 4 O C, or at ambient/room temperatures for 96 hours, and beyond unti at east 7 or 8 days. The device shoud be suitabe for respiratory or skin esion specimens that require to be transported from remote sites, even if they cannot be frozen or transported with dry ice.

17 MWE Σ-Virocut Swab Look-Back Study G.J.A. Etringham 1, A. Rudsdae 1, C. Hi 2 1) Department of Viroogy, Heath Protection Agency, Newcaste-upon-Tyne, UK. 2) Medica Wire & Equipment Co Ltd. Abstract Positive Resuts/Location Midands Channe Isands Introduction The use of ess invasive methods for producing cinica sampes has progressed rapidy in recent years, with the production of more speciaised samping techniques that cause itte or no distress to the patient but yied high quaity cinica materia that may be examined for the presence of microbioogica pathogens. These sampes must end themseves to the ever increasing nove methodoogies that are increasingy becoming routine in the diagnostic aboratory. The detection of vira pathogens reies heaviy on moecuar technoogies that require extraction of target nuceic acid that must be of high quaity, pure and free of inhibitors. Therefore, the generation of the primary sampe is very important. Σ- Virocut from MWE (Medica Wire and Equipment Co Ltd) provides a combined vira specimen coection and transport system that achieves these goas providing high quaity, stabe sampes. 1,2,3. The Σ-Virocut swab system is routiney used in many aboratories for the detection of a range of vira agents, both DNA or RNA, that typicay cause infection of the respiratory and genita tract. A ook-back exercise of data generated from 2009 Infuenza A H1N1v pandemic and respiratory virus detection is presented here. Ampification Target Nose and Throat (n=189) Infuenza A 5 Infuenza B Parainfuenza 1 A + H A + N1 4 2 H1 + N1 3 0 Infuenza B 0 0 Tabe 1. distribution of resuts when screening respiratory swabs for Infuenza A,H1N1v and Infuenza B Of the 294 sampes submitted from the oca trust, 8 different vira pathogens were detected in 42 sampes (14.3%). Tabe 2. shows the distribution of vira targets detected in the cohort of 294 respiratory sampes submitted from the oca trust for compete vira screening. Throat (n=92) Site Nose (n=11) Nasopharynx (n=2) Parainfuenza 2 Methods Swabs taken for respiratory sites were sent to the Newcaste Heath Protection Agency Laboratory from three main sources. During summer 2009, 453 respiratory swabs were submitted from sites in the Midands and 603 from the Channe Isands. These were ony screened for the presence of Infuenza A, Infuenza A H1v (and N1 if resuts were discordant) and Infuenza B, in an attempt to detect the circuating Infuenza A H1N1v pandemic virus. From 02/01/10 30/04/10, 294 respiratory swabs from symptomatic patients aged 6 days to 85 years were submitted from the oca trust (189 nose/throat, 92 throat, 11 nasa and 2 nasopharyngea). These were screened by a number of rea-time PCR assays designed to detect a range of vira pathogens incuding Infuenza A and B, RSV A and B, Parainfuenza 1,2,3 and 4, Rhinovirus (hrv), human Metapneumovirus (hmpv) and Adenovirus. These swab sampes were first inactivated at Containment Leve III and spiked with an extraction contro target prior to undergoing an automated tota nuceic extraction method on either the Biomerieaux easymag or Qiagen QIAsymphony instrument as per the manufacturers protoco. The resutant euate was then subjected to a number of PCR reactions in an attempt to detect one or more of the respiratory targets. A respiratory PCR assays were performed using the ABI 7500 FAST Rea-Time PCR Patform, empoying TaqMan hydroysis chemistry with fuorescenty abeed probes that aow detection of the vira targets. Data anayses were performed post-pcr using the instrument software and resuts coated. Positive contro materia was derived from wit type target virus grown in tissue cuture prior to extraction. Σ-Virocut swabs were aso chaenged with the same positive contro materia and examined as with cinica sampes. Resuts Positive resuts were obtained for a targets when swabs were chaenged with known contro materia, the resuts were comparabe with the positive contro resuts. Vira Target Discussion On examination of those resuts generated from the Σ-Virocut swabs taken from respiratory sites and submitted for Infuenza A H1N1v screening, it can ceary be seen that a stabe, high quaity sampe has been presented for anaysis. The difference in percentage positivity rate does not represent any inadequacies of the sampe or transport system but merey refects epidemioogica distribution of the virus. The data obtained from sampes taken in the oca trust catchment area compare we with that of previous years, and when testing different sampe types e.g. nasopharyngea secretions or washings. A wide variety of vira targets were detected, with itte difference in percentage positivity rate based on site. The detection of the extraction contro in a euates provides evidence that the constituents of the swab and transport media did not inhibit the PCR reaction. In concusion, the Σ-Virocut swabs, when used as directed, are abe to provide high quaity cinica materia that may be anaysed with confidence in downstream moecuar assays foowing nuceic acid extraction. The purified nuceic acid derived from the swabs may be examined for both RNA and DNA targets. References Parainfuenza 3 1 Parainfuenza 4 1 RSV A 2 1 RSV B 5 9 Adenovirus 5 1 hmpv 3 hrv 8 1 Tabe 2. distribution of vira targets when screening respiratory swabs from different respiratory sites for common respiratory viruses. Swabs submitted for Infuenza A, H1N1v and Infuenza B screening from the Midands and the Channe Isands yieded 170 (37.5%) and 42 (6.9%) confirmed Infuenza A H1N1v positive resuts respectivey as shown in tabe 1. No positive resuts were obtained for other Infuenza A strains or for Infuenza B. Tabe 1. shows the resuts based on target combination to provide a confirmed resut. Caption: Ange of the North This image is suppied for singe usage and non-commercia Purposes ony. It must be correcty credited and captioned. Photographer: Graeme Peacock ID: 107

18 POSTER M42, Cinica Viroogy Symposium 2009, Daytona Beach INVESTIGATION OF THE SUITABILITY OF THE VIROCULT SWAB TRANSPORT DEVICE FOR INFLUENZA A SPECIMENS WHICH ARE TO BE ANALYZED BY CELL CULTURE OR MOLECULAR TECHNIQUES Andrew Rudsdae1 and Dougas Shedden2 1Regiona Virus Laboratory, Heath Protection Agency, Newcaste, United Kingdom and 2Medica Wire & Equipment, Corsham, United Kingdom ABSTRACT Introduction: In recent years many cinica microbioogy aboratories have discontinued the use of ce cuture for the identification of viruses, reying instead on the more rapid moecuar techniques now widey avaiabe. Nevertheess it is often necessary to submit specimens to reference aboratories for further identification or confirmation using ce cuture. The present study is intended to investigate whether Virocut swabs coud be used as a singe source for both types of testing, thus avoiding the need for mutipe specimens and coection devices. Methods: A new ce cuture based study was performed for Infuenza A virus, using the methods described in CLSI Quaity Contro of Microbioogica Transport Devices Approved Standard M40-A, measuring recovery for up to 8 days hoding time on Virocut swabs, both at ambient and refrigeration temperatures. The resuts were compared with cinica studies using moecuar techniques for the identfication of Infuenza A on Virocut -coected specimens. Resuts: It was shown that Virocut -coected specimens recovered Infuenza A virus for at east 8 days, both at ambient and refrigeration temperatures, and in addition worked acceptaby with each of the moecuar techniques assessed. From this study, it is shown that for Infuenza A, an important respiratory pathogen, the Virocut -coected specimens coud be used for both ce cuture and moecuar testing. INTRODUCTION Infuenza is an acute upper respiratory tract infection, normay associated with the winter months in temperate cimates. Athough normay sef-imiting in otherwise heathy aduts, there can be a high mortaity and morbidity for vunerabe groups incuding the edery and the very young. There is aso a considerabe economic burden in terms of oss of productivity due to absence in business, and the considerabe cost of hospitaisation. Traditiona methods for detecting infuenza virus incude ce cuture, compement fixation, and haemaguttinin inhibition. Such methods, however, are sow and often of itte vaue in determining treatment for the patient. They are of more reevance in providing epidemioogica data for monitoring the spread of particuar strains. The more recent deveopment of rapid methods of infuenza detection such as Reverse Transcriptase PCR (RT-PCR), and Direct Antigen Immunofuorescence aows more rapid detection and identification of the infecting virus, providing the strain is aready known, and its characteristics are aready stored within the test s database. Athough ce cuture is being or has been phased out in many aboratories, there is sti a need in reference aboratories for cuture as the god standard fina identification and confirmation step, and for the isoation and characterisation of new strains. This is particuary important for infuenza virus which is inherenty variabe due to antigenic drift, the resut of the high frequency of point mutations within certain

19 POSTER M42, Cinica Viroogy Symposium 2009, Daytona Beach genes, and the more drastic antigenic shift when genetic re-assortment occurs between different coinfecting subtypes. For many years Virocut swabs have been used for the coection of respiratory specimens for investigation by ce cuture. The device incude a transport medium which wi keep many species of virus viabe for many days. One of the advantages of the Virocut device is that it can be transported at ambient temperatures. In a recent survey of methods in 20 European countries, it was reported that in 16 countries vira specimens were submitted under ambient conditions, incuding 13 where submission was by post. Specimens were taking between 24 and 48 hours in transit. For many of the new moecuar techniques, it is not required to keep the viruses aive, but the tests invove antibodies or enzymes which coud be sensitive to interference from components of a transport medium. In most situations it woud be preferabe to have a transport device that woud be compatibe with both methods, aowing the specimen to be initiay tested by a rapid method, with the resut providing the basis for any recommended treatment of the patient. Subsequenty the device coud be forwarded to a reference aboratory for isoation of virus by cuture and further characterisation. The present study was designed to assess the suitabiity of Virocut for the transport of Infuenza Type A specimens for cuture, using the CLSI M40-A standard for transport devices. In addition, a survey was made of iterature references over the previous 15 years comparing overa identification rates for Infuenza Type A, and Infuenza Types A and B combined, for a methods, with those obtained when Virocut was used as the main coection device. METHODS - Ce Cuture Infuenza A Strain 3524/08 (Cinica Isoate) Strain 3524/08 (H1N1) 250μ into 4 tubes PLC ces O C Observe daiy unti Cytopathic effect observed (5 days) Virus identity confirmed as Infuenza A (Light Diagnostics Infuenza Reagent 5017 Simofuor Reagent 5296) Virus diuted (0.3m Virus suspension + 2.7m EMEM (Biowhittaker BE12-136F) 4 Virocut swabs immersed in each diution for 10 seconds, then immediatey paced in Virocut medium Then hed as foows: A O C B O C C 3 RT (19-21 O C) D 8 RT (19-21 O C) After hoding time Add 4m EMEM to Virocut tube. Vortex 10 seconds 100μ from tube into ce cuture (PLC ces maintained in 1m EMEM + 1% Foeta Caf Serum) (x2) O C Observe daiy for appearance of CPE RESULTS Limit of detection (owest starting concentration / eariest fu CPE) A O C B O C C 3 RT(19-21 O C) D 8 RT(19-21 O C) 7 days confirmed by immunofuorescence 7 days confirmed by immunofuorescence 11 dayconfirmed by immunofuorescence 10 8 days confirmed by immunofuorescence

20 POSTER M42, Cinica Viroogy Symposium 2009, Daytona Beach METHODS- Moecuar An anaysis was made of reports pubished since 1995 where non-cuture methods were used to detect infuenza virus in specimens from patients presenting with respiratory symptoms. Some of the studies were epidemioogica, with arge numbers of patients, with the objective of monitoring the spread of infuenza, incuding particuar serotypes. Others were studies devised to assess the performance of various tests. In some of the studies Virocut swabs were used as the coection device, whie in the others the devices were throat swabs, nasopharyngea swabs or nasopharyngea aspirates. The intention of this study is to demonstrate whether the overa proportion of positive specimens of infuenza type A, or infenza types A & B, observed when using moecuar techniques was simiar or significanty different between specimens coected using Virocut, specimens coected using other devices, and for a specimens. The resuts of a the studies were coated, adding together the tota numbers of vaid specimens, and the tota number of specimens shown to be positive for infuenza virus by the methods being used or assessed. Overa specimens were examined, incuding 4310 specimens coected and transported using the Virocut device, Virocut transported specimens, and 6647 specimens where other devices were used for coection. RESULTS Tabe 1 Infuenza Types A & B Method of coection Number of specimens Number of specimens positive for Infuenza virus Detection rate for infuenza A methods % A methods excuding Virocut % A methods using Virocut as coection device % Tabe 2 Infuenza Type A Method of coection Number of specimens Number of specimens positive for Infuenza virus Detection rate for infuenza A methods %

21 POSTER M42, Cinica Viroogy Symposium 2009, Daytona Beach A methods excuding Virocut % A methods using Virocut as coection device % CONCLUSIONS Ce cuture technique Live virus was detected by the appearance of cytopathic effect, confirmed by immunofuorescence, in the ce ayer inocuated from Virocut swabs after hoding periods of 3 days and 8 days at ambient temperatures or refrigeration temperatures. This exceeded the requirements of Standard M40-A. Moecuar techniques There was a remarkabe convergence of the overa detection rates for infuenza Type A, and for Types A & B, from diverse popuations of respiratory patients, with amost identica rates being demonstrated whether sampes were obtained by Virocut swabs, or by other methods, or by a methods. Whie further statistica anaysis may be required to assess the true significance of the convergence of the moecuar resuts, it does seem evident that the Virocut swab is a reiabe specimen coection device for infuenza Type A virus, whether investigation is by traditiona cuture methods, or by the newer rapid moecuar techniques. References 1. Amano, Y. & Q. Cheng, 2005, Detection of Infuenza Virus: Traditiona Approaches and Devekopment of Biosensors, Ana. Bioana Chem, 381: Chan, K.H., et a, 2002, Evauation of the Directigen FuA + B Test for Rapid Diagnosis of Infuenza Virus Type A and B Infections, J. Cin. Microbio., 40: Covaciuc, K. A., 1999, Comparison of Four Cinica Specimen types for Detection of Infuenza A and B Viruses by Optica Immunoassay (FLU OIA Test) and Ce Cuture Methods, J. Cin. Microbio., 37: Ghebremedhin, B., et a, 2009, Comparison of the Performance of the rapid Antigen Detection actim Infuenza A&B Test and RT-PCR in Different Respiratory Specimens, J. Med. Microbio. 58: Habib-Bein, N. A., 2003, Comparison of SmartCycer Rea-Time Reverse Transcrition-PCR Assay in a Pubic Heath Laboratory with Direct Immunofuorescence and Ce Cuture Assays in a Medica Center for Detection of Infuenza A Virus, J Cin. Microbio., 41: Hermann, B., 2001, Simutaneous Detection and Typing of Infuenza Viruses A and B by a Nested Reverse Transcription-PCR: Comparison to Virus Isoation and Antigen Detection by Immunofuorescence and Optica Immunoassay (FLU OIA), J. Cin. Microbio., 39: Hindiyeh, M., 2000, Evauation of BioStar FLU OIA assay for rapid detection of infuenza A and B viruses in respiratory specimens, J. Cin. Viro., 17:

22 POSTER M42, Cinica Viroogy Symposium 2009, Daytona Beach 8. Lambert, S. B., 2007, Community Epidemioogy of Human Metapneumovirus, Human Coronavirus NL63, and other Respiratory Viruses in Heathy Preschoo-Aged Chidren Using Parent-Coected Specimens, Pediatrics, 120: Lambert, S. B., 2008, Comparing Nose-Throat Swabs and nasopharyngea Aspirates Coected From Chidren With Symptoms for Respiratory Virus Identification using Rea-Time Poymerase Chain Reaction, Pediatrics 122: Liao, R.S., et a, 2009, Comparison of Vira isoation and Mutipex Rea-Time reverse Transcription-PCR for Confirmation of Respiratory Syncytia Virus and Infuenza Virus Detection by Antigen Immunoassays, J. Cin. Microbio., 47: Lina, B. et a, 1996, Surveiance of Community-Acquired Vira Infections Due to Respiratory Viruses in Rhone-Apes (France) during Winter 1994 to 1995, J. Cin. Microbio., 34: Lina, B., 2005, Test Evauation Report Medix Biochemica Infuenza A & B (Ref 32832ETMB) Actim Infuenza A & B (Ref 32832ETAC), Word Heath Organization Coaborating Centre for Virus Reference and Research 13. Magnard, C., et a, 1999, Comparison of Two Nested PCR, Ce Cuture, and Antigen Detection for the Diagnosis of Upper Respiratory Tract Infections due to Infuenza Viruses, J. Medica Viro., 59: Mehmann, M., et a, 2007, Comparison of the Mchip to Vira Cuture, Reverse Transcription-PCR, and the QuickVue Infuenza A+B Test for Rapid Diagnosis of Infuenza, J. Cin. Microbio., 45: Pachucki, C. T., et a 2004, Utiity of Reverse Transcriptase PCR for Rapid Diagniosis of Infuenza A Virus Infection and Detection of Amantadine-Resistant Infuenza A Virus Isoates, J. Cin. Microbio.42: Perez-Ruiz, M., et a2007, Testing of Diagnostic Methods for Detection of Infuenza Virus for Optima Performance in the Context of an Infuenza Surveiance Network, J. Cin. Microbio. 45: Poehing, K. A., et a, 2002, Bedside Diagnosis of Infuenzavirus Infections in Hospitaized Chidren, Pediatrics 110: Rezza, G., et a, 2006, Respiratory Viruses and Infuenza-Like Iness: A Survey in the Area of Rome, Winter , Eurosurveiance 11 (10), 01 October Ruest, A., et a, 2003, Comparison of the Directigen Fu A+B Test, the QuickVue Infuenza Test, and Cinica Case Definition to Vira Cuture and Reverse Transcription-PCR for Rapid Diagnosis of Infuenza Virus Detection, J. Cin. Microbio., 41: Schweiger, B., et a, 2000, Appication of a Fuorogenic PCR Assay for Typing and Subtyping of Infuenza Viruses in Respiratory Sampes, J. Cin. Microbio. 38: Vabret, A., et a, 2000, Comparison of three non-nested RT-PCR for the detection of infuenza A viruses, J. Cin. Viro. 17: Vaassina, M., et a, 1997, Rapid Detection of Different RNA Respiratory Virus Species by Mutipex RT- PCR: Appication to Cinica Specimens, Cin. Diagn. Viro. 8: Waace, L. A., et a, 1999, Infuenza diagnosis: from Dark Isoation into the Moecuar Light. West of Scotand Respiratory Virus Study Group. J. Infect. 39: Wright, K. E., et a, 1995, Typing and Subtyping of Infuenza Viruses in Cinica sampes by PCR, J. Cin. Microbio. 33: Yamada, A. et a, 1991, Detection of Infuenza Viruses in Throat Swab by Using Poymerase Chain Reaction, Microbio. Immuno. 35: Zhang, W. D., & D. H. Evans, 1991, Detection and Identification of Human Infuenza Viruses by the Poymerase Chain Reaction, J. Viro. Methods. 33: Zitterkopf, N., et a, 2006, Reevance of Infuenza A Virus Detection by PCR, She Via Assay, and Tube Ce cuture to Rapid Reporting Procedures, J. Cin. Microbio. 44: NCCLS, 2003, Quaity Contro of Microbioogica Transport Systems: Approved Standard NCCLS Document M40-A 29. CLSI, 2006, Vira Cuture; Approved Guideine. CLSI Document M41A

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