APHL seeks to select 3 public health laboratories to serve as a National Influenza Surveillance Reference Centers providing influenza virus isolation, neuraminidase inhibition and whole genome sequencing testing services.
Proposals will be evaluated based on the laboratory’s existing and/or potential capability and capacity to address the following testing services and requirements:
- Influenza virus isolation (with in-house cell culture)
- Neuraminidase inhibition testing
- Influenza whole genome sequencing (WGS)
- Ability to access and use the APHL AIMS environment for WGS data transfer and analysis
- Database Management
Request for Proposal Materials
National Influenza Surveillance Reference Center RFP provides details on how to complete this RFP. The RFP contains a copy of the scoring criteria that will be used for evaluation of proposals in Appendix C.
Selected Evaluation Team
The National Influenza Surveillance Reference Center RFP evaluation team has been selected and approved, as indicated in the RFP, by APHL’s Senior Director of Public Health Systems.
There are four (4) selected reviewers from CDC’s Influenza Division:
- Three (3) laboratory team leads that are CDC subject matter experts in the test methods included in the RFP
- One (1) acting Associated Director of Laboratory Science for the Influenza Division
There are four (4) selected reviewers from APHL’s membership:
- One (1) Deputy Laboratory Director
- Two (2) virology supervisors/experts from public health laboratories
- One (1) APHL Influenza Subcommittee member that was formerly at a public health laboratory and remains an expert in the field of influenza test methods and laboratory leadership
Questions and Answers
Q: Can we get a copy of the relevant protocols for this project?
A: Yes, please contact
Stephanie Chester directly to get a copy of the protocols. Please note that we are only able to share these protocols for informational purposes for the RFP (i.e., to inform budgetary and technical expertise requirements). These protocols are subject to change at any time; selected reference centers will be given updated protocols at the beginning of each season and at other times as necessary.
Budget and Training
Q: When drafting a year 1 budget, should it include both indirect and direct costs?
A: Both the VIP and NI projects are reimbursed on per specimen rates which include both direct and indirect costs. During initial implementation of WGS, reimbursement will be based on the direct costs required to implement the test method as well as flat rate reimbursements to cover staff time and indirect costs. Once a PHL has established WGS capabilities and starts surveillance testing, it will be reimbursed on a per specimen basis with direct and indirect costs included.
Q: For the application on page 6 item 11, it only asks for a Year 1 budget. However, on page 4 under Award, it states that the #2 reference center may be granted up to $300,000 additional for WGS. For laboratories that do not have existing influenza whole genome sequencing capacity, do we ask for any WGS sequencing needs in the Year 1 budget, or do we ask for those in a separate budget, or is that something that would be negotiated with the lab that is chosen as the #2 lab?
A:We would like to understand what you already have in place that could be leveraged for the project so itemizing that out as existing infrastructure and capabilities is important. For the additional support up to $300,000 that would be negotiated with the chosen laboratory based on what they have in place and what equipment, software, etc. they need to meet the requirements in Appendix A. Therefore if you do not feel you have everything in place to perform sequencing in Year 1, you do not need to include a budget for Year 1 for WGS, but please clearly outline what you would need to bring the capacity on board.
Q: Are laboratories able to use supplies available in the Influenza Reagent Resource (IRR) for reference center testing?
A:No, Reference Center services are to be performed using a per-specimen billing model to account for the costs directly associated with the work.
Q: Should the year 1 budget be based on 1000 specimens?
A:Yes. This will help to compare proposals.
Q: Does the budget for implementing WGS for reference center #2 include IT infrastructure and staff support for building the data transfer pipeline?
A:The financial support listed on page 13 is intended to cover both direct and indirect expenses related to the implementation of WGS including but not limited to equipment, IT support and staff time.
Q: Are FedEx fees and specimen shipping materials included in the budget?
A:The reference centers will use a CDC FedEx account to cover shipping costs. The reference center is responsible for providing shipping containers but CDC will return shippers each week.
Q: Will training for selected reference centers be at CDC or site visits to the selected laboratories?
A:Initial training for laboratories will involve a site visit by the CDC to the selected reference center(s).
Virus Isolation and Neuraminidase Inhibition
Q: If a laboratory submits both viral isolates and matching clinical material, which gets inoculated for virus isolation?
A: In the coming season, the reference centers will inoculate the original clinical material. The reference centers will send the virus isolate along with remaining original clinical material to CDC.
Q: If a lab currently produces culture medium in-house is it acceptable to use the in-house medium rather than the specific vendor and catalog number(s) listed in the CDC SOPs?
A: Yes, if the culture medium maintains acceptable growth, in-house products may be used.
Q: Our lab doesn’t usually heat-inactivate serum as described in the SOP. Is this necessary?
A: As long as the FBS provides acceptable growth for cells, heat-inactivation may not be necessary.
Q: At what point in the cell culture and virus isolation procedures are antibiotics used in the cell culture protocol?
A: The media used during maintaining and passaging un-inoculated flasks does not need antibiotics. However once the flasks are inoculated with specimens/isolates, the media used should contain pen/strep as specified in the SOP.
Q: We routinely test all cells for mycoplasma contamination. May we continue this safeguard to help maintain a mycoplasma-free environment?
A: CDC has no objections to the reference centers performing mycoplasma testing, but it is not required and not directly compensated in the per specimen rate.
Q: The recommended split ratio is 1:10. Is this a hard and fast rule?
A: There can be some flexibility in the split ratio and it may vary between the MDCK and MDCK SIAT 1 cells. In the past, most of the reference centers split and seeded flasks allowing 2-3 days for the monolayers to become confluent before using for inoculations, which is similar to how CDC flasks are prepared. Selected reference centers would be expect to follow a similar schedule when they are preparing flasks for inoculation.
Q: There is a specific vendor named in the SOPs for the T75 flasks. Provided they are specifically those made for cell culture work, is acceptable to use T75 flasks from a different vendor?
A: Any T75 flasks designed for cell culture work should be appropriate.
Q: If an isolate is not submitted, will the reference center attempt to grow the original specimen?
Q: Do the virus isolation protocols require any Mycoplasma contamination monitoring?
A:CDC does not require this in their protocols.
Q: Can selected reference centers use their own MDCK cells or are they required to get new cells from CDC?
A:At the beginning of each season, CDC provides new flasks of low passage MDCK and MDCK SIAT-1 cells. Reference centers are requested to use these new stocks so that all reference centers are using similar passage levels.
Q: Do all specimens get tested in the NI assay?
A:All viruses that grow are expected to be tested in the NI assay. Viruses can be batched on a weekly basis.
Q: What brand of fluorescent plate reader is acceptable for NI testing?
A:There is not a specific brand required.
Records and Reporting
Q: Does CDC provide worksheet templates or are the reference centers expected to create worksheets? Are the worksheet completed electronically or by hand?
A: CDC provides all necessary templates. Most worksheets are completed electronically. For the virus isolation project, there are some forms that require handwriting (e.g. daily CPE checks).
Q: Are there standard templates for weekly and monthly reports?
A:For the weekly reports, there is currently a query built into the Access database that will generate the report. For the monthly reports, the reference centers have to generate their own reports but there is a standard list of data to report on.
Q: To prevent duplicate work, can you upload a spreadsheet from the Access database or FluLIMS to the internal PHL LIMS or vice versa?
A:FluLIMS has the ability to ingest data from several different sources because CDC utilizes messaging to transfer data. Whether the PHL LIMS can upload a spreadsheet will depend on the capability of the participating laboratory LIMS. CDC will utilize time saving data management processes involving LIMS and messaging when practical and feasible.
Q: What correspondence with submitting laboratories are the reference center responsible for?
A:Reference centers are responsible for sending an acknowledgement of specimen receipt. The acknowledgement of receipt is currently a Word letter generated with data inserted from the Access database and then manually emailed to the submitter. There are some ad hoc communications that will occur between the reference centers and submitting laboratories to clarify specimen submission information. CDC is responsible for sending final reports including antigenic characterization to submitting laboratories.
Whole genome sequencing (WGS)
Sequencing and analysis
Q: For WGS, is the goal to sequence original clinical material or viral isolates?
A: The goal is to sequence original clinical material. Viral isolates should be sequenced if it is the only available material.
Q: What extraction platform is required?
A: No specific kit or platform is required at this time. CDC is still exploring the best option for extraction performance and cost effectiveness. This will be determined during the pilot phase. For proposal and budgetary purposes, you can include your routine extraction methods either manual or automated.
Q: Why are the PCR extension times so long in the WGS protocol? Are increasing extension times necessary?
A: The long extension times dramatically improve the larger segments and the ratio of larger to smaller segments. The amplification protocol was designed to not be too specific in order to potentially accommodate other needs, such as emerging viruses. Any changes made to the PCR protocol would be made across all reference centers. CDC is amenable to working with selected reference centers to explore methods that improve efficiency and effectiveness during pilot phases and over time, but consistency across centers will be required.
Q: What equipment is needed for checking PCR fragment sizes and library quantification?
A: CDC uses QIAxcel, which has broad range capillaries that can be used for verifying that there are 8 amplified segments and determining the size of the library. QIAxcel is not necessary for the protocol; but some kind of fragment analyzer is needed.
Q: Will submitters provide the Ct value of each specimen?
A:Specimen submitters will send a specimen submission form. Currently, the Ct value can be added in the comment column on this form, but with the addition of WGS CDC and APHL may make Ct values a required field.
Q: Is there specific criteria for accepting sequencing results based on depth of coverage?
A:Ideally we desire 200-500 X coverage over every position (at least 100X).
Q: Are the MiSeq instruments expected to be dedicated exclusively to Influenza during runs?
A:While, it may not be critical to have a dedicated instrument for this task, the influenza WGS protocol is streamlined and it may be more difficult to coordinate multiple protocols and sample indices and data transmission. Thus we prefer that the run used is dedicated to the influenza projects. This is something that CDC and the selected reference centers can further evaluate/coordinate.
Q: How much bioinformatics analysis will be required of the selected reference centers?
A:Existing bioinformatics expertise is not a requirement to be selected as a reference center. APHL and CDC are planning to make bioinformatics software available to reference centers in the AIMS environment for limited analysis in the future. We anticipate that reference center involvement in bioinformatics analysis will evolve over time.
Data storage and transfer
Q: What size file is generated by WGS?
A: An approximately 13 GB file is generated for each MiSeq Run (i.e., 96 samples).
Q: What would labs use for data transfer at the beginning of the project if AIMS streaming is unavailable?
A: Labs can use a FTP site to get data to AIMS as a temporary solution.
Q: How much local storage is required for WGS?
A:Local storage is only necessary to serve as a backup if there is an issue accessing AIMS. Therefore, a terabyte of local storage is sufficient.
Q: How automated will WGS data transfer be?
A:The goal is to automate streaming of data from the MiSeq to AIMS. Specifications for this capability will be defined as the project continues.
Q: Until WGS data streams automatically to AIMS, is the entire data set expected to be uploaded to AIMS weekly?
A:The data is expected to be uploaded within 48 hours of completing a run. However, it should be noted that reference centers are allowed to batch on a bi-weekly basis for WGS testing.
Q: How many datasets do you estimate will be transmitted per week? How many genomic datasets do you create currently?
A:We estimate one dataset from a full run per week. However, reference centers will be permitted to batch specimens and perform WGS on a bi-weekly basis when specimens are limited.
Q: Does the WGS raw data file stay the same size while being transferred?
A:The WGS raw data files change in size throughout the transport process. This is due to encryption and decryption. The file will be the same size when it is delivered to the intended destinations.
Q: Can you define the connection required to access AIMS? Specifically, do these methods utilize a domain or a static IP address?
A:To access AIMS, a static IP or specific domain are not required. Several levels of authentication are required for incoming connections including an authentication certificate provided by the AIMS Platform Team.
Q: Will the S3 client be installed on a server or individual computer?
A:The AIMS S3 Client can be installed on either an individual computer or server. A lightweight utility, similar to an SFTP client is installed and used for secure data transport.
Q: At what stage of development are FluLIMS and the WGS data transfer system?
A:FluLIMS will only be used internally by CDC. Reference centers will be able to use a web-based accessioning tool that will be hosted in AIMS and accessible via Internet or web browser. Eventually, sample accessioning information will be transferred from the tool to FluLIMS via the AIMS messaging hub. Both CDC and reference centers will be able to login to the web-based tool to track and process samples and associated meta-data. Individual reference centers will only be able to access their center’s data.
Q: Is there any standardized health messaging occurring in relation to the WGS testing?
A:No, at this time data transmission only involves the sharing of raw sequence data files (e.g. FASTQ files). There is no HL7 messaging required for this project at this time.
Q: How long do you need to store the WGS datasets online? If you archive the datasets, how long do you need to keep the archived data?
A:CDC and APHL will maintain archives of the data on AIMS, it is not a responsibility of the reference centers. Data will be moved to archives after it has been sufficiently processed and analyzed, likely 2-3 weeks after it is uploaded to AIMS. Both CDC and the reference center will have the ability to call back data from archives (i.e., Amazon Glacier).
Can you please send me the technical specifications and implementation guides for all 3 of the data transmission options for AIMS as reference on page 13 of the RFP?
A:Connection to the AIMS Platform is required due to the need to utilize the web tool for accessioning and the Clarity LIMS for data management and Quality Control. The preferred method for transport is the AIMS S3 Client, but there are additional options if desired as described below.
PHINMS (Public Health Information Network Messaging System) is the CDC-provided software- employing Electronic Business using Extensible Markup Language (ebXML) technology, PHINMS can securely send and receive any message type over the Internet, facilitating interoperability among myriad public health information systems. PHINMS is widely used by all jurisdictions for use cases like PHLIP, LIMSi and ELR. Ease of use and status of current implementation is a plus but lack of large data file transfer speeds is a critical factor to consider when selecting the transport protocol.
PHINMS Implementation Guides and Additional Information
Direct Secure Messaging:
Direct secure messaging (DSM) is a secure, encrypted web-based communication system for physicians, nurse practitioners, physician assistants, and other healthcare providers to share protected health information (PHI). Although not optimal for this project, it is available as a transport option.
Additional information on Direct Secure Messaging and the ONC Direct Project