WAVLD Symposium Handbook_V4.indd - csiro

Introduction
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
A REVIEW OF TRAINING IN MOLECULAR DIAGNOSTIC TECHNIQUES
FOR AVIAN INFLUENZA IN SOUTH EAST ASIA
L. I. Pritchard 1 , A. Foord 1 , T. Taylor 1 , A. Axel 1 , R. Lunt 1 , J. Hammond 1 ,
C. Groocock 3 , L.H. Lauerman 2 and P.W. Daniels 1
1 CSIRO Livestock Industries, Australian Animal Health Laboratory (AAHL) Geelong, Australia
2 P.O. Box 189 East Olympia, WA 98540-0189, USA
3 1030 West Creek Ave Cutchogue NY 11935, USA
The ability to control avian influenza (AI) is critically dependant on the speed and accuracy of both
epidemiology and diagnosis. For this reason AusAid and APHIS/USDA funded projects were initiated to
provide training for diagnosis and epidemiology to scientists from South East Asian countries. Starting in
early 2006, training courses for molecular diagnosis of AI were developed and conducted in Australia,
Indonesia, Philippines and Taiwan. Under these projects diagnostic staff were trained in epidemiological,
serological and molecular techniques (including real-time qRT-PCR and conventional RT-PCR). The
scientific participants involved came from Indonesia, Cambodia, Thailand, Philippines, Vietnam, Sri Lanka,
India and Taiwan.
Methods
The setup and workflows for real-time PCR laboratories were the major focus of the technology transfer and
training, however advice was also given with regards to other procedures such as biosecurity. The training
was both in-house and multiple visits to each Indonesian DIC lab. The real-time RT-PCR techniques were
designed at AAHL and run on Applied Biosystems 7500 real-time machines as described by Heine et al,
2007.
Results
During this short period we were able to;
1. Provide training to about 80 scientific staff from South East Asian countries, including Indonesia,
Cambodia, Thailand, Philippines, Vietnam, Sri Lanka, India and Taiwan.
2. Develop a proficiency test program in AI real-time RT-PCR for use in Indonesian labs.
3. Implement a novel purchasing system for Indonesian labs to enable them to access reagents
for molecular diagnosis of AI.
4. Evaluate different real-time PCR methodologies and machines for AI diagnosis.
Discussion and Conclusions
This AusAid/USDA-funded project for the technology transfer of Avian Influenza (AI) real-time PCR tests was
developed at the Australian Animal Health laboratory (AAHL). The project involved; an initial gap analysis of
the facilities at eight laboratories in Indonesia, and training of South East Asian scientific staff at AAHL, in
Indonesia and in Taiwan.
Training for scientific staff from other South East Asian countries was also sponsored by the USDA – FAS, -
APHIS, American Institute in Taiwan (AIT), AAHL/CSIRO and Animal Health Research Institute in Taiwan. In
Indonesia a program was carried out between February and June 2007 for the diagnosis by real-time PCR of
AI Type A, H5 and H7 viruses. Initially training was done at AAHL for scientific staff from Indonesia and
proficiency test programs were developed and dispatched. Many factors were shown to affect the accuracy
of molecular diagnosis with laboratory setup, workflows, quality controls, difficulties with reagents and the
competency of the staff being the major focus areas. We also undertook a limited evaluation of the methods
used for AI training carried out in Taiwan between May and June 2007. Scientists from Indonesia, India, Sri
Lanka, Vietnam, Cambodia, Thailand, Philippines and Taiwan undertook an intensive, two week course
which involved both real-time and conventional RT-PCR, serology and virus isolation.
Findings revealed that the accuracy of molecular diagnosis can be significantly affected by a number of
factors including sample workflows and storage conditions, but especially the methods used for nucleic acid
extraction, cDNA synthesis and PCR amplification. We present the results of this evaluation and training.
Wed 14 November
Wed 14 November
World Association of Veterinary Laboratory Diagnosticians – 13 th International Symposium, Melbourne, Australia, 11-14 November 2007
DEVELOPMENT AND CONSOLIDATION OF AN EQA PROGRAMME FOR THE MOLECULAR
DIAGNOSIS OF AVIAN INFLUENZA
Halpin, K., Heine, H., Johnson M., Stevens, V., Trinidad, L., Davies, K. and Selleck, P.
CSIRO
Australian Animal Health Laboratory
Background: In 2006, avian influenza (AI) TaqMan RT-PCR assays which were developed at the Australian
Animal Health Laboratory were transferred from AAHL to several international and state veterinary
diagnostic laboratories. In order to support the implementation of these assays and provide the laboratories
with the ability to assess their capability to perform the assay to an outcome consistent with other
laboratories performing the same test, a proficiency testing program was established.
Methods: Participating laboratories were provided with protocols, technical information and support to
implement the AI TaqMan assays. All laboratories implemented the Influenza Type A TaqMan assay and the
H5 TaqMan assay. Over the next 8 months, four proficiency testing panels were distributed to ten
laboratories, including state, national and international laboratories. Each panel contained blind coded
samples representing a range of concentrations of the pathogen, as well as material derived from an
uninfected source for test evaluation. Test results were analysed and critical parameters from the different
instrument platforms were identified in consultation with the participating laboratories.
Results: A national proficiency testing program containing high and low positive, negative and replicate
samples has been implemented to monitor the performance of AI TaqMan RT-PCR. Management of this
proficiency program has been transferred to the Australian National Quality Assurance Program (ANQAP).
Standard operating procedures for these assays and test validation data collected from this program have
been submitted to the Australian Subcommittee on Animal Health Laboratory Standards (SCAHLS).