NIH Human Microbiome Project - Core Microbiome Sampling Protocol A (HMP-A)

The Human Microbiome Project, a program initiated under the NIH Roadmap, is sponsoring studies to characterize the human microbiome and analyze its role in human health and disease.

The NIH Human Microbiome Project is sponsoring this study. The HMP Core Microbiome Sampling clinical protocol team leaders at the National Institute of Dental and Craniofacial Research (NIDCR) and the National Human Genome Research Institute (NHGRI) will work with the study sites to develop and implement the study in accordance with Good Clinical Practices. Contact information for these individuals and other facilities providing study support is listed on the protocol roster and in Appendix B of this MOP.

The Study Steering Committee is composed of the Study Chair, the Principal Investigator at each of the two clinical sites, and representatives from the NIH.

Study Management lies primarily in the hands of the Steering Committee. The Principal Investigators (responsibilities listed in Section 1.4) will make decisions regarding conduct of the trial with the endorsement and approval of the Steering Committee.

The Steering Committee will hold regular discussions via conference calls on the overall strategy and progress of the project. The information discussed on these calls will be used to generate reports used to document the overall progress of the study. The Steering Committee will review the progress of specimen collection, DNA isolation, and analysis of the sequence data.

The Steering Committee has the responsibility for developing protocol amendments. Protocol amendments will only be implemented after a formally amended version of the protocol has been approved and written IRB approval for the amendment is obtained (see Section 1.5).

The Steering Committee has responsibility for publishing the results of this study. The publication will be in accordance with the publication policy listed in Section 1.6.

• To ensure that he/she has sufficient time to conduct and complete the study and have adequate staff and appropriate facilities which are available for the duration of the study and to ensure that other studies do not divert essential subjects or facilities away from the study at hand.
• To submit an up-to-date curriculum vitae and other credentials (e.g. medical license number in the United States) to the NIH and, where required, to relevant authorities.
• To submit relevant IRB documentation and correspondence to the NIH.
• To acquire the normal ranges for laboratory tests performed locally and, if required by local regulations, obtain the Laboratory License or Certification.
• To conduct the study in compliance with the protocol and appendices.
• To cooperate with the clinical site monitors in the monitoring process of the study.

No changes to the study protocol will be allowed unless they have been discussed in detail and have received the concurrence of the Steering Committee.

Any amendment/modification to the protocol will be adhered to by the participating center(s) and will apply to all subjects. Written IRB approval of protocol amendments is required prior to implementation; modifications are submitted to the IRB for information only.

Timely communication with the scientific community is one of the essential functions of the Steering Committee, and is accomplished by the publication of manuscripts in scientific literature and oral or poster presentations at scientific meetings. Several publications and presentations will emanate from the Steering Committee, but most are of the following types: 1) the core manuscripts reporting results of the study, i.e., those that cover primary, secondary, and other objectives of the study (core protocol publications/presentations); 2) specialized laboratory studies or other investigations that are not central to the objectives of the study (ancillary study publications/presentations); and 3) general reviews.

The publication policy of the Steering Committee is meant to be flexible and to facilitate rapid and accurate publication of results. Investigators are responsible for drafting the publications and presentations with meaningful input from sponsors. Internal review of manuscripts and abstracts is deemed necessary to ensure accuracy and consistent representation of concepts and data from the clinical trials. The procedures outlined herein are guidelines and all publications of the Steering Committee must meet the criteria for authorship, disclosure, scientific integrity and other requirements of peer-reviewed scientific journals.

The Steering Committee members will be the authors of the core manuscripts. Other manuscripts and abstracts are drafted by writing teams whose members must meet guidelines pertinent to the scientific standards of authorship. Masthead authorship should reflect those individuals whose scientific contributions to the publication were essential to its conduct. Contribution of the laboratory, the Clinical Data Coordinating Center and NIH should also be reflected.

The Steering Committee has the primary responsibility for carrying out editorial review of publications. While the sponsors may require up to 30 days for review of proposed publications, efforts will be made to accommodate the timely review of manuscripts and abstracts.

Subject Study IDs are not to be used in any publications.

Media inquiries and press releases should be referred to the NHGRI Communications and Public Liaison Branch:

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This office should approve any press releases and responses to inquiries. Local media activity should be coordinated with the NHGRI Office of Communications.

Manuscripts/publications derived from this study are to be sent to PubMed Central in accordance with NIH Public Access Policy (Consolidated Appropriations Act 2008).

This manual is prepared and distributed by the CDCC. Contact the Data Project Manager, ████████████████████ or by phone at: ██████████, with any questions about this manual.

This manual will be reviewed at least annually and updated as necessary. When the MOP is updated, the new version will be posted on the study web site, accompanied by a memo describing the revisions. If there is a discrepancy between the current MOP and the protocol, refer to the current protocol and notify the NIH representative and the CDCC. See the study web site for previous archived versions of the MOP.

This NIH-sponsored study will be conducted in accordance with its protocol, the policies and procedures described in this document, in compliance with Good Clinical Practice (GCP) as laid out in the International Conference on Harmonisation (ICH) E6 GCP Consolidated Guidance (ICH 1996), and in accordance with applicable regulatory requirements.

NIH studies will be conducted according to the principles of respect for persons, beneficence, and justice as stated in the Belmont Report. This study will also embrace the principles set forth in the Declaration of Helsinki. Investigators will comply with the provisions for the protection of the rights and welfare of human research subjects set forth in the U.S. Code of Federal Regulations, Title 45 Part 46, and in compliance with determinations of all Institutional Review Boards (IRBs) overseeing the research. All institutions participating in the protocol will have in place a Federal Wide Assurance (FWA) with the DHHS Office for Human Research Protections (OHRP). The assurance documents the institution’s commitment to the human subjects regulations.

The study will also be conducted under Good Clinical Practice (GCP) as laid out in the International Conference on Harmonisation (ICH) E6 GCP Consolidated Guidance (ICH 1996). Investigator responsibilities are set out in Section 4 of the E6 Guideline (as published in the Federal Register May 1997). Sponsor responsibilities are set out in Section 5 of the E6 ICH Guideline (as published in the Federal Register May 1997).

Investigators should understand their commitments set forth in these regulations and standards. Investigators should also be familiar with the reporting requirements of their IRBs. We recommend that investigators maintain a copy of their IRB policies as part of the study file.

The study protocol, informed consent documents and all types of subject recruitment or advertisement information must be submitted to the IRB for review and must be approved prior to study initiation. Any amendments to the protocol and/or consent form must also be approved by the IRB prior to implementing any changes in the study.

The investigator is responsible for keeping the IRB apprised of the progress of the study and of any changes made to the protocol as deemed appropriate, but in any case, at least once a year. The investigator must also keep the IRB informed of any serious adverse events, unanticipated problems, or protocol deviations resulting in serious or severe adverse events.

To protect the privacy of study subjects, the Study ID code list should be maintained in a secure location that is separate from the regulatory binder. Copies of any identifying documents, e.g., documents containing the subject’s name, should also be maintained in a secure location that is separate from both the regulatory binder and the subjects’ individual study binders.

If a revised protocol or amendment is submitted to the IRB, the version number and date must be cited and the submission must be recorded in the study regulatory file. All protocol submissions must include the full protocol. Protocol submission should follow local IRB policies.

Any reports of SAEs or unanticipated problems received by the site from NIH must be submitted to the IRB.

Informed consent is an ongoing process that begins with the first contact with a prospective subject and continues until the study is completed. The consent form provides information about the study, including the rights of the subject and the risks and benefits involved in participating in the study. The consent form also documents the subject’s agreement to participate. All procedures, subject obligations, and subject rights should be explained to the subject in easily understood language. During the explanation of the study and during the actual study, the subject is entitled to privacy and respect. The investigator or a designee may present the information and administer the consent. The investigator/designee should be well versed in the protocol and able to answer questions about the study procedures. The investigator/designee presenting the study should encourage the prospective subject to ask questions during this introduction to the study and anytime during his/her participation. Following the information presentation, the administrator should feel confident that the subject understands the study before the consent form is signed and before final inclusion into the study.

A sample consent form is provided for the protocol. Sites may modify the sample consent form as necessary for submission to the local IRB, but no information from the risk section may be deleted from the sample consent form. The site specific consent form will be reviewed and approved by NIH, prior to submission to the site IRB.

A copy of the current IRB-approved consent form will be used to obtain informed consent from the subject. The consent form must be signed by the subject before participation in any study-related activities. A copy of the signed and dated consent form must be provided to the subject. Signed consent forms must remain in each subject’s study file and must be available for verification by study monitors at all times.

• Subjects should be re-consented if information is changed that might have an impact on their continued participation.
• The signature must be the subject’s legal name.
• The subject should not use initials.
• The signature must be in ink.
• The subject must record the date of his/her own signature. It is not acceptable for the research staff to complete the date for the subject.

A protocol deviation is any noncompliance with the study protocol, Good Clinical Practice (GCP), or protocol-specific Manual of Procedures requirements. The noncompliance may be either on the part of the subject, the Investigator, or the study site staff. As a result of deviations, corrective actions are to be developed by the site and implemented promptly.

These practices are consistent with ICH Good Clinical Practice:

4.5 Compliance with Protocol, sections 4.5.1, 4.5.2, and 4.5.3

5.1 Quality Assurance and Quality Control, section 5.1.1

5.20 Noncompliance, sections 5.20.1, and 5.20.2.

Each investigator must adhere to the study protocol as detailed in the study protocol document. Each investigator will be responsible for enrolling only those subjects who have met all protocol eligibility criteria.

A physician may implement a deviation from, or a change in the protocol to eliminate immediate hazard(s) to study subjects without prior NIH and/or IRB approval. As soon as possible, the implemented deviation or change, the justification and if appropriate, the proposed protocol amendment(s) must be submitted to:

• The IRB for review and approval/favorable decision
• NIH for agreement; and if required
• The regulatory authority(ies)

All deviations from the protocol must be addressed in the study subject source document. The documentation should include the reasons for the deviation and all attempts to prevent or correct them. A completed copy of the Protocol Deviation (PD) Form must be maintained in the Regulatory File, as well as in the subject’s source document file.

It is the responsibility of the site to use continuous vigilance to identify and report deviations within 5 working days of identification of the protocol deviation, or within 5 working days of the scheduled Protocol-required activity.

Protocol deviations must also be reported to the IRB, following the local IRB’s instructions, and documented in the study records. The site PI and study staff are responsible for knowing and adhering to their IRB requirements.

All required regulatory paperwork must remain at the study site (as indicated in the site registration packet received prior to study start) and must be accurately maintained and may be verified during study monitoring visits.

Site Regulatory Documents should include the following:

1. Study Personnel signature/responsibility list
2. Monitor Log and Monitoring Reports
3. Subject Screening/Enrollment log
4. Study ID Code List
5. Original Protocol and Revisions/Amendments
6. Consent Forms (all versions)
7. Blank Case Report Forms (CRFs)
8. Advertisements and Subject Information Materials
9. MOP
10. CVs and Practitioner Licenses
11. IRB Approval - Regulatory Review History
12. Copies of SAE Reports and Unanticipated Problem Reports
13. Copies of Protocol Deviation Reports
14. Laboratory normal values and accreditations
15. Specimen Retention Records
16. Sponsor Correspondence
17. Internal Correspondence
18. Notes to File

The Study Task Delegation and Staff Signature List should be updated regularly for the duration of the study. The objective of this form is to identify site personnel, to describe the responsibilities that the investigator delegates to each person, to establish dates of participation for every person involved in the study, to document signatures and initials of study personnel, and to document the proper training of each team member. The initials of personnel providing training should also be recorded. All personnel listed on the Investigator of Record Form must be listed on this form. Changes include additions or changes in site staff or changes in the responsibilities of already existing staff. More than one person can be responsible for a given task. The original, completed forms should be kept in the site regulatory binder until the closeout of the study. At study close out, a copy of this form should be sent to NIH.

Records and documents pertaining to the conduct of the study, including CRFs, source documents, consent forms, and laboratory test results must be retained by the investigator for a minimum of seven years after the investigation is discontinued or until the NIH authorizes transfer or destruction of study records. No study records will be destroyed without prior authorization from the NIH.

For the purpose of compliance with Good Clinical Practice and Regulatory Agency Guidelines, it may be necessary for NIH or their designees to conduct a site monitoring visit. This may occur at any time from the start of the study to after conclusion of the study.

The source document is defined as the first place the data are recorded. The CDCC will provide source documents derived from the eCRFs. Blank source documents are posted to the study web site. In some instances, staff might need documentation from their own or other institutions (e.g., laboratory reports or a hospital report for an SAE). In this case, please request a copy of the record from the institution. It is also recommended that copies of records from outside the clinical research site be added to the subject’s binder.

All source documents should be completed by the clinician (or other appropriate study personnel). Data entries into source documents should be made in blue or black ink. Corrections should be made with a single line through the entry and the change initialed and dated. Original entries should remain legible (i.e., they should never be erased or covered with correction fluid to obscure the original entry). Late entries, e.g., laboratory results on the Eligibility Checklist, should be initialed and dated at the time entered.

Data should be handled in accordance with GCP, U.S. federal regulations, local regulations (if applicable), and instructions from NIH. All source documents should be filled out completely by the examining personnel or the study coordinator and should be signed by the person collecting the data on that form. The source documents are reviewed, signed and dated by the principal investigators or study staff designated by the principal investigators.

Source documents for subjects who are screened but not enrolled must be retained following the same guidelines as other study source documents.

Data will be entered electronically over the Internet by site study staff into AdvantageEDC, developed and maintained by The EMMES Corporation. Instructions for use of the system and completion of the data screens (eCRFs) for each study are included in the AdvantageEDC User’s Guide, which is posted to the study web site. Data entry should be current to within 72 hours of the last visit for each subject unless otherwise specified.

AdvantageEDC includes an individual Forms Grid, which indicates the current status of forms submitted for each subject. In addition, a Forms Submission Schedule is posted for each protocol on the study web site.

The CDCC reviews data for quality and provides a number of quality assurance reports to ensure that study data are clean and complete. Quality assurance reports will include, but are not limited to, the following: missing forms, missing and out of range values, automated data queries, and targeted manual reviews of study data. Data queries will be posted to the study web site and must be addressed within 1 week after posting unless otherwise specified.

Records and documents pertaining to the conduct of this study, including CRFs, source documents, consent forms, and laboratory test results, must be retained by the investigator for a minimum of 7 years after study completion. Electronic data including clinical information, LIMS (Laboratory Information System) and sequencing data will be transferred to the database of Genotype and Phenotype (dbGaP). No study records shall be destroyed without prior authorization from NIH. The CDCC will provide a copy of the locked data set to NIH upon study completion.

In order to ensure confidentiality of data the following procedures will be followed:

• Label both the source documents and the specimens with code numbers.
• Enter data from the source documents in a controlled-access database on the Internet, identified only by code number. Only researchers who have been approved to look at the information by an NIH Data Access Committee will be able to see this information.
• Store all source documents and consent forms in a locked file cabinet. Only members of the study team will have access to this file cabinet.

The Office for Human Research Protections (OHRP), Department of Health and Human Services (DHHS) considers unanticipated problems, in general, to include any incident, experience, or outcome that meets all of the following criteria:

• unexpected (in terms of nature, severity, or frequency) given (a) the research procedures that are described in the protocol-related documents, such as the IRB-approved research protocol and informed consent document; and (b) the characteristics of the subject population being studied;
• related or possibly related to participation in the research (possibly related means there is a reasonable possibility that the incident, experience, or outcome may have been caused by the procedures involved in the research); and
• suggests that the research places subjects or others at a greater risk of harm (including physical, psychological, economic, or social harm) than was previously known or recognized.

An incident, experience, or outcome that meets the three criteria above generally will warrant consideration of substantive changes in the research protocol or informed consent process/document or other corrective actions to protect the safety, welfare, or rights of subjects or others. Examples of corrective actions or substantive changes that might need to be considered in response to an unanticipated problem include:

• changes to the research protocol initiated by the investigator prior to obtaining IRB approval to eliminate apparent immediate hazards to subjects;
• modification of inclusion or exclusion criteria to mitigate the newly identified risks;
• implementation of additional procedures for monitoring subjects;
• suspension of enrollment of new subjects;
• suspension of research procedures in currently enrolled subjects;
• modification of informed consent documents to include a description of newly recognized risks; and
• provision of additional information about newly recognized risks to previously enrolled subjects.

Institutions engaged in human subjects research conducted or supported by DHHS must have written procedures for ensuring prompt reporting to the IRB, appropriate institutional officials, and any supporting department or agency head of any unanticipated problem involving risks to subjects or others (45 CFR 46.103(b)(5)). Furthermore, for research covered by an assurance approved for federal wide use by OHRP, DHHS regulations at 45 CFR 46.103(a) requires that institutions promptly report any unanticipated problems to OHRP.

Incidents or events that meet the OHRP criteria for unanticipated problems require the completion of an unanticipated problem report form. OHRP recommends that investigators include the following information when reporting an adverse event, or any other incident, experience, or outcome as an unanticipated problem to the IRB:

• appropriate identifying information for the research protocol, such as the title, investigator’s name, and the IRB project number;
• a detailed description of the adverse event, incident, experience, or outcome;
• an explanation of the basis for determining that the adverse event, incident, experience, or outcome represents an unanticipated problem;
• a description of any changes to the protocol or other corrective actions that have been taken or are proposed in response to the unanticipated problem.

For multicenter research protocols, if a local investigator at one institution engaged in the research independently proposes changes to the protocol or informed consent document in response to an unanticipated problem, the investigator should consult with the study sponsor or coordinating center regarding the proposed changes because changes at one site could have significant implications for the entire research study.

Forms describing unanticipated problems will be submitted to the CDCC and the IRB per site requirements. The CDCC will notify the NIH study representative of the unanticipated problem. Unanticipated problems will be reported immediately to the NIH Clinical Study Oversight Committee (CSOC). The CSOC may convene an ad hoc meeting for review of the unanticipated problem and consideration of corrective actions. Other supporting documentation of the problem may be requested by the CSOC and should be provided as soon as possible. A summary of the CSOC meeting and proposed actions will be provided to the study Steering Committee. The study PIs will then submit the CSOC review and any protocol changes to the IRBs.

Investigators will be responsible for reporting unanticipated problems from the time that the first subject is enrolled until one year after the final subject is enrolled.

The OHRP, DHHS defines an Adverse Event (AE) as any untoward or unfavorable medical occurrence in a human subject, including any abnormal sign (for example, abnormal physical exam or laboratory finding), symptom, or disease, temporally associated with the subject’s participation in the research, whether or not considered related to the subject’s participation in the research (modified from the definition of adverse events in the 1996 International Conference on Harmonisation E-6 Guidelines for Good Clinical Practice). A Serious Adverse Event/Experience (SAE) is any adverse event/experience that meets any of the following criteria:

• Results in death
• Is life-threatening (places the subject at immediate risk of death from the event as it occurred)
• Requires inpatient hospitalization or prolongation of existing hospitalization
• Results in a persistent or significant disability or incapacity
• Results in congenital anomaly/birth defect
• Any other adverse event that, based upon appropriate medical judgment, may jeopardize the subject and may require medical or surgical intervention to prevent one of the other outcomes listed in this definition.

For those events meeting the previously described definition of Serious Adverse Events, the completion of an SAE form is required.

OHRP considers adverse events that are unexpected, related or possibly related to participation in research, and serious to be the most important subset of adverse events representing unanticipated problems, because such events always suggest that the research places subjects or others at a greater risk of physical or psychological harm than was previously known or recognized, and routinely warrant consideration of substantive changes in the research protocol or informed consent process/document or other corrective actions in order to protect the safety, welfare, or rights of subjects. Furthermore, OHRP notes that IRBs have authority to suspend or terminate approval of research that, among other things, has been associated with unexpected serious harm to subjects (45 CFR 46.113). In order for IRBs to exercise this important authority in a timely manner, they must be informed promptly of those adverse events that are unexpected, related or possibly related to participation in the research, and serious (45 CFR 46.103(b)(5)).

All serious adverse events will be:

• recorded on the appropriate Serious Adverse Event case report form and faxed to the CDCC at 301-251-1355. [The CDCC will notify the NIH study representative of the serious adverse event within one business day.]
  • All deaths and immediately life-threatening events, whether related or unrelated, will be reported via fax within 24 hours of site awareness.
  • Serious adverse events other than death and immediately life-threatening events, regardless of relationship, will be reported via fax within 72 hours of site awareness.
• followed until satisfactory resolution or until the Principal Investigator or Sub Investigator deems the event to be chronic or the subject to be stable;
• reported to the IRB per site requirements;
• entered in AdvantageEDC on the appropriate eCRF;
• reported to and evaluated by the Independent Safety Monitor (ISM).

  SAEs that are related to study participation will be reported immediately to the NIH CSOC. The CSOC may convene an ad hoc meeting for review of the SAE and consideration of corrective actions. Other supporting documentation of the event may be requested by the CSOC and should be provided as soon as possible. A summary of the CSOC meeting and proposed actions will be provided to the study Steering Committee. The study PIs will then submit the CSOC review and any protocol changes to the IRBs.

  Serious adverse events that are considered unrelated to study participation will be provided to the CSOC in a line listing to be reviewed at regularly scheduled meetings.

  This study will employ an unsolicited SAE reporting system. Subjects will be counseled to report SAEs as listed in Section 4.3.

Questions about SAE reporting can be referred to █████, the NIDCR Safety Coordinator or █████, Medical Monitor. Contact information for these individuals and for the study site independent safety monitors (ISM) follows:

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The Clinical Study Oversight Committee (CSOC), an independent group of experts, will advise NIH and study investigators on this study. The responsibilities of the CSOC are to 1) monitor human subject safety by reviewing and evaluating the accumulated study data, 2) review study conduct and progress, and 3) make recommendations to NIH concerning the continuation, modification, or termination of the study. The CSOC considers study-specific data as well as relevant background information about applicable procedures and progress of the study.

Prior to the first data review and preferably prior to study initiation, the CSOC will define its deliberative processes. These may include event triggers or a process that would call for an ad hoc review, milestones expectations, endpoint analysis, and voting procedures. The CSOC is responsible for maintaining the confidentiality of its internal discussions and activities as well as the contents of reports provided to it.

The CSOC will review the protocol, including the oversight and risk monitoring plan, and identify any major concerns prior to implementation. During the study the CSOC will review:

• Real-time and cumulative safety data for evidence of procedure-related serious adverse events
• Unanticipated problems involving risks to subjects or others (for additional information, see the online guidance document from the Office for Human Research Protections, available at http://www.hhs.gov/ohrp/policy/AdvEvntGuid.htm)
• Adherence to the protocol
• Factors that might affect the study outcome or compromise the study data, such as protocol violations, losses to follow-up, breach of subject confidentiality
• Unexpected barriers, if any, to study progress or completion, such as slow enrollment, new data or findings, other milestones, change in resources, futility of endpoints, etc.

The CSOC will conclude each review with each member’s recommendation to NIH as to whether the study should continue, be modified, or be terminated. Recommendations regarding modification of the design and conduct of the study may include corrective actions when performance is unsatisfactory, recommendations to suspend or terminate enrollment, or recommendations to modify consent documents.

Confidentiality must always be maintained during all phases of CSOC review and deliberations.

The membership of the CSOC will reflect the disciplines and clinical specialties, such as dental, medical, and nursing, necessary to interpret the data from the clinical study and to fully evaluate subject safety. The CSOC will consist of at least three voting members. Membership will include an Independent Safety Monitor (ISM) from each of the participating sites, an individual with expertise in the clinical aspects of the subject population being studied or procedures being performed, and an individual with expertise in current clinical research conduct and methodology.

Consideration will be given to including other voting or ad hoc members, such as a bioethicist, if it is considered that the study design or subject population would benefit from this expertise. CSOC and ad hoc members may be from the principal investigator’s institution or from other participating sites, but will not be directly involved with the study or under the supervision of the study investigator. Furthermore, the CSOC members should be in a different organizational group than the Principal Investigator (PI).

NIH staff not involved in the study may also participate as voting members. NIH staff involved in the study may participate as ex officio, non-voting members. Individuals with vested interests in the outcome of the study are not eligible to serve on the CSOC as ex officio or voting members.

The voting members of the CSOC for this study are:

See Section 4.4 for contact information for the ISMs.

The initial CSOC meeting will occur before the start of the study or as soon thereafter as possible. NIH staff may discuss the NIH perspective on and expectations for the study at this initial meeting. At this meeting the CSOC will discuss the protocol, set triggers for data review, define a quorum, and establish guidelines for monitoring the study. The CSOC will decide which member(s) should receive reports of serious adverse events and unanticipated problems in real time and determine if on-site review of clinical records might be needed. Guidelines for stopping the study for concerns of risk or other factors will be established. At this meeting, the CSOC will also develop procedures for conducting business (e.g., data required for review, voting rules, attendance, etc.). Teleconference calls may be an appropriate means for conducting meetings.

Based on initial discussions, the CSOC will decide whether to meet on a regular basis or only with the occurrence of serious adverse events or unanticipated problems involving risks to subjects. It may be appropriate for meetings to be convened on an ad hoc basis, in response to the occurrence of serious adverse events or unanticipated problems involving risks to subjects. Scheduling of meetings will be based on the magnitude of the perceived risks, rate of subject enrollment, or problems that occur during the progress of the study.

The NIH is responsible for convening meetings or conference calls as needed. However, meetings may be requested for cause by any member of the CSOC, the investigators, IRB, or NIH. The investigators will be responsible for ensuring the distribution of materials for review to CSOC members and other meeting participants.

It is the responsibility of the PI to ensure that the CSOC is apprised of all new safety and risk information relevant to the study. Summary safety data, enrollment data, and other progress reports (e.g., protocol deviations, site monitoring summaries) will be forwarded periodically to the CSOC. The CSOC will receive all protocol revisions and may receive other documents relating to the study.

Reports will be prepared by the study investigators. The general content for reports to the CSOC is as determined by the CSOC at the initial meeting. The CSOC and NIH must also review and approve the actual data elements to be presented. At each meeting, additions or modifications to these reports may be directed by the CSOC on a one-time or continuing basis. Distribution of written reports should allow sufficient time for review.

Reports for meetings of the CSOC will consist of the Open Session Report. Open Session reports are distributed to CSOC members, selected NIH staff, and other appropriate persons as directed by the CSOC.

Safety and enrollment data will be forwarded periodically to all CSOC members. The CSOC will also receive all protocol revisions and may receive other documents relating to the study, such as annual reports, protocol deviations, manuscripts, and newsletters.

Verbal Report: At the conclusion of a CSOC meeting, the CSOC will discuss its findings and recommendations with NIH representatives and the study investigators. If NIH is not represented at the meeting, the CSOC Chair will contact NIH immediately after the meeting to brief the NIH staff.

Summary Report: The CSOC will periodically issue a written summary report that identifies topics discussed by the CSOC and describes their individual findings, overall safety assessment, and recommendations. This would generally occur after each meeting, but CSOCs that meet on a more frequent basis may summarize more than one meeting in each report. The rationale for recommendations will be included when appropriate. This report will not include confidential information. The CSOC Chair or designee is responsible for preparing and distributing the report. Unless otherwise specified, the summary report will be forwarded through the NIH to a designated study team representative (usually the Principal Investigator) and to other appropriate NIH staff. The study team representative is responsible for disseminating the CSOC summary report to site investigators. Site investigators must, in turn, submit the reports to their respective Institutional Review Boards (IRB) in accordance with local IRB policy.

Immediate Action Report: The CSOC Chair will notify the NIH study representative of any findings of a serious and immediate nature, such as if the CSOC recommends that all or part of the study be discontinued. The NIH study representative will immediately inform appropriate NIH staff, including the Director of OCTOM and the NIH Medical Monitor. In addition to verbal communications, recommendations to discontinue or substantially modify the design or conduct of a study must be conveyed to NIH in writing on the day of the CSOC meeting. This written, confidential briefing may contain supporting data and should include the CSOC members’ rationale for its recommendations. The written briefing should be submitted to OCTOM.

Once the CSOC is established, each of the relevant IRBs will be informed of the operating procedures with regard to data and safety monitoring (e.g., who, what, when, and how monitoring will take place). This information will serve to assure the IRB that the safety of the research subjects is appropriately monitored. If the IRB is not satisfied with the monitoring procedures, it should request modifications. While it is recognized that it may not be possible to satisfy every IRB completely, IRB comments should be considered seriously.

A Screening and Enrollment Log with the list of valid subject Study IDs will be provided to the site by the Clinical Data Coordinating Center (CDCC). Subjects who have documented informed consent by signing a study Consent Form will be assigned study IDs sequentially as they are screened. The Study ID includes a protocol identifier, a site identifier, and a subject identifier.

Examples: 01DBA0001, 01DBA0002

• Protocol identifier: 01D
• Site identifier: Baylor College of Medicine: BA
• Subject identifier: 0001, 0002, 0003, etc.

The Study ID must be entered on all source documents and will be used to identify all subject data records in AdvantageEDCSM. Subject names or other personally identifying information should NOT be recorded on any documents sent to NIH, the Coriell Institute, or the CDCC (e.g., supporting documents for reporting adverse events).

Screening must take place between 2 and 30 days before sampling commences. The time from the first date of screening to the last date of screening may not exceed 30 days. The time from the first date of screening to the date of the final body site specimen collection may not exceed 44 days (allowing 14 days to complete all specimen collections).

• The subject screening visit may occur over multiple days; in this event the date of the first visit should be used for entry into the eligibility checklist as the screening date; study enrollment must occur within 30 days of this date.
• Subjects who fail any screening criterion that, in the opinion of the clinician, could be remediated within the allowed 30-day window between first date of screening and first date of sampling, may be rescreened for that parameter within 30 days. If found eligible, the subject may be enrolled and appointed for sample collection.
  • Example 1: A subject who has untreated dental caries may obtain the necessary dental treatment and return to the study clinic within 30 days. In this case, only the oral screening examination would need to be repeated to determine eligibility for the subject.
  • Example 2: At initial screening, a female subject has a posterior fornix pH of 4.6. The clinician may feel that proximity to the tail-end of menstruation contributes to this pH and that a repeat measurement within the next 30 days has a high probability of falling in the required range of pH below 4.5, thereby making the subject eligible for enrollment and sample collection.

  In both of the above examples, the clinician may elect to defer all of the screening for this subject, or may allow screening of other body sites and schedule a return screening visit as soon as practical (but within the 30-day window) to repeat the screening exam of the body site(s) that failed.

  Appendix M contains a list of topics that should be discussed with female subjects prior to scheduling the screening visit, in order to minimize the number of screening failures related to vaginal pH.

• Subjects whose first date of screening is more than 30 days prior to enrollment must be re-screened before they can be enrolled; consent to participate in the study must be re-confirmed.
• Subjects who fail screening due to time-limited exclusion criteria may be rescreened at a later date to determine eligibility for enrollment; consent to participate in the study must be re-confirmed.

Subjects will be assigned a new Study ID if they are re-screened outside of the allowed 30-day window for completion of screening.

The subject screening process will verify a subject’s eligibility based on all inclusion and exclusion criteria listed in the protocol, and will include the following procedures:

All subjects who are screened for this study should be recorded on the Screening and Enrollment Log provided on the Study Materials page of the study web site. All screened subjects will be entered into the AdvantageEDC Internet Data Entry System (IDES). Those who will participate in the study will be enrolled into the protocol in AdvantageEDC; those who will not participate will be entered as “Screening Failures” in AdvantageEDC.

Follow all safety guidelines of your local safety committee for collection and handling of all biological specimens. Observe Universal Precautions guidelines, including the wearing of gloves, safety glasses or face shield, and a lab coat.

If a subject has a body piercing within the area from which a specimen will be obtained (e.g., tongue, nose), the subject should remove any piercing jewelry or hardware on the day of specimen collection. Study staff should record the presence of a piercing in the applicable comments section of the visit documentation form.

During sampling, each specimen collection tube and container (See App. K) will be labeled with the appropriate, body site-specific, pre-printed clinic label bearing the following information (See App. N for an example of a single subject label set.):

Site staff will write the subject’s study ID number and visit date, and circle the appropriate visit number in the space provided.

Body Site		Specimen	Acronym	Collection Tube
Oral Cavity		1. Saliva	SAL	Screw-top, conical 50 mL tube
	Soft	2. Tongue Dorsum	TONG	750 μL MoBio buffer in MoBio tubes, 2 mL
		3. Hard Palate	HPAL
		4. Buccal Mucosa	BUCC
		5. Keratinized Gingiva (attached)	GING
		6. Palatine Tonsils	PTON
		7. Throat	THRO
	Hard	8. Supragingival Plaque	SUPRA
		9. Subgingival Plaque	SUB
Skin		10. Retroauricular Crease – Left	RAC_L	750 μL MoBio buffer in MoBio tubes, 2 mL
		11. Retroauricular Crease – Right	RAC_R
		12. Antecubital Fossa – Left	ACF_L
		13. Antecubital Fossa – Right	ACF_R
Nasal Cavity		14. Anterior Nares (L & R pooled)	NASAL	750 μL MoBio buffer in MoBio tube, 2 mL
GI Tract		15. Stool	STOOL	Stool kit
Vagina		16. Vaginal Introitus 17. Posterior Fornix	INTRO PFORN	750 μL MoBio buffer in MoBio tubes, 2 mL
		18. Mid-Vagina	MIDVAG
Blood		Whole Blood	COR-1 COR-2	10mL Yellow Tops with ACD Solution A (2)
		Blood for Immune assays	SERUM	10mL Red Top Vacutainer Tube with Anticoagulant

7.3.1. Specimen Collection from the Oral Cavity

7.3.1.1. Sampling Schedule

Specimens will be collected from the oral cavity of each subject at the Baseline Sampling Visit and at the Re-Sampling Visit.

7.3.1.2. Specimen Labeling

Label the specimen collection tubes with the appropriate body site-specific, pre-printed clinic label.

7.3.1.3. Materials Needed

1. Standard dental chair with good lighting.
2. Examination kit (See App. J for kit contents) containing at least one long-handled mouth mirror, dental explorer, surgical scissors, cotton pliers, 2 × 2 gauze pads, cotton rolls, cotton pledgets, wooden tongue depressor.
3. Small (2 mL) screw-top (external thread) MoBio tubes containing 750 μL of specimen collection fluid (MoBio buffer).
4. 50 mL sterile conical polypropylene tube (Falcon) for saliva collection.
5. Body site-specific, pre-printed clinic label set for all oral cavity sites to be collected.
6. Sterile Catch-All™ Specimen Collection Swabs (Epicentre Biotechnologies, Madison WI) to collect specimens from soft tissue surfaces.
7. Multiple sterile Micro Mini Five Gracey curettes (one curette will be used to sample and pool the supragingival plaque from the index teeth, and one curette will be used to sample and pool subgingival plaque from the same index teeth; additional sterile curettes should be available in case they are needed).
8. Stopwatch with second hand.
9. Ziploc plastic bag to hold the MoBio tubes containing the collected specimens.
10. Small ice chest with ice for transport to the clinical laboratory for processing.

7.3.1.4. Collection Methods

Aseptic technique will be used for collection of all specimens (See App. O).

Sequence of collection:

1. Saliva
2. Soft tissue sites*
3. Hard tissue sites

*Sampling of the throat is difficult because of the gag reflex. It is suggested that this sample be obtained as the very last specimen, after all the other soft tissue and hard tissue sites have been sampled.

[Figure from Cochrane Systematic Reviews on Oral Cancer, May 6, 2007]

After each specimen is collected, place the tube in a Ziploc bag and place over ice. Within 4 hours of collection, take to the clinical lab for processing.

7.3.1.5. Saliva

1. Subject is asked to let saliva collect in the mouth for at least 1 minute. The subject is then asked to drool into the labeled 50 mL collection tube (Falcon, sterile conical polypropylene tube with flat-top screw cap). This process may be repeated multiple times in order to collect larger volumes of saliva (2–5 mL).
2. Alternatively, saliva production can be stimulated by administration of a standard piece of unflavored gum base (1.0–1.5 g; Wrigley Co., Peoria, IL). This piece of gum may be placed in the subject’s mouth. The subject is asked to swallow any accumulated saliva and instructed to chew the gum at a regular rate. Upon sufficient accumulation of saliva in the oral cavity, the saliva is drooled into the labeled collection tube. This process may be repeated multiple times in order to collect larger volumes of saliva (2–5 mL).
3. Put the tube in a Ziploc bag and place over ice. Within approximately 4 hours take to the clinical lab for processing.

7.3.1.6. Soft tissue sites

1. All soft tissue sites are sampled using Catch-All™ Sample Collection Swabs. Immediately after swabbing, each swab must be swirled in 750 μL of MoBio buffer in the MoBio tube. The swab sponge should be pressed against the tube wall multiple times for 20 seconds to ensure transfer of bacteria from swab to solution. The specimen in the MoBio buffer should be kept cold until processing.
   1. Tongue dorsum:

      Swab 1 cm2 of the center of the tongue for 5 seconds.

   2. Hard palate:

      Swab the entire hard palate for 10 seconds.

   3. Buccal mucosa:

      Swab the entire area of both left and right buccal mucosa for 10 seconds each. Take care not to touch the teeth.

   4. Keratinized (attached) gingiva:

      Swab the maxillary anterior attached gingiva for 10 seconds.

   5. Palatine Tonsils:

      Swab left and right palatine tonsils for 5 seconds each, concentrating on fissures and pits.

   6. Throat:

      This site is difficult because of the gag reflex. It is suggested that this sample be obtained as the very last specimen, after all the other soft tissue and hard tissue sites have been sampled. Have the subject open mouth and relax tongue; insert depressor all the way to the rear of tongue and depress entire tongue out of the way. Gently swab across the rear of the oropharynx for up to 5 seconds. Do not swab tonsils.

Each of the above soft tissue sites is sampled and vialed separately and labeled with the appropriate body site-specific, pre-printed clinic label.

7.3.1.7. Hard tissue sites

1. Supragingival plaque

   The goal is to sample supragingival plaque from the 6 index teeth: 2 molar teeth (#3 and #19), 2 premolar teeth (#12 and #28) and 2 incisor teeth (#9 and #25).

   Molar teeth: 2 molar teeth must be sampled: #3 – first molar in upper right quadrant, and #19 – first molar in lower left quadrant. If the index first molar has an unrestored mesial surface, sample this tooth. If the index first molar has a restored mesial surface, and the second molar in the same quadrant has an unrestored mesial surface, the second molar should be sampled instead, as the goal is to sample unrestored surfaces as often as possible. If both molars in an index molar quadrant have restored mesial surfaces, then sample the index first molar.

   Premolar teeth: 2 premolar teeth must be sampled: #12 – first premolar in upper left quadrant, and #28, the first premolar in the lower right quadrant. If the index premolar has an unrestored mesial surface, sample this tooth. If the index first premolar has a restored mesial surface, and the second premolar in the same quadrant has an unrestored mesial surface, the second premolar should be sampled instead, as the goal is to sample unrestored surfaces as often as possible. If both premolars in an index premolar quadrant have restored mesial surfaces, then sample the index first molar. If a premolar is missing because of orthodontic extraction, sample the residual premolar regardless of whether the mesial surface is restored or not.

   Incisor teeth: 2 incisor teeth must be sampled: #9 – central incisor in the upper left quadrant, and #25 – central incisor in the lower right quadrant.

   Procedure: For the first tooth, isolate the site to be sampled with cotton rolls and dry with a gentle stream of air from an air-water syringe. With a Gracey curette remove all of the supragingival plaque from the mesial surface of the selected index tooth with as many strokes as necessary. Immerse the curette tip in the MoBio buffer in the MoBio tube for 4–5 seconds. Wipe off the face of the curette on the inside edge of the collection tube. The site can be immediately sampled again using the same procedure. Repeat the process for all the selected index teeth.

   If the subject has little plaque, the arch counterparts to the index molar and premolar teeth should also be sampled. For example, #14 – first molar in upper left quadrant, and # 30 – first molar in lower right quadrant (mesial to distal ends) and #5 – first premolar in upper right quadrant, and #21 – first premolar in lower left quadrant should also be collected (a total of 10 teeth). Replace the lid on the MoBio tube and shake for 4–5 seconds in an attempt to maximize dispersion of the specimen in the fluid.

   Record on the CRF the resulting selection of teeth for sampling.

   Put the tube in a Ziploc bag and place over ice. Within approximately 4 hours take to the clinical lab for processing.

2. Subgingival plaque

   Prior to collection of the subgingival specimens, place cotton rolls, dry the area, and wipe off any residual supragingival plaque. Collect subgingival plaque from the mesio-buccal surface of the selected index teeth with a sterile Gracey curette. Immerse the curette tip in the MoBio buffer in the MoBio tube vial for 4–5 seconds. Wipe off the face of the curette on the inside edge of the collection tube. Discard any specimens with significant amounts of blood, based on clinical judgment (i.e., if flooding occurs). Because of the risk of bleeding, immediate re-sampling of the same site is not recommended. If the pooled plaque is considered insufficient (from subjects with very clean mouths), sample the counterpart index teeth as described above. If the pooled plaque is still considered insufficient, then sample mesio-buccal subgingival plaque of second molars and pool these specimens with the first collected specimens. Replace the cap on the tube and shake for 4–5 seconds in an attempt to maximize dispersion of the specimen in the fluid. Put the tube in a Ziploc bag and place over ice. Within approximately 4 hours take to the clinical lab for processing.

   The supragingival plaque samples are pooled in one MoBio tube. The subgingival plaque samples are pooled in a second tube. Each tube is labeled with the appropriate body site-specific, pre-printed clinic label.

7.3.2. Specimen Collection from the Skin and Nasal Cavity

7.3.2.1. Sampling Schedule

Specimens will be collected from the skin and nasal cavity of each subject at the Baseline Sampling Visit and at the Re-Sampling Visit.

7.3.2.2. Specimen Labeling

Label the specimen collection tubes with the appropriate body site-specific, pre-printed clinic label.

7.3.2.3. Materials Needed

1. Small (2 mL) screw-top (external thread) MoBio collection tubes containing 750 μL of specimen collection fluid (MoBio buffer)
2. Pre-printed clinic label set for all body sites to be collected
3. Sterile Catch-All™ Sample Collection Swabs (Epicentre Biotechnologies, Madison WI)
4. Vial of sterile SCF-1 (50 mM Tris buffer [pH 7.6], 1 mM EDTA [pH 8.0], and 0.5% Tween-20) to pre-moisten Catch-All swabs prior to collection
5. Ziploc plastic bag to hold the MoBio collection tubes containing the collected specimens
6. Small ice chest with ice

7.3.2.4. Collection Methods

Aseptic technique will be used for collection of all specimens (See App. O). A single pair of gloves may be worn to collect all of the skin and nasal cavity specimens from a subject, with care taken to avoid contamination of the gloves. If the gloved hand used to stretch the skin for sampling comes in contact with the sampling area, the glove(s) should be changed before sampling the next site.

Sequence of collection:

• Retroauricular crease (behind ears, left & right sides to be kept separate)
• Antecubital fossa (inner elbow, left & right sides to be kept separate)
• Anterior nares (left & right sides to be combined)

Note: It is helpful for the subject to be comfortably lying down for these sampling procedures.

7.3.2.5. Retroauricular crease

The area of interest begins where the top of the ear joins the face and extends to where the ear lobe connects to the face. Skin surface specimens will be collected with a Catch-All™ Sample Collection Swab, moistened with sterile SCF-1 solution. To access the site, fold the ear forward with one hand to expose the crease. With the other hand, hold the shaft of the swab parallel to the surface of the skin and rub the swab back and forth along the retroauricular crease approximately 50 times, applying firm pressure (this requires approximately 30 seconds). Insert the swab head into the tube containing MoBio buffer. The head of the swab should then be aseptically cut from the handle, and the tube cap should be screwed back in place.

Note: To obtain optimal skin surface specimens, observe the key aspects of the sampling technique: use of the moistened swab, application of firm pressure, and consistency in rubbing (50 times back and forth on the sampling site during 30 seconds).

Right and left sides are sampled and stored separately and labeled with the appropriate body site-specific, pre-printed clinic label. Put the tubes in a Ziploc bag and place over ice. Within approximately 2 hours, take to the clinical lab for processing.

7.3.2.6. Antecubital fossa (inner elbow)

This area is located exactly at the bend of the inner elbow at the junction of the arm and the forearm. Skin surface specimens will be collected with a Catch-All™ Sample Collection Swab, moistened with sterile SCF-1 solution. With one hand, stretch the antecubital skin taut. With the other hand, hold the swab so the shaft is parallel to the skin surface and rub the swab back and forth approximately 50 times along the antecubital crease, applying firm pressure (this requires approximately 30 seconds). Insert the swab head into the tube containing MoBio buffer. The head of the swab should then be aseptically cut from the handle, and the tube cap should be screwed back in place.

Note: To obtain optimal skin surface specimens, observe the key aspects of the sampling technique: use of the moistened swab, application of firm pressure, and consistency in rubbing (50 times back and forth on the sampling site during 30 seconds).

Right and left sides are sampled and stored separately and labeled with the appropriate body site-specific, pre-printed clinic label. Put the tubes in a Ziploc bag and place over ice. Within approximately 2 hours, take to the clinical lab for processing.

7.3.2.7. Anterior nares

With a twisting motion, gently rub the mucosal surfaces of the anterior nares with a sterile Catch-All specimen collection swab, going round the area 2 times.

Right and left sides are sampled and pooled together as a combined specimen and labeled with the appropriate body site-specific, pre-printed clinic label.

Immediately after swabbing, each swab must be swirled in 750 μL of MoBio buffer in the tube. The swab sponge should be pressed against the tube wall multiple times for 20 seconds to ensure transfer of bacteria from swab to solution.

Put the tubes in a Ziploc bag and place over ice. Within approximately 2 hours take to the clinical lab for processing.

7.3.3. Specimen Collection from the Gastrointestinal Tract

7.3.3.1. Sampling Schedule

Subjects will be provided with a stool specimen collection kit at both the screening (for baseline sample collection) and baseline sampling (for re-sampling collection) visits. Subjects will collect stool specimens within a 24-hour period before each sampling visit. Subjects will bring the specimens to the clinic staff at the time of the visit.

7.3.3.2. Specimen Labeling

• Label the specimen collection containers with the appropriate body site-specific, pre-printed clinic label at the time of provision to the subject. (This label should be placed on the side wall of the container, near the tapered bottom. Avoid placing the label in the area where the container will be seated in the collection frame.)
• Label the specimen transport box with the pre-printed stool box label bearing the following information at the time of provision to the subject*:

  e.g.

  Subject ID #: 01DBA0001

  Below to be completed by subject

  Stool collection date: 09/25/08 (ddMMMyyyy)

  Stool collection time: 0830 AM or PM

  Below to be completed by clinical staff

  Visit date: 09/26/08 (ddMMMyyyy)

  Time of receipt: ___ ___: ____ ____ (24 hour clock)

  Staff Initials: _________________

• Upon return of the specimen, site staff will write the date and time the specimen is received and confirm that receipt is within the 24 hour period of collection.

7.3.3.3. Materials Needed

1. One stool collection kit with two stool collection containers (one is for back-up)
2. One Ziploc bag
3. One Thermosafe shipping container
4. Eight to ten polar packs for transport of specimen
5. One roll of packing tape
6. Specimen collection instructions
7. Body site-specific, pre-printed clinic label
8. Stool box label

7.3.3.4. Collection Methods

The following information and the kit will be provided to the subjects. The stool collection containers will be labeled with a body site-specific, pre-printed clinic label at the time of provision to the subject. The containers will be inspected for integrity and cleanliness prior to clinic labeling.

Instructions For Stool Specimen Collection

**Before you collect your specimen, please place all gel packs in your freezer for at least 12 hours. **

Specimen should be collected no more than 24 hours before your clinic visit.

STEP 1. Raise the toilet seat. Place the stool collection frame on the back of the toilet bowl (see Figure 1). All four corners of the collection frame should be supported by the toilet bowl. Place collection bowl in frame (see Figure 2).

Figure 1

Figure 2

STEP 2. Place toilet seat down (see Figure 3). Do not urinate into the collection container*. Deposit your stool directly into the collection container.

If accidental urination occurs, discard the stool and collect a new sample using the second (back-up) container provided.

Figure 3

STEP 3. After collecting your specimen, remove the container from the frame (see Figure 4). Place the container on a flat surface and firmly press the lid closed (see Figures 5 and 6).

Figure 4

Figure 5

Figure 6

STEP 4. Place the closed container into the Ziploc bag (see Figure 7) and seal the bag.

Figure 7

STEP 5. Discard collection frame in trash.

STEP 6. Package your specimen immediately following the instructions below. Write the date and time of collection on the outer box label, and bring it in with you for your baseline sampling visit or re-sampling visit. Your specimen may be stored in the Styrofoam box with gel packs (see below) up to a maximum of 24 hours, prior to your clinic visit.

**Stool Packaging Instructions**

1. Place all seven gel packs in your freezer (see Figure 1). Do not collect your specimen until the gel packs have been in your freezer for at least 12 hours.	FIGURE 1
2. Remove the gel packs from your freezer and place two of the gel packs in the bottom of the Styrofoam box (see Figure 2).	FIGURE 2
3. Place the sealed Ziploc bag containing your stool specimen on top of the two frozen gel packs in the Styrofoam container (see Figure 3).	FIGURE 3
4. Place four of the frozen gel packs around the specimen container so that the container is completely surrounded (see Figures 4 and 5).
FIGURE 4	FIGURE 5
5. Place one gel pack on top of the container (see Figure 6).	FIGURE 6
6. Place the Styrofoam lid on the Styrofoam container (see Figure 7) and close cardboard box.	FIGURE 7
7. Using the tape dispenser provided, seal the middle of the box and both sides of the box (see Figures 8 and 9).
FIGURE 8	FIGURE 9
8. Bring the box with you to your sample collection appointment. [Note: Subject ID# and body site-specific, pre-printed clinic label will be applied to the container at the time of provision to the subject]

7.3.3.5. Specimen Receipt

When subject arrives in the clinic for the specimen collection visit, the stool collection container should be retained in the Styrofoam box with the gel packs while being transported to the clinical lab.

7.3.4. Specimen Collection from the Vagina

7.3.4.1. Sampling Schedule

Specimens will be collected from the vagina of each female subject at the Baseline Sampling Visit and at the Re-Sampling Visit.

7.3.4.2. Specimen Labeling

Label the specimen collection tubes with the appropriate body site-specific, pre-printed clinic label.

7.3.4.3. Materials Needed

1. Standard gynecologic exam table with good lighting
2. Examination kit containing at least one medium Pederson and one large Pederson speculum, microelectrode pH meter (Oakton, model pH Spear) and 3 sterile Catch-All™Sample collection swabs (Epicentre Biotechnologies, Madison WI)
3. Small (2 mL) screw-top (external thread) MoBio collection tubes containing 750 μL of specimen collection fluid (MoBio buffer)
4. Pre-printed clinic label set for all body sites to be collected
5. Stopwatch with second hand
6. Ziploc Biohazard-labeled plastic bag to hold the MoBio collection tubes containing the collected specimens
7. Small ice chest with ice

7.3.4.4. Collection Methods

Aseptic technique will be used for collection of all specimens (See App. O).

1. Collect information regarding menstrual cycle by inquiring about last menstrual period (LMP) and cycle length.
2. Record use of all contraception within the preceding 12 months on the Concomitant Medications or Medical History forms as applicable:
   1. For oral contraceptives, record brand name of oral contraceptive, date initiated (month/year), and whether it is used “continuous dose” (placebo pills not taken, such that menstruation does not occur) or “traditional dose.”
   2. For intrauterine devices, record type of device used (Mirena® versus Paragard® versus other) and date of insertion (month/year).
   3. For progesterone-based injections and insertion rods, record delivery device (Depo-Provera® versus Implanon™ versus other) and date of insertion or last injection (month/year).
   4. For permanent sterilization methods (tubal ligation and Essure® device), record type and date of procedure (month/year).
3. Spread labia to visualize vaginal introitus immediately posterior to hymenal ring/tissue.
4. Repeat vaginal pH determination of vaginal introitus immediately prior to sampling (each and every time sampling is performed). Follow the same procedure used for screening subjects, as described in Section 6.3.7 Vaginal Examination and pH Determination.
5. Proceed with obtaining vaginal introitus specimen: place one Sterile Catch-All™ Sample Collection Swab (Epicentre Biotechnologies, Madison WI) at the vaginal introitus posterior to the hymenal ring/tissue and rotate swab along the lumen with a circular motion five times. Immediately after swabbing, the swab is swirled in 750 μL of MoBio buffer in the labeled specimen collection tube. The swab should be pressed against the tube wall multiple times for 20 seconds to ensure transfer of bacteria from swab to solution.
6. Insert pre-warmed speculum. It is preferable that the speculum be used without lubricant or water. If, in the opinion of the clinician, it is not feasible to insert the dry speculum without causing extreme discomfort to the subject, the exterior of the tip of the speculum may be moistened with tap water, taking care to use as little water as possible to avoid diluting the vaginal fluids being collected and to avoid any impact on pH measurement.
7. Visualize cervix and posterior fornix.
8. Determine pH of posterior fornix immediately prior to sampling (each and every time sampling is performed). Follow the same procedure used for screening subjects, as described in Section 6.3.7 Vaginal Examination and pH Determination. If posterior fornix pH>4.5 at sampling visit but was ≤ 4.5 at screening, proceed with sampling the posterior fornix.
9. Proceed with obtaining posterior fornix specimen: place one Sterile Catch-All™ Sample Collection Swab (Epicentre Biotechnologies, Madison WI) in the posterior fornix and rotate the swab along the lumen with a circular motion five times. Immediately after swabbing, the swab is swirled in 750 μL of MoBio buffer in an appropriately labeled specimen collection tube. The swab should be pressed against the tube wall multiple times for 20 seconds to ensure transfer of bacteria from swab to solution.
10. After obtaining posterior fornix specimen, proceed with collecting vaginal midpoint specimen using one Sterile Catch-All™ Sample Collection Swab (Epicentre Biotechnologies, Madison WI) to gently rub the mid-vaginal wall. Immediately after swabbing, the swab is swirled in 750 μL of MoBio buffer in an appropriately labeled specimen collection tube. The swab should be pressed against the tube wall multiple times for 20 seconds to ensure transfer of bacteria from swab to solution.
11. Store all the tubes on ice and deliver to the clinical laboratory within approximately two hours.

Avoid sampling of the vaginal area where there has been contact with the speculum.

Vaginal introitus, Posterior fornix and Mid-vagina areas are sampled and stored separately and labeled with the appropriate body site-specific, pre-printed clinic label.

7.3.5. Collection of Blood Specimens (Baseline collection only)

7.3.5.1. Sampling Schedule

Thirty mL of whole blood will be collected from all subjects at the Baseline Sampling Visit.

7.3.5.2. Specimen Labeling

The phlebotomist will label the specimen collection vials with the appropriate body site-specific, pre-printed clinic label.

NOTE: Subject’s gender will be indicated on the Coriell samples by the addition of an M or F on the clinic label.

e.g.

01DBA

Visit: 01 / 02

DT:

COR-1 M or F (indicate one)

7.3.5.3. Materials Needed

• Blood collection kit from Coriell Institute for Medical Research containing two yellow top tubes
• Red top vacutainer tubes (without anticoagulant)
• Pre-printed clinic label set for all body sites to be collected

7.3.5.4. Collection Methods

Aseptic technique will be used for collection of all specimens (See App. O).

Coriell Specimens

1. 20 mL (2 × 10 mL) will be collected in the Coriell-provided yellow-top collection tubes (vacutainer with ACD Solution A), labeled with the pre-printed clinic label and stored at room temperature. These tubes will be sent to the clinical laboratory for bar-coding, packaging and same day shipment to the Coriell Institute for Medical Research. Follow preparation, packaging and shipping instructions as provided by Coriell Institute and contained in App H.
2. Blood will be processed and stored at the Coriell Institute for Medical Research for future genotyping, DNA sequencing, and creation of cell lines.

Serum Sample (Serum)

1. 10 mL will be collected in a red-top tube without anticoagulant (for future serum immune response measurements) and labeled with the pre-printed clinic label.
2. Serum will be processed (per site SOP). Samples (up to 5 × 1 mL) will be bar-coded by the clinical lab and stored at −20ºC in the clinical laboratory for future antibody assays.

Bar-code labeling and GlobalTrace data entry of all specimens will be done exclusively in the clinical laboratory at each site.

Labeling Supplies

• Rolls of paired sets of identical barcodes (provided by the CDCC/EMMES)
• PC-compatible barcode scanner
• GlobalTrace Source Document (provided by the CDCC/EMMES)

Procedures

• Samples will be transported (per site SOP) from the clinical site to the appropriate clinical laboratory personnel for processing within two hours of collection.
  • – For each sample, a GlobalTrace source document will be completed, capturing the subject’s Study ID, visit number and date of collection, and YES to the question of future use.
• After the clinical lab has processed and/or aliquoted the sample into the appropriate tube, the following procedure will be followed FOR EACH SAMPLE:
  • – One barcode label, of two identical barcodes found in pairs on the roll of barcode labels, will be applied to the tube. The other accompanying barcode will be placed on the GlobalTrace source document for that subject’s specimen type. The source document will be signed and dated by the study personnel labeling the tube.
  • – This labeling process will be repeated for each tube into which the sampling specimen has been collected/aliquoted. All of the subject’s specimens of the same type should be included on the same GlobalTrace source document for any given visit.
    • Barcoded tubes (MoBio) will then be placed in the freezer for storage.
    • Barcoded yellow-top collection tubes of whole blood will be shipped to Coriell (per Coriell SOP).

The barcodes from the GlobalTrace source document will be scanned into the GlobalTrace system and associated with the subject’s Study ID, visit number, and visit date, noting the specimen type as well as the future use choice in the Purpose field. Select Future Use as YES for all enrolled subjects in this study.

For details on the GlobalTrace System, see the GlobalTrace System User’s Guide.

Note: Specimens can be entered in GlobalTrace only for subjects who have already been enrolled in AdvantageEDC. Therefore, specimens collected at sampling visits will not be able to be entered into GlobalTrace until after the subject has been enrolled in AdvantageEDC at Visit 01.

7.7. Initial Processing of Saliva and Stool Specimens

Aseptic technique will be used for processing of all specimens (See App. O).

7.7.1. Saliva

1. Centrifuge Falcon tubes containing collected saliva at 2600g for 15 minutes at room temperature. If the solids are not sufficiently separated, centrifuge again for 20 minutes.
2. Using a 1 mL Pipetman, transfer the saliva supernatant to two MoBio 2 mL tubes containing 750μL of lysis buffer.
3. Tubes will then be barcode labeled (see section 7.4) and frozen at −80ºC prior to DNA extraction.

7.7.2. Stool

1. From each sampling visit, four aliquots of stool lysate will be generated for purification of DNA and two aliquots of stool will be generated for future purification of RNA.
2. Upon receipt of the stool specimen, the clinical laboratory will aliquot approximately 2 mL of specimen into one 50 mL Falcon tube. Stool may be distributed using a disposable spatula or a metal spatula that is subsequently wiped clean and ethanol flamed for sterilization. 5 mL MoBio lysis buffer is added to the Falcon tube, and the sample is homogenized by vortexing 30–40 seconds. Note that the laboratory will need to have extra MoBio lysis buffer for this step, as this volume is not supplied with the DNA extraction kit.
3. Centrifuge the Falcon tube 5 minutes at 1500 rcf and pipet 1 mL of supernatant into 4 barcoded MoBio Garnet Bead tubes containing 750μL of MoBio buffer (this tube with buffer comes with the PowerSoil™ kit).
4. Heat at 65°C for 10 minutes, then at 95°C for 10 minutes. Samples may be stored frozen at −80ºC prior to DNA extraction.
5. Process per MoBio PowerSoil™ DNA Isolation Kit directions (see Section 7.9.2), and store the extracted and barcoded DNA at −80ºC.
6. For the future RNA extraction, add 500 μL of stool specimen (as noted above) to each of two 2 mL external thread cryovials (Nunc or Nalgene). Add 1 mL of RNAlater® (Ambion, Inc., AM7020), which will help stabilize the nucleic acids during long-term storage. Barcode label, and freeze cryovial at −80ºC.

Between 18 May 2009 and 15 June 2009, clinical specimens were collected in SCF-1 buffer instead of in MoBio buffer. For laboratory processing of these specimens, the following procedures will be followed:

1. Centrifuge the sample at 13,000 rpm for 10 minutes and remove supernatant.
2. Use approximately 500 μL of MoBio Powersoil Bead Solution provided in the MoBio Bead tube to resuspend the pellet.
3. Transfer the resuspended sample back to the MoBio Bead tube from which you took approximately 500 μL solution, and proceed with step one from the MoBio PowerSoil DNA Isolation Kit.

7.10. Specimen Processing for Extraction of Microbial RNA

Aseptic technique will be used for processing of all samples (See App. O).

[THIS SECTION IS UNDER DEVELOPMENT]

For each tube containing extracted nucleic acid sample, a GlobalTrace source document will be completed, capturing the subject’s Study ID, visit number (of corresponding sampling material), date of processing, and YES to the question of future use.

After the clinical lab has processed and/or aliquoted the sample into the appropriate tube, the following procedure will be followed FOR EACH SAMPLE:

• One barcode label, of two identical barcodes found in pairs on the roll of barcode labels, will be applied to the tube. The other accompanying barcode will be placed on the GlobalTrace source document for that subject’s specimen type. The source document will be signed and dated by the study personnel labeling the tube.
• This labeling process will be repeated for each tube into which the sampling specimen has been collected/aliquoted. All of the subject’s specimens of the same type should be included on the same GlobalTrace source document for any given visit.
• Barcoded tubes will then be placed in the freezer for storage or prior to shipment to a sequencing center.

The barcodes from the GlobalTrace source document will be scanned into the GlobalTrace system and associated as an aliquot or pool with the subject’s source specimen material, Study ID, visit number, and visit date, noting the specimen type as well as the future use choice in the Purpose field. Select Future Use as YES for all enrolled subjects in this study.

For details on the GlobalTrace System and its aliquoting features, see the GlobalTrace System User’s Guide.

This section is under development and will describe how the specimens will be coded and inventoried at the clinical laboratories and sequencing centers.

Nucleic acid samples will be stored in Tris-EDTA buffer and shipped to the sequencing centers in 1.5–2 mL sterile polypropylene tubes. The tubes will be packaged and shipped with cold gel packs (−20ºC) specifically made to maintain the specimen integrity and packages will be marked as required by the current regulations.

Insert detailed shipping instructions here including frequency of shipments and how specimens will be designated to each site. Also include LIMs methodologies for maintenance of EMMES ID through sequencing and library creation.

Include a description of what the different laboratories will do with specimens, i.e., microbial DNA quantification and evaluation, sequencing.

The extracted nucleic acid samples will be quantified and checked for DNA integrity and relative purity. These samples will be stored immediately in the processing laboratories at −70°C. Aliquots of the samples will be transferred to the sequencing centers for metagenomic sequencing. The clinical labs will manage requests to submit/transfer samples from these sites to the genome sequencing centers.

[Insert any DNA QC to be done at seq. centers.]

When subjects sign the consent form to participate in the study, they agree to the storage and future use of their specimens.

If a subject later chooses to withdraw consent for future use of stored specimens, the PI at the site or the sponsor will notify the Coriell Institute for Medical Research and any clinical laboratories housing their specimens or extracted nucleic acids to discard all remaining materials from that subject. These materials will be discarded in the appropriate manner and the disposition will be documented. (See Appendix F.)

The CDCC prepares source documents to match the AdvantageEDC eCRF screens. These forms are posted to the Study web site:

https://web.emmes.com/study/dnt/hmp/index.htm

The forms for each study are accessed under the Protocols tab, by protocol number, selecting Study Materials, then Source Documents. Sites should print the current version of the study source document visit forms only as needed to keep up with enrollment.

If a Study Status Change form has been completed, sites should not retain any blank source document visit forms for a subject beyond the effective date of the change form.

Blank visit forms related to an incomplete/partial visit or missed evaluations should have the page header completed, lined through, and initialed/dated.

9.1.4. Supplemental Visit Numbering

• Supplemental visits that are not scheduled in the protocol are assigned a study number with the trailing alpha, “S” (for supplemental). For example:
  • If a subject has an unscheduled visit (e.g., return for additional sampling) that occurs between Visits 01 and 02, the visit would be numbered 01S.
  • If more than one supplemental visit occurs between two scheduled visits, the alpha designation proceeds from “S” to “Z” (e.g., three visits between the scheduled Visits 01 and 02 would be named 01S, 01T, and 01U, respectively).

  If the clinical lab determines that insufficient or inadequate specimen material has been obtained, they may request that the subject return for a supplementary sampling of the required material.