Suitability of virocult for influenza a specimens using cell culture or molecular techniques.indd

1Regional Virus Laboratory, Health Protection Agency, Newcastle, United Kingdom and 2Medical Wire & Equipment, ABSTRACT
respiratory specimens for investigation by cell culture. The device include Introduction: In recent years many clinical
a transport medium which will keep many species of virus viable for many microbiology laboratories have discontinued the use days. One of the advantages of the Virocult® device is that it can be of cell culture for the identifi cation of viruses, relying transported at ambient temperatures. In a recent survey of methods in 20 instead on the more rapid molecular techniques now European countries, it was reported that in 16 countries viral specimens were submitted under ambient conditions, including 13 where submission widely available. Nevertheless it is often necessary was by post. Specimens were taking between 24 and 48 hours in transit.
to submit specimens to reference laboratories for For many of the new molecular techniques, it is not required to keep the further identifi cation or confi rmation using cell culture. viruses alive, but the tests involve antibodies or enzymes which could be The present study is intended to investigate whether sensitive to interference from components of a transport medium. In most Virocult® swabs could be used as a single source situations it would be preferable to have a transport device that would be compatible with both methods, allowing the specimen to be initially for both types of testing, thus avoiding the need for tested by a rapid method, with the result providing the basis for any multiple specimens and collection devices. recommended treatment of the patient. Subsequently the device could be forwarded to a reference laboratory for isolation of virus by culture and Methods: A new cell culture based study was
performed for Infl uenza A virus, using the methods described in CLSI Quality Control of Microbiological The present study was designed to assess the suitability of Virocult® for the transport of Infl uenza Type A specimens for culture, using the CLSI Transport Devices Approved Standard M40-A, M40-A standard for transport devices. In addition, a survey was made measuring recovery for up to 8 days holding time on of literature references over the previous 15 years comparing overall Virocult® swabs, both at ambient and refrigeration identifi cation rates for Infl uenza Type A, and Infl uenza Types A and B combined, for all methods, with those obtained when Virocult® was used temperatures. The results were compared with clinical studies using molecular techniques for the identfi cation of Infl uenza A on Virocult®-collected METHODS - Cell Culture
Infl uenza A Strain 3524/08 (Clinical Isolate) Strain 3524/08 (H1N1) Results: It was shown that Virocult®-collected
Observe daily until Cytopathic effect observed (5 days) specimens recovered Infl uenza A virus for at least 8 Virus identity confi rmed as Infl uenza A (Light Diagnostics Infl uenza days, both at ambient and refrigeration temperatures, and in addition worked acceptably with each of the molecular techniques assessed. From this study, it is (0.3ml Virus suspension + 2.7ml EMEM (Biowhittaker BE12-136F) shown that for Infl uenza A, an important respiratory pathogen, the Virocult®-collected specimens could be 4 Virocult swabs immersed in each dilution for 10 seconds, then immedi- used for both cell culture and molecular testing.
Infl uenza is an acute upper respiratory tract infection, normally associated with the winter months in temperate climates. Although normally self- limiting in otherwise healthy adults, there can be a high mortality and morbidity for vulnerable groups including the elderly and the very young. There is also a considerable economic burden in terms of loss of productivity due to absence in business, and the considerable cost of 100μl from tube into cell culture (PLC cells maintained in 1ml EMEM + 1% Traditional methods for detecting infl uenza virus include cell culture, complement fi xation, and haemagluttinin inhibition. Such methods, however, are slow and often of little value in determining treatment for the patient. They are of more relevance in providing epidemiological data for monitoring the spread of particular strains. The more recent development of rapid methods of infl uenza detection such as Reverse Transcriptase Limit of detection (lowest starting concentration / earliest full CPE) PCR (RT-PCR), and Direct Antigen Immunofl uorescence allows more rapid detection and identifi cation of the infecting virus, providing the strain 10-3 @ 7 days confi rmed by immunofl uorescence is already known, and its characteristics are already stored within the 10-2 @ 7 days confi rmed by immunofl uorescence C 3 days @ RT(19-21OC) 10-3 @ 11 day confi rmed by immunofl uorescenceD 8 days @ RT(19-21OC) 100 @ 8 days confi rmed by immunofl uorescence Although cell culture is being or has been phased out in many laboratories, there is still a need in reference laboratories for culture as the gold standard fi nal identifi cation and confi rmation step, and for the METHODS- Molecular
isolation and characterisation of new strains. This is particularly important An analysis was made of reports published since 1995 where non- for infl uenza virus which is inherently variable due to antigenic drift, the culture methods were used to detect infl uenza virus in specimens from result of the high frequency of point mutations within certain genes, patients presenting with respiratory symptoms. Some of the studies were and the more drastic antigenic shift when genetic re-assortment occurs epidemiological, with large numbers of patients, with the objective of between different co-infecting subtypes.
monitoring the spread of infl uenza, including particular serotypes. Others were studies devised to assess the performance of various tests. In some For many years Virocult® swabs have been used for the collection of of the studies Virocult® swabs were used as the collection device, while
in the others the devices were throat swabs, nasopharyngeal swabs or Hindiyeh, M., 2000, Evaluation of BioStar FLU OIA assay for rapid detection of infl uenza A and B viruses in respiratory specimens, J. Clin. Virol., 17: 119-126 Lambert, S. B., 2007, Community Epidemiology of Human Metapneumovirus, Human Coronavirus NL63, and other Respiratory Viruses in Healthy Preschool-Aged Children Using The intention of this study is to demonstrate whether the overall proportion Parent-Collected Specimens, Pediatrics, 120: 929-937 of positive specimens of infl uenza type A, or infl enza types A & B, Lambert, S. B., 2008, Comparing Nose-Throat Swabs and nasopharyngeal Aspirates Col-lected From Children With Symptoms for Respiratory Virus Identifi cation using Real-Time observed when using molecular techniques was similar or signifi cantly Polymerase Chain Reaction, Pediatrics 122: 615-620 different between specimens collected using Virocult®, specimens Liao, R.S., et al, 2009, Comparison of Viral isolation and Multiplex Real-Time reverse collected using other devices, and for all specimens.
Transcription-PCR for Confi rmation of Respiratory Syncytial Virus and Infl uenza Virus Detection by Antigen Immunoassays, J. Clin. Microbiol., 47: 527-532 Lina, B. et al, 1996, Surveillance of Community-Acquired Viral Infections Due to Respiratory The results of all the studies were collated, adding together the total Viruses in Rhone-Alpes (France) during Winter 1994 to 1995, J. Clin. Microbiol., 34: 3007- numbers of valid specimens, and the total number of specimens Lina, B., 2005, Test Evaluation Report Medix Biochemica Infl uenza A & B (Ref 32832ETMB) shown to be positive for infl uenza virus by the methods being used or Actim Infl uenza A & B (Ref 32832ETAC), World Health Organization Collaborating Centre for assessed. Overall 10812 specimens were examined, including 4310 specimens collected and transported using the Virocult® device, Virocult® Magnard, C., et al, 1999, Comparison of Two Nested PCR, Cell Culture, and Antigen Detec-tion for the Diagnosis of Upper Respiratory Tract Infections due to Infl uenza Viruses, J. transported specimens, and 6647 specimens where other devices were Mehlmann, M., et al, 2007, Comparison of the Mchip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Infl uenza A+B Test for Rapid Diagnosis of Infl uenza, J. Clin. Microbiol., 45: 1234-1237 Pachucki, C. T., et al 2004, Utility of Reverse Transcriptase PCR for Rapid Diagniosis of Infl uenza A Virus Infection and Detection of Amantadine-Resistant Infl uenza A Virus Isolates, J. Clin. Microbiol.42: 2796-2798 Perez-Ruiz, M., et al2007, Testing of Diagnostic Methods for Detection of Infl uenza Virus for Optimal Performance in the Context of an Infl uenza Surveillance Network, J. Clin. Microbiol. Poehling, K. A., et al, 2002, Bedside Diagnosis of Infl uenzavirus Infections in Hospitalized Rezza, G., et al, 2006, Respiratory Viruses and Infl uenza-Like Illness: A Survey in the Area of Rome, Winter 2004-2005, Eurosurveillance 11 (10), 01 October 2006 Ruest, A., et al, 2003, Comparison of the Directigen Flu A+B Test, the QuickVue Infl uenza Test, and Clinical Case Defi nition to Viral Culture and Reverse Transcription-PCR for Rapid Diagnosis of Infl uenza Virus Detection, J. Clin. Microbiol., 41:3487-3493 Schweiger, B., et al, 2000, Application of a Fluorogenic PCR Assay for Typing and Subtyping of Infl uenza Viruses in Respiratory Samples, J. Clin. Microbiol. 38: 1552-1558 Vabret, A., et al, 2000, Comparison of three non-nested RT-PCR for the detection of infl u-enza A viruses, J. Clin. Virol. 17: 167-175 Valassina, M., et al, 1997, Rapid Detection of Different RNA Respiratory Virus Species by Multiplex RT-PCR: Application to Clinical Specimens, Clin. Diagn. Virol. 8: 227-232 Wallace, L. A., et al, 1999, Infl uenza diagnosis: from Dark Isolation into the Molecular Light. West of Scotland Respiratory Virus Study Group. J. Infect. 39: 221-226 Wright, K. E., et al, 1995, Typing and Subtyping of Infl uenza Viruses in Clinical samples by Yamada, A. et al, 1991, Detection of Infl uenza Viruses in Throat Swab by Using Polymerase Chain Reaction, Microbiol. Immunol. 35: 259-265 Zhang, W. D., & D. H. Evans, 1991, Detection and Identifi cation of Human Infl uenza Viruses by the Polymerase Chain Reaction, J. Virol. Methods. 33: 165-189 Zitterkopf, N., et al, 2006, Relevance of Infl uenza A Virus Detection by PCR, Shell Vial As- say, and Tube Cell culture to Rapid Reporting Procedures, J. Clin. Microbiol. 44: 3366-3367 NCCLS, 2003, Quality Control of Microbiological Transport Systems: Approved Standard CLSI, 2006, Viral Culture; Approved Guideline. CLSI Document M41A CONCLUSIONS
Cell culture technique
Live virus was detected by the appearance of cytopathic effect,
confi rmed by immunofl uorescence, in the cell layer inoculated
from Virocult® swabs after holding periods of 3 days and 8 days
at ambient temperatures or refrigeration temperatures. This ex-
ceeded the requirements of Standard M40-A.

Molecular techniques
There was a remarkable convergence of the overall detection
rates for infl uenza Type A, and for Types A & B, from diverse
populations of respiratory patients, with almost identical rates
being demonstrated whether samples were obtained by Virocult®
swabs, or by other methods, or by all methods.

While further statistical analysis may be required to assess the true signifi cance of the convergence of the molecular results, it does seem evident that the Virocult® swab is a reliable specimen collection device for infl uenza Type A virus, whether investigation is by traditional culture methods, or by the newer rapid molecular techniques. References
.Amano, Y. & Q. Cheng, 2005, Detection of Infl uenza Virus: Traditional Approaches and Deveklopment of Biosensors, Anal. Bioanal Chem, 381: 156-164 Chan, K.H., et al, 2002, Evaluation of the Directigen FluA + B Test for Rapid Diagnosis of Infl uenza Virus Type A and B Infections, J. Clin. Microbiol., 40: 1675-1680 .Covalciuc, K. A., 1999, Comparison of Four Clinical Specimen types for Detection of Infl u-enza A and B Viruses by Optical Immunoassay (FLU OIA Test) and Cell Culture Methods, J. Clin. Microbiol., 37: 3971-3974 Ghebremedhin, B., et al, 2009, Comparison of the Performance of the rapid Antigen Detec-tion actim Infl uenza A&B Test and RT-PCR in Different Respiratory Specimens, J. Med. Microbiol. 58: 365-370 .Habib-Bein, N. A., 2003, Comparison of SmartCycler Real-Time Reverse Transcrition-PCR Assay in a Public Health Laboratory with Direct Immunofl uorescence and Cell Culture As-says in a Medical Center for Detection of Infl uenza A Virus, J Clin. Microbiol., 41: 3957-3601 .Hermann, B., 2001, Simultaneous Detection and Typing of Infl uenza Viruses A and B by a Nested Reverse Transcription-PCR: Comparison to Virus Isolation and Antigen Detection by Immunofl uorescence and Optical Immunoassay (FLU OIA), J. Clin. Microbiol., 39: 134-138



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