Development of a global HPV laboratory network
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1 Annual Report on Development of a global HPV laboratory network (TSA: V ) Period: 15 July to 15 May 2010 Principal Investigator: Joakim Dillner Institution: Laboratory Medicine Skåne Clinical Microbiology Entrance 78 University Hospital MAS Malmö, Sweden Point of Contact: Joakim Dillner Contract Administration: Helena Persson, Date of this report submitted: 23/
2 Introduction A brief introduction of the activities being conducted in your laboratory relating to HPV LabNet. The activities of the reference lab are considered under the following headings which were part of the Technical Services Agreement with WHO. In this report, we describe our progress* in each of these Terms of Reference. Scientific and technical advice 1. In coordination with WHO, contribute to the development of a global HPV laboratory manual, in collaboration with other HPV experts and HPV network laboratories. Report: The first version of the HPV laboratory manual has been submitted to IVB WHO for external expert review. The manual includes chapters on surveillance; nucleic acid amplification technique (NAT) assays and how to assess their performance and make data internationally comparable through the use of international standards; serology assays including EIA and PsV neutralization assays; quality assurance as well as standard operating procedures (SOP); and guidance on some of the specific techniques which had been evaluated through collaborative studies in which HPV LabNet members had participated. Global Reference Laboratory (GRL) Sweden started the project and coordinated the first draft. GRL Sweden has throughout participated as one of the Editors and actively contributed to the finalization of this project. 2. Provide scientific advice to the respective WHO regional office or WHO/Headquarters regarding laboratory techniques, including virological and serological detection of HPV infections, for surveillance purposes. Report: GRL Sweden has participated in monthly telephone conferences with the WHO Headquarters, NIBSC and GRL USA. These HPV LabNet participants have in effect constituted an intensively collaborating working group to ensure the progress of the LabNet. Advice has also been given at in-person visits to WHO Headquarters and in conjunction with 2 HPV LabNet international training workshop (August 2009 in Bangkok, Thailand, in April 2010 in Lausanne, Switzerland) 3. Collaborate with local and regional public health authorities and research institutions, and other international agencies, who may request advice on monitoring HPV vaccination programs. Report: Staff from the national reference laboratory in Norway made a site visit at our laboratory to study HPV DNA typing methods, national quality assurance programs and vaccination surveillance strategies. We have collaborated with the Swedish Institute for Infectious Disease Control to launch an HPV vaccination registry in Sweden as well as piloting a monitoring system, that uses both 2
3 condyloma incidence/typing, monitoring of HPV prevalences among teenagers using urine samples taken during the Chlamydia screening program and systematic typing of cases of HPVassociated diseases. The PI (Joakim Dillner) has also been expert advisor to the Swedish government on HPV surveillance system design. GRL Sweden has also participated in an international meeting convened at WHO HQ on HPV monitoring and has further presented on the HPVLabNet and on HPV monitoring at the meeting at the WHO HQ of the HPV vaccine advisory committee. 4. Participate in the international collaborative studies to develop HPV laboratory reference reagents for HPV testing, coordinated by NIBSC, disseminating knowledge on, and the use of, HPV international standard reagents to improve the accuracy of DNA and serological measurements. Report: We have in collaboration with the RRL in Thailand tested candidate sera for an HPV 18 antibody reference reagent that will be developed in collaboration with NIBSC. Sera from two women who had high antibody titers against HPV18, but were negative against all other HPV types, have been collected in sufficiently large volumes to now constitute a candidate international standard. An international collaborative study to characterize the candidate international standard is currently being initiated by NIBSC. We have evaluated a series of HPV VLPs that were produced in sufficiently large amounts for possibly becoming reference reagents for high quality, internationally standardized HPV serology. Differences in VLP quality has been identified as a major source of variability in HPV serology and establishment of a carefully validated reference reagent of VLPs for serology would significantly contribute to better and more comparable HPV serology. HPV 16 VLPs from two commercial companies and three LabNet laboratories as well as HPV 18 VLPs from two of the sources and HPV 6, 11 VLPs from one source were characterized and validated. A summary report of the validation is attached. We have also made several lectures about the importance of these standards at both national and international conferences. Conclusion, if any: GRL Sweden has taken on a substantial part of the task to promote the LabNet and the standardization work for both HPV DNA typing and HPV serology. Quality assurance 1. In coordination with WHO, contribute to the creation of a HPV quality assurance program for the LabNet by establishing HPV proficiency panels, and assist with analysis of proficiency study results. Report: To continue the quality assurance program for HPV DNA testing and typing, we manufactured, distributed and evaluated the 2009 HPV LabNet HPV DNA typing proficiency panel, consisting of 43 samples for typing and 3 extraction controls. The panel was distributed to all HPV LabNet laboratories. The summary report of the 2009 WHO HPV DNA proficiency panel testing is attached. 3
4 A scientific paper with the results of the 2008 WHO HPV DNA proficiency panel study has been submitted to Journal of Clinical Microbiology. The 2010 HPV DNA typing proficiency panel will be made at a larger scale and will be available for testing for about 100 laboratories worldwide. The work to accomplish this has been planned and distribution will be done during the second half of 2010 as part of a new Technical Service Agreement with WHO. We organized and completed the first international HPV serology proficiency study according to a serology standard operating procedure developed within the LabNet. HPV 16 and HPV 18 VLP-coated ELISA plates together with 90 serum samples and reagents were distributed to all LabNet members. The final report on the HPV serology proficiency study is enclosed. 2. At the request of the WHO regional office or WHO/HQ, serve as a resource for preparation, distribution, and/or storage of standard reagents, proficiency panels and cell lines to other laboratories. Report: We are storing a bulk stock of purified plasmids at minus 80 C, that we have prepared for use in the HPV DNA typing proficiency panels (a new one annually is planned). We have sent 2 μg HPV DNA of HPV 6, 11, 31, 33, 45, 52 and 58 plasmid preparations to NIBSC to be used as candidate international standards for HPV DNA. As International Standards was a new use of these plasmids, we had to negotiate new Material Transfer Agreements (MTAs) with all the proprietors of these plasmids. This was a sizable and rather complicated task, that we have now successfully completed. We have also started the collection of serum samples to be used for upcoming annual HPV serology proficiency panels. 3. Perform confirmatory testing on samples from other laboratories as requested, to ensure that all assays perform at acceptable levels of sensitivity, specificity and reproducibility. Report: We have received samples for confirmatory testing from the almost all regional LabNet laboratories during this time period: RRL Thailand 99 samples, RRL India 86 samples, RRL Australia 239 samples, RRL Argentina 154 samples, RRL Japan 100 samples, RRL Switzerland 107 samples, RRL Tunisia 13 samples. These samples were selected to contain all HPVnegative cervical cancers and in situ cancers (possible false negatives) as well as a random sample of positives and problem samples deemed to not be possible to type. The samples have been tested both by our laboratory and by the GRL USA. Many important findings have emerged, e.g. the existence of HPV16 variants that are missed by several common HPV detection systems. A summary report of the testing is prepared by GRL USA. 4. Participate in on site visits to other countries/ provinces as part of the WHO evaluation team, if requested. 4
5 Report: This has not been requested by WHO during this year. We did visit the RRL Thailand and the RRL Europe in Switzerland for the 2009 and 2010 WHO HPV LabNet International Training Workshops. For the Thailand visit, we sent technical personell in advance to perform training of the course host lab in advance of giving the course. Conclusion, if any: There has been remarkable progress with the work regarding HPV DNA testing and typing QA in terms of proficiency panel preparation and global spreading of the awareness of the QA principles. Both the projects on International Standards, international proficiency testing panels and confirmatory testing schemes have progressed well and are now comprehensively implemented. The work on HPV serology is following suit closely after the work on HPV DNA typing, with a candidate international standard for HPV 18 now having been sourced and characterized, with the first international HPV serology proficiency study having been completed and with a new project initiated for sourcing of international standards for HPV serology for the additional oncogenic HPV types that are likely to be part of second generation HPV vaccines (HPV31,33, 45, 52, 58). Training 1. Coordinate or participate in WHO training workshops for strengthening the HPV laboratory testing capacity. Report: Both PI Joakim Dillner and lab manager Carina Eklund from GRL Sweden were participating as invited speakers in the training workshop arranged by RRL Thailand in Bangkok in August Carina Eklund also participated in setting up, rehearsing and performing the lab training sessions. Joakim Dillner participated as invited speaker in the WHO HPVLabNet training workshop organized by the RRL Switzerland in Lausanne in March Assure that sufficient trained and qualified personnel and facilities are available to fulfill the tasks related to HPV DNA and antibody laboratory detection. Report: Trained personnel available for HPV DNA typing and detection; Eight qualified persons (Carina Eklund, Kia Sjölin, Aline Marshall, Christina Cavala, Anna Söderlund-Strand, Cecilia Wahlström, Herman Palm and Ingrid Lindström) Trained personnel available for HPV serology, 3 persons (Carina Eklund, Christina Cavala, Helena Faust) Qualified Supervisory personnel; Joakim Dillner (professor), Carina Eklund (lab manager), Ola Forslund (associate professor), Lena Dillner (director) 3. In conjunction with WHO, provide corrective advice to laboratories within the laboratory network with deficiencies identified by the proficiency studies. Report: Individual advice has been sent to non proficient laboratories. Additional technical advise has been given by upon requests from LabNet members. 5
6 Conclusion, if any: In our opinion, the 2009 WHO HPVLabNet International Training Workshop in Bangkok was a great success. It significantly improved the situation for the RRL, both by providing improved expertise, providing political support and by establishing network contacts with a large number of regional laboratories who were entering the field but were not previously aware of QC issues or the existence of the international standardization efforts. The 2010 WHO HPVLabNet International Training Workshop had a somewhat different focus, as the participants were selected by the WHO regional offices and thus represented the entire world. While this was also very interesting, I have the impression that the regional course had a greater impact in creating new collaborative networks and strengthening regional research. I would recommend the organization of annual WHO HPVLabNet International Training Workshops, but the format of regional participants and international teaching faculty should be favoured. Communication 1. Promote the exchange of information with laboratories at national and regional level and the HPV LabNet. Report: When WHO HQ opened a Share point for the WHO HPV LabNet, we were transferring all our SOPs to the share point. We did take part in two LabNet training workshops, several meetings at WHO Headquarters as well as national meetings. 2. Provide an annual report to WHO/HQ and respective regional office presenting a critical appreciation of the activities regarding the participation in the global HPV laboratory network. Report: This is this report. We are also submitting the report to the Regional Office for Europe (att: Galina Lipskaya). 3. Disseminate relevant information through contributions to a HPV laboratory network quarterly news letters. Report: We have been contributing with requested material regarding LabNet studies to the newsletter in communication with Dr. Garland, who is the editor. Conclusion, if any: Communication strategies were initially slow, but are now well underway. We perceive the information within the LabNet and to the HPV scientific community as efficient, but have repeatedly noticed that information strategies used are insufficient to reach many important stakeholders. We need the help of WHO to communicate about the existence of the LabNet as well as inform health authorities, infectious disease control agencies about the WHO HPVLabNet and the possibilities of Laboratory Surveillance for promoting effective HPV control by vaccination. 6
7 Specific activities conducted by Global Reference Laboratory, Sweden.Please indicate and report by referring to the specific TOR of your laboratory in the TSA. 1. Prepare the 2009 HPV proficiency panel for DNA genotyping and organize the 2009 HPV LabNet proficiency study: Completed, see above and attached report. A manuscript with the results of the 2008 HPV DNA LabNet proficiency study has been submitted to JCM in May Complete confirmatory testing: during this period we have retested samples from 7 RRL, the compilation of results is done in collaboration with GRL USA. 3. Sourcing of sera for developing the IS of anti-hpv 18 antibodies has been done in collaboration with the RRL in Thailand, where GRL Sweden performed the characterization of the sera and RRL Thailand enrolled and bled the donors. The candidate standard was sent to NIBSC in the end of May Evaluation of VLPs for use as reference reagents in LabNet studies has been performed, see above and attached report. 5. The first HPV serology proficiency study was organized; the finalized SOP is included in the WHO HPV Lab manual. A report of the study has been prepared and submitted to WHO, attached. 6. The work on a pilot study for a standardised data-reporting format is ongoing. Experiences from different LabNet members are compiled in collaboration with GRL USA. 7. The HPV Laboratory Manual has been submitted to WHO for review 8. We have taken the lead in HPV LabNet activities regarding International Standards, Proficiency Panels, Confirmatory Testing Schemes, Development of SOPs for inclusion in an HPV Laboratory manual, standardized data reporting format and for performing pilot studies on practical and cost-efficient HPV surveillance strategies based on laboratory testing. We have systematically been transferring the lead of these activities to GRL USA, wherever and whenever possible. GRL USA is today leading the finalization of the Laboratory Manual, Confirmatory Testing Schemes and standardized data reporting format such that we now have a balanced division of work between the GRLs. The collaboration between the GRLs is excellent and the labs work closely together not only on the PI level but also on the Lab Manager level. 9. Provide technical services on surveillance & long-term monitoring of vaccination studies. This is done in close collaborations with the authorities in our country (see above). We aim at designing a very ambitious program that could serve as a role model for other countries. This includes systematic condyloma surveillance and typing, epidemiological studies on HPV prevalence in teenagers and cervical screening populations, routine HPV typing of all cases of vaccine-preventable cancers and safety and effectiveness monitoring. Recommendations to WHO HPV LabNet Please provide any comments, conclusions on this years progress, make any recommendations to WHO for the next year activities. These will be very helpful for understanding the current status of the subject, identifying the gaps/needs, and planning for the next steps in the whole pipeline. Points for improvement: 7
8 -We need to collect a supply of critical reference reagents for HPV antibody testing. These are too difficult and cumbersome to prepare in every lab. Specifically: 1. More well characterized serum samples for annual serology proficiency panels is needed and sourcing needs to be intensified. 2. Virus-like particles of serological grade. These exist for HPV16 in sufficient amounts for HPV LabNet use. VLPs as reference reagents for serological testing for additional HPV types would be essential. In addition, production of the VLPs in amounts that would enable making them globally available would be advantageous. -Extension of the WHO HPV LabNet to include National HPV Reference Laboratories and prioritization of Communication. Many countries have already started vaccination programs and these programs may either be without HPV surveillance programs or may have quite ambitious national HPV surveillance programs that are not part of the WHO LabNet and are not internationally standardized making results from different countries difficult to compare. The possibilities of internationally standardized HPV Laboratory surveillance needs to be communicated to stakeholders and national HPV Reference Laboratories as efficiently as possible and through the most relevant and authoritative channels. Although the LabNet can help in writing scientific publications that appear in MedLine, by the NewsLetter and by posting material at the LabNet homepage at HQ, more authoritative and professional communication strategies would be required, particularly when it comes to reaching infectious disease control agencies, health policy makers and official national HPV reference laboratories that are designing HPV laboratory surveillance programs. Proposed specific activities for the next year Please propose next year workplan regarding "specific activities" your laboratory will conduct pertain to the mission of HPV LabNet. This will be subject to discussion/modification and servicing as the basis for the next year TSA. For any activities have not been implemented against the TORs, please indicate any reasons and further action. We have tried to structure the proposed tasks in a Table, below: 8
9 SUB- PROJECT What should be done PROJECT Time-line DNA proficiency panel 2010 Preparation of panel The 4 th DNA proficiency panel 2010 will have the same content as the 3 rd panel 2009 with differences in the mixed samples and randomisation. It will also include 3 DNA extraction controls. April May 2010 Distribution The panel should be distributed in June June 2010 SOP for HPV LabNet proficiency study AND for confirmatory testing Preparation of report Draft Circulation in LabNet Finalization All laboratories should send the results back within 4 weeks. Report to WHO It's now mature to formalize a SOP for conducting the HPV LabNet proficiency study as a formal document/practice which shall include principles, panel preparation, evaluation, distribution, study organization, data reporting, analysis, criteria, summary report, feedback to labs, i.e. all critical elements involved in the study. This is to keep consistency of the studies conducted in a standard manner. September 2010 December 2010 June 2010 Laboratory manual Submit to WHO as a final version The manual has been submitted to WHO for review. If changes are required, to take part in the editing and revision of the manual. Done by Dec 2010 International reference sera for HPV 18 VLP-ELISA HPV 18 Neutralisation assay Format for data reporting Evaluation in collaborative study Candidate serum pool sent to NIBSC Collaborated with NIBSC in design and evaluation of international collaborative study to validate the HPV 18 International Standards serum Stability study of selected reference reagent of HPV 18 VLPs, both when stored in solution and when coated on plates. Organised the first international proficiency study for HPV 18 serology, within the LabNet Testing using serum from the HPV16 serology proficiency study. Comparison of results with other LabNet labs (study coordinated by GRL CDC) Assist GRL/CDC in finalizing SOP A common format to register and compile HPV surveillance data from different countries will be developed at the global reference laboratories and trialled in LabNet. Report form to be optimized and finalized. Submit to WHO. Task transferred to GRL USA, but we intend to collaborate with GRL USA in ensuring that the project is progressing. May 2010 December 2010 December 2010 September 2010 Initiated in May 2009 Confirmatory testing The goal is to report data to WHO on a yearly basis (in a standardised format) Will retest samples that have been sent in by LabNet labs. All laboratories should send; all HPV negative cervical cancers, all HPV X and 100 random CIN 3 or CxCa. Additional tasks: *Assist WHO and NIBSC in efforts to develop standard supplies of 1. Positive and negative control monoclonal antibodies. 2. Virus-like particles of serological grade. We can do advise and quality control testing. 9
10 LIST OF ENCLOSURES 1. Report of the First International HPV 16 serology proficiency study 2. VLP reference reagent validation report HPV DNA proficiency study report HPV DNA proficiency study scientific article 10
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