Established in December 2002, PulseNet Asia Pacific is picking up its pace as a molecular subtyping network for infectious diseases surveillance in the region. Pulling together efforts from thirteen countries and areas, members meet annually to share their advancement in local foodborne/infectious diseases surveillance system and discuss key issues to attaining our common goal to facilitate the timely exchange of DNA fingerprinting data of these pathogens.
With this network of people, key issues such as formation of steering committee to lead the development of the network, setting up of work groups to address major concerns, recognizing sources of funding for supporting countries/areas projects and laboratory capacity building, and commitment to expand and sustain the network were addressed in annual meetings in Honolulu (2002), Hong Kong (2004), Tokyo (2005) and Nanjing (2006).
Since 2004, significant progress has been achieved in laboratory capacity building, training, quality assurance, protocol evaluation, and communication enhancement.
Initially, PFGE laboratory facilities in PulseNet Asia Pacific members ranged from fully equipped with hardware and software to those start from scratch. Hence, laboratory capacity building is the first priority so that our target of a regional network for infectious diseases surveillance will be achievable. At least one PFGE system is installed in all the participating laboratories with tremendous effort of Laboratory Resources and Support Work Group that are coordinated by Dr. Watanabe of Japan. Having common software for PFGE data analysis and management in all countries/areas will take us a significant step closer to our target.
Training is another key to the success of the network. Consequently, four PFGE workshops were held at Public Health Laboratory Centre (PHLC) in Hong Kong (HK) from 2004 to 2007. The workshops are organized with joint efforts by Center for Diseases Control and Prevention and Association of Public Health Laboratories of USA, National Institute of Infectious Diseases of Japan and PHLC of HK. A total of 35 scientists from the region attended the PFGE workshops in the past four years.
Laboratory theories as well as hands-on practices of the standardized Salmonella serotypes PFGE protocol and data analysis were taught during the first three workshops. With increasing interests in data analysis and data management, a software training workshop was delivered in mid February 2007.
The purpose of having a Platform for Interlaboratory Comparison Work Group (PICWG) is to ensure comparability of PFGE patterns among different laboratories in our network. Its main features include data shared will still be owned by individual members, and each participant will respect the confidentiality of the information.
PICWG started with Phase I in August of 2004 in which two freeze-fried ampoulesof Salmonella strains were tested and distributed to seven participant laboratories by coordinating laboratory (PHLC of Hong Kong). In return, PFGE gel images from members were submitted through email for further analysis. Finally, consensus patterns were resulted and mailed back to individual participants with comments on gel quality and suggestions for improvement. The distribution of Phase I in 2005 was identical with previous one except increasing number of participating laboratories (from seven to nine) and two different Salmonella strains were included.
With the success of Phase I, members agreed to take part in Phase II which started in 2006. During Phase II, XbaI-PFGE patterns of top three Salmonella serotype from 11 countries/areas were collected and compared. Thirty distinguishable patterns were identified from 35 XbaI-pattern submitted. Both Phases I and II will continue in 2007.
To ensure the reproducibility and robustness of standardized PFGE protocols, external validation of protocols in different laboratories are important. In view of public health importance of Vibrio species in this region, some PulseNet Asia Pacific laboratories and PulseNet USA collaborated closely in external validation and evaluation for the rapid one-day standardized PFGE protocol for subtyping Vibrio cholerae and Vibrio parahaemolyticus respectively.
Future collaboration of the network with other PulseNet international networks will be undertaken for evaluating rapid standardized PFGE protocol for subtyping pathogens of significant public health concerns such as Shigella flexneri.
Email has been the major communication tool among members of the network since 2002. Web-based discussion forums were subsequently developed by Dr. Brent Gilpin of New Zealand. The PulseNet Asia Pacific WebBoard is a convenient means of contact as well as user-friendly. It also provides different level of security to ensure confidentiality of information exchange.
Discussion forums are categorized systemically as follows:
PulseNet Asia Pacific laboratories will continue to work on a common Software platform for inter-laboratory comparison, and addressing the training need of member laboratories. There is also the possibility of expanding the organism coverage in the PICWG, and further encourage discussion and information exchange. With provision of adequate resources, it is envisaged that there will be enhanced collaboration among different laboratories and among different PulseNet International Networks.
For further information please contact:
Dr Kai-Man Kam, Consultant Medical Microbiologist, Department of Health, Hong Kong.
PulseNet Asia Pacific group photo, Kolkata, India, February 2008
On September 24, 2004, PulseNet China held the first meeting in Beijing. The central laboratory is in National institute for Communicable Disease Control and Prevention, and State Key laboratory for Infectious Disease Prevention and Control, China CDC. Within these years PulseNet China were working on central laboratory establishment, technical learning, protocol optimization and evaluation of some special pathogens, database construction, PFGE training to other laboratories, and practice in response to outbreaks.
Other bacteria pathogens:
Nesseria meningitidis, Yersinia pestis, Leptospira interrogans, Streptococcus suis.Up to March 2007, high-quality patterns of more than 2,600 strains were recorded in the database of the central laboratory:
All the Isolates recorded in the central laboratory database are collected from more than 20 provinces. In the database, isolates came from Strain Banks in China CDC (collected from different provinces in different years), recent outbreaks in some provinces (the isolates were transferred into the lab in China CDC, the central lab, to run PFGE). The data of other bacteria, and of other provincial laboratries are under collected and estimated.
PulseNet China is trying to optimize the PFGE protocol of Leptospira interrogans. Based on the other PFGE protocols of other pathogens provided by PulseNet, the methods for chromosome DNA purification, restriction endonuclease digestion and the parameters for running PFGE were optimized. The Not I patterns of partial isolates of serogroup icterohaemorrhagiae in the surveillance in Sichuan province were analyzed.
More than 10 provincial CDC laboratories have been equiped with PFGE instruments (Bio-Rad CHEF Mapper or DR III). Up to now the PFGE pattern form half of these laboratories can be analyzed directly with BioNumerics. Fifty-eight professional technicians from 29 province- and city-level CDC have been trained in the central laboratory, including specialized training, to do PFGE analysis of the strains collected historically in surveillance, and the strains from response to outbreaks.
In 2007 the key work in PulseNet China is focusing on network management, regulations, data exchange and training. The regulations include procedure of the analysis, validation and certification of the new laboratories when joining the network, cooperation agreement (memorandum of understanding) to the network laboratories, and construction of QA/QC system of the network. The network will be managed based on these regulations.
Data exchange is the challenge in PulseNet China. Now the primary manner of data exchange is email. The possibilty of discussion forum of PulseNet China is under evaluation.
The PulseNet standardized PFGE protocols were adopted by the laboratories of Taiwan Centers for Disease Control (Taiwan CDC) in 2002 for routine subtyping and building a DNA fingerprint database of bacterial pathogens. After 4-year practice, PulseNet Taiwan, the National Molecular Subtyping Network for Infectious Disease Surveillance, was formally inaugurated by the Department of Health, Taiwan, on 3 October 2006.
| Bacterial species | Number of isolates | Number of PFGE patterns |
| Salmonella spp. | 10,238 | 1,669 |
| Shigella spp. | 2,043 | 516 |
| Vibrio parahaemolyticus | 89 | 45 |
| Vibro cholerae (most are reference strains) | 26 | 19 |
| E. coli O157 | 12 | 11 |
| Listeria monocytogenes | 5 | 5 |
Salmonellosis is estimated to be the most prevalent foodborne disease. To monitor this disease, a National Salmonella reference laboratory was established by Taiwan CDC in 2004. More than 2,000 Salmonella isolates were collected each year from the collaborative hospitals for determination of serotypes, PFGE fingerprints and traits of antimicrobial susceptibility. To date, the Salmonella DNA fingerprint database has contained more than 10,000 PFGE fingerprint entries representing for over 70 serotypes. For some serotypes with abundant PFGE genotypes, the serotype of a new isolate can be predicted by comparison of PFGE fingerprint with those in the fingerprint database. Since 2007, we have applied this database for serotype prediction and serotypes of 98.44% (3,034/3,082) isolates of the year were determined by PFGE fingerprint comparison. Taiwan PulseNet has been applied to confirm an international outbreak of Salmonella Agona infections linked to infant food and rule out many salmonellosis outbreaks occurred in USA.
S. flexneri is mainly circulating among aboriginal tribes in the mountainous area; while S. sonnei infections mainly occurs in the industrialized western Taiwan and most are associated with imported clones. All the Shigella isolates are sent to the laboratories of Taiwan CDC and routinely analyzed by PFGE with NotI and XbaI. S. sonnei isolates are also routinely analyzed by MLVA method. The Shigella DNA fingerprint database has been used to rule out some international shigellosis outbreaks including the recent outbreak linked to baby corn exported from Thailand.
V. parahaemolyticus is the leading causal agent for foodborne disease outbreaks in Taiwan. Since most of the outbreaks of V. parahaemolyticus infections are easily identified, routine PFGE analysis is not applied to this organism.
To date, Taiwan has no indigenous case of E. coli O157:H7 infection; only an imported case was identified in 2001. V. cholerae infection was rare for the past 40 years. Listeriosis is not in the list of notifiable diseases in Taiwan. Although the incidence rate is unknown, L. monocytogenes is infrequently identified from patients in this country.
| Bacterial species | Number of Isolates | Number of PFGE patterns |
| Streptococcus pyogenes | 1,188 | 109 |
| Klebsiella pneumoniae | 684 | 499 |
| Neisseria meningitidis | 254 | 122 |
| Bordetella pertussis | 92 | 71 |
The PulseNet platform is also applied to build a DNA fingerprint database for some infrequent as foodborne or non-foodborne bacterial pathogens. Streptococcus pyogenes DNA fingerprint database contains PFGE fingerprints and emm sequences. Neisseria meningitidis database contains PFGE fingerprints, MLVA profiles, and MLST, proA and proB sequences; whereas, Bordetella pertussis database contains only PFGE fingerprints of the organism.
An automatic plug washer has been invented by a local company, which was an improved model of the primordial plug washer originally designed by US CDC. The quality of DNA prepared by using the new plug washer is good and stable. We also evaluated the restriction enzyme and protocol for PFGE analysis of V. parahaemolyticus. The study indicated that the PulseNet standardized PFGE protocol for enterobacteria with some modifications is applicable to PFGE analysis of V. parahemoltyicus and NotI is more cost-effective than SfiI. Our data indicated that NotI should be used as the primary enzyme and SfiI as the second enzyme for PFGE analysis of V. parahaemolyticus.
With grant support partly from the National Institute of Infectious Diseases, Japan, Taiwan CDC has successfully developed a MLVA method with 26 VNTRs for S. sonnei. The MLVA method exhibits higher level of discriminatory power than PFGE. The method is potentially a useful tool for phylogenetic analysis of S. sonnei. Development of MLVA methods for S. flexneri and Salmonella Typhimurium are one of the current focuses of Taiwan CDC.
The bacterial laboratories of Taiwan CDC have been equipped with 11 PFGE instruments (Bio-Rad CHEF Mapper), 3 sequencers (ABI 3130) and more than 10 sets of BioNumerics software. PFGE analysis of some bacterial pathogens is routinely performed in the laboratories. Since 2004, more than 3,000 bacterial isolates were analyzed each year. With the aid of automatic plug washer, the PFGE analysis capacity of the laboratories can be up to 8,000 isolates per year.
PulseNet Taiwan has been adopted by DOH as an early detection tool in the national food safety surveillance system. In this regard, the PulseNet Taiwan Task Force will include partners from food and agriculture sectors. In the future, PulseNet Taiwan will focus on the surveillance of shigellosis and to be an active member in the PulseNet International community.
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