Annotated-Protocol Patel

Feasibility of using saliva samples to assess measles Principal Investigator: Pooja Patel
susceptibility in Chandigarh, India
Feasibility of using saliva samples to assess measles susceptibility in

children: a cross-sectional study in Chandigarh, India
Principal Investigator:
Pooja Patel
Faculty Advisor:
Dr. Matthew Boulton
Version Number: 1.0
1 March 2019
Version 1.0 Page 1 of 16

1 March 2019
Table of Contents
List of Abbreviations 3
Protocol Summary 4
1 Key Roles 5
2 Background Information and Scientific Rationale 6
2.1 Background Information 6
2.2 Scientific Rationale 6
2.3 Potential Risks and Benefits 6
3 Objectives 7
4 Study Design 8
5 Study Population 9
5.1 Selection of the Study Population 9
5.2 Inclusion and Exclusion Criteria 9
6 Study Procedures and Evaluations 10
6.1 Study Procedures 10
6.2 Data Management 10
7 Statistical Considerations 11
7.1 Study Outcome Measures 11
7.2 Sample Size Considerations 11
7.3 Participant Enrollment and Follow-Up 11
7.4 Analysis Plan 11-12
8 Subject Confidentiality 13
8.1 Future Use of Stored Specimens 13
9 Informed Consent Process 14
10 Literature References 15
Supplements and Appendices 16

1 March 2019
List of Abbreviations
Acronym Full name
HIPAA Health Insurance Portability and Accountability Act

IgG Immunoglobulin G
IRB Institutional Review Board
N Number (typically refers to subjects)
PGIMER Post Graduate Institute of Medical Education and Research
PI Principal Investigator

1 March 2019
Protocol Summary
Title: Feasibility of using saliva samples to assess measles susceptibility

in children: a cross-sectional study in Chandigarh, India
Population: The study population will consist of a subset of children from the
larger study consisting of 1,500 people in Chandigarh. The subset
will include 350 children aged 0-15, randomly selected from three
different settings in Chandigarh: rural, urban, and resettlement
colonies. This sampling method will aim to select a subset of
participants to be representative of the population.
Number of Sites: 30
Study Duration: Data collection will be conducted from May 2019 until the following
year.
Study Design: Upon consent, trained field staff will administer a questionnaire and
collect both a saliva sample and a serum sample from each child.
Samples will then be processed at PGIMER’s laboratory and results
will be uploaded to a spreadsheet to be paired with demographic
information from the questionnaire. We will use the sensitivity and
specificity from saliva samples as the outcome of this study, in
comparison to serum samples. Review of the research project will
be requested from both the PGIMER and University of Michigan
(UM) institutional review boards (IRB).
Objectives:
Primary Objective: Compare detection of antibodies from saliva to that from serum.
Secondary Objective: Characterize the amount of false positives and negatives by various
groups and socio-demographic strata.

1 March 2019
1 Key Roles
Principal Investigator: Pooja Patel
MPH Candidate, Global Health Epidemiology
University of Michigan
1415 Washington Heights
Ann Arbor, MI 48109
404-386-5365 (Phone)
poojaptl@umich.edu
Faculty Advisor: Matthew L. Boulton, MD, MPH

Senior Associate Dean, Global Public Health
Professor of Epidemiology;
Professor of Internal Medicine
University of Michigan
1415 Washington Heights SPH II M5218
Ann Arbor, MI 48109
734-936-1623 (Phone)
mboulton@umich.edu

1 March 2019
2 Background Information and Scientific Rationale

2.1 Background Information
Measles continues to be one of the most highly communicable diseases in the world1,
with ten percent of measles-related deaths occurring in India2. Despite the existence of
an effective vaccine, measles is a leading cause of the nation’s childhood mortality
among vaccine-preventable diseases. India has set a goal to eliminate measles by
20203. Assessing measles susceptibility in a population by testing antibodies may serve
to inform vaccination campaigns and other control efforts.
2.2 Scientific Rationale

Current testing of measles antibodies relies heavily on collection of serum; this may not
always be feasible in low resource settings and parents may hesitate to have their
children pricked for blood collection, skewing the study population4. Other methods of
antibody testing, including samples collected from saliva, may provide a more simple,
low-cost alternative.
2.3 Potential Risks and Benefits

Risks: There is a possibility of risk for participants, and the likelihood of such risk ranges
from rare to infrequent. The main source of risk would be from a break of confidentiality.
We have minimized the risk of a confidentiality breach by omitting the collection of
identifiable information (i.e. names, addresses, and numbers) and only recording a
questionnaire ID. Surveys will be handled with care and kept in a safe location.
Participants may also have an emotional response to survey questions. We can
minimize this risk by allowing an option to not respond to any question that elicits
feelings of discomfort.
Benefits: The generalizable knowledge gained from this research outweighs the minimal
social risk that may arise from a confidentiality breach or personal discomfort
experienced. The results of the study will likely provide benefits to society and future
patients.

1 March 2019
3 Objectives
Primary Objective Compare detection of antibodies from saliva to that from
serum (gold standard) and calculate the sensitivity and
specificity to assess feasibility of using saliva samples for
measles antibody testing.
Primary Outcome Measures Saliva specimens will be collected by swabbing the child’s
mouth for ~1 minute. Serum specimens will be collected by
venipuncture; For subsequent analyses, 1ml is needed for
children <1 year of age and 2ml of blood is needed for
children 1 year or older. Both types of specimens (saliva
and serum) collected from each individual will be sent to
PGIMER’s laboratory for testing. Using their standard
protocol for each type of specimen, the lab will measure
measles IgG antibodies for each sample.
Antibody results will be categorized according to the

following thresholds:
 Negative: <150UI/ml
 Borderline: 150-200IU/ml
 Positive: > 200IU/ml
NOTE: these thresholds are subject to change depending
on the diagnostic testing kits used.
Secondary Objective Characterize the amount of false positives and negatives in

by various groups and socio-demographic strata including
age and income.
Secondary Outcome Measures A questionnaire will be administered by the trained field

worker (ie nurse, anganwadi worker) to the parent or
caregiver of the child. The form aims to capture additional
variables of interest to complement antibody testing
results.
Some variables include: sociodemographic information,
socioeconomic status, and measles vaccination history,
mother’s measles history (vaccination or disease).

1 March 2019
4 Study Design
This cross-sectional study is supplemental to an earlier study that was conducted via an
ongoing collaboration between UM and PGIMER. The larger, population based study of
1,500 study participants aimed to evaluate and characterize of susceptibility to measles in
both children and adults in Chandigarh, India.
The current research will select a subset of 350 children from the initial study population to
assess the feasibility of using saliva samples to measure measles antibodies in children.
Upon consent, trained field staff will administer a questionnaire and collect both a saliva
sample and a serum sample from each child. Samples will then be processed at PGIMER’s
laboratory and results will be uploaded to a spreadsheet to be paired with demographic
information from the questionnaire. Review of the research project will be requested from
both the PGIMER and University of Michigan (UM) institutional review boards (IRB).

1 March 2019
5 Study Population
5.1 Selection of the Study Population
The study population will consist of a subset of children from the larger study consisting
of 1,500 people in Chandigarh. The subset will include 350 children aged 0-15, randomly
selected from three different settings in Chandigarh: rural, urban, and resettlement
colonies. We will select participants so that each of the following age quintiles has an
even number of children: 0-9 months, 9 months to 2 years, 2 to 5 years, 6-10 years, and
11-15 years.
5.2 Inclusion and Exclusion Criteria

Inclusion Criteria:
 Individuals living in Chandigarh
 Individuals who were enrolled in the previous study
 Individuals between 0 and 15 years of age
Exclusion criteria
 Individuals with a health condition that prohibits them from participating in normal
daily activities
 Individuals with known hemophilia or other blood dyscrasias characterized by
potential for excessive bleeding
 Individual with acute febrile illness

1 March 2019
6 Study Procedures and Evaluations

6.1 Study Procedures
Data collection
Trained field workers will walk the study participants (or their parent/caregiver in this
situation) through the informed consent form to ensure they understand its contents.
They will answer any questions or address concerns that the parent or caregiver may
have. Since participants are children aged 0-15, informed consent (assent in this case)
will be obtained from the parent or caregiver. Participants will receive a copy of the
informed consent form. Once the consent form has been signed, the questionnaire will
be administered. The saliva and serum specimens will then be collected from the child.
Questionnaire
The trained field workers will administer the short questionnaire. In this case, a parent or
caregiver will answer the questions on behalf of the study participant. Answers will be
recorded on a tablet and will be reviewed for completeness. Completed questionnaires
will be uploaded within 24 hours to the study database.
Quality Assurance and Management

All involved study staff will be appropriately and fully trained. Trainings for specimen
collection and testing will be conducted prior to the start of the data collection period.
The study will have ongoing quality control checks to monitor and evaluate the study
procedures.
6.2 Data Management

Informed consent forms and other paper documents will be kept at PGIMER and will be
handled according to the institution’s standard protocol.
Questionnaires will be administered via a tablet. Responses will be uploaded to a local

server and saved in M-box.
The laboratory will export diagnostic testing results as a spreadsheet which will then be
uploaded and saved to M-box. The laboratory will keep all specimens for a year to
ensure completion of testing and quality assurance.
Only those who have granted access will be able to access the collected data and
specimens.

1 March 2019
7 Statistical Considerations
7.1 Study Outcome Measures
IgG measles antibodies will be tested using both saliva and serum specimens collected
from 350 children in Chandigarh, India.
A questionnaire will be administered by the trained field worker (ie nurse, anganwadi
worker) to the parent or caregiver of the child. The form aims to capture additional
variables of interest to complement antibody testing results. Some variables include:
sociodemographic information, socioeconomic status, and measles vaccination history,
mother’s measles history (vaccination or disease).
7.2 Sample Size Considerations

A total of 350 participants will be selected for this study. The sampling
Method mentioned in section 5 aims to select a subset of participants to be
representative of the population.
7.3 Participant Enrollment and Follow-Up

Participants will be enrolled at their homes and will not have any follow-up visits since
this is a cross-sectional study to compare specimens collected at one point in time. All
data collection will be collected at the initial visit during which the participant is enrolled.
7.4 Analysis Plan

Quality checks will conducted for all data. Data will first be checked for completeness to
ensure we have all appropriate datasets, variables, and observations. In order to check
for inappropriate or unusual values, we will run descriptive statistics to check distribution
of each variable. We will also check consistency of related variables and recalculate any
measures that may have been automatically calculated.
Data analysis will be conducted using a statistical software program, such as SAS or R.
Primary Objective
Antibody results for both saliva and serum specimens will be categorized according
to the following thresholds:
 Negative: <150UI/ml
 Borderline: 150-200IU/ml
 Positive: > 200IU/ml
IgG antibody results from both methods will then be compared for each individual. In
this case, results from serum will serve as our gold standard to test for sensitivity
and specificity. We will compare results from the saliva samples to the gold standard
to categorize each as a false positive, true positive, false negative, or true negative.

1 March 2019
Secondary Objective
We will use various multiple linear regression models to compare the amount of false
positives and false negatives across age groups; models will include variables that were
collected as part of the questionnaire.

1 March 2019
8 Subject Confidentiality
All staff with access to study data, including those collecting data and processing lab
specimens, will undergo both research ethics and confidentiality training. This
confidentiality training will be consistent will US regulations for protection of personal
health information (according to the Health Insurance Portability and Accountability Act
(HIPAA) of 1996). As with all training, research ethics and confidentiality training will be
documented for each member of the research team.
8.1 Future Use of Stored Specimens

Specimens will be stored at PGIMER for up to one year for quality assurance purposes.
After this time, specimens will be discarded according to the institution’s standard protocol.
We will not use specimens for purposes outside of those agreed upon in the informed
consent form.

1 March 2019
9 Informed Consent Process

We will request informed consent from a parent or caregiver since participants in this study
are children. The trained field worker will provide a paper copy of the informed consent
form to the parent or caregiver and walk them through each section to ensure proper
comprehension of the contents.
Informed consent will be obtained in the form of written permission from the parent/legal
guardian. Data collection will be conducted in the presence of the parent/legal guardian or
their designee. For children too young to answer questions, the parent/legal guardian will
be interviewed.

1 March 2019
10 Literature References
1. World Health Organization. WHO | Immunization, Vaccines and Biologicals.
https://www.who.int/immunization/diseases/measles/en/. Accessed February 2, 2019.
2. World Health Organization | WHO vaccine-preventable diseases: monitoring system.

2015 global summary. http://apps.who.int/immunization_monitoring/globalsummary.
Accessed February 15, 2019.
3. World Health Organization India | Measles.

http://www.searo.who.int/india/topics/measles/about/en/. Accessed February 11, 2019.
4. Ostler, M. W., Porter, J. H., & Buxton, O. M. (2014). Dried blood spot collection of health
biomarkers to maximize participation in population studies. Journal of visualized
experiments: JoVE, (83), e50973. doi:10.3791/50973

1 March 2019
Supplements and Appendices
Questionnaire
Informed consent form
(not actually attached)

1 March 2019

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Feasibility of using saliva samples to assess measles Principal Investigator: Pooja Patel

susceptibility in Chandigarh, India

Feasibility of using saliva samples to assess measles susceptibility in

Dr. Matthew Boulton

Version Number: 1.0

Version 1.0 Page 1 of 16

Supplements and Appendices 16

Version 1.0 Page 2 of 16

Acronym Full name

HIPAA Health Insurance Portability and Accountability Act

Version 1.0 Page 3 of 16

Title: Feasibility of using saliva samples to assess measles susceptibility

Version 1.0 Page 4 of 16

Faculty Advisor: Matthew L. Boulton, MD, MPH

Version 1.0 Page 5 of 16

2 Background Information and Scientific Rationale

2.2 Scientific Rationale

2.3 Potential Risks and Benefits

Version 1.0 Page 6 of 16

Antibody results will be categorized according to the

Secondary Objective Characterize the amount of false positives and negatives in

Secondary Outcome Measures A questionnaire will be administered by the trained field

Version 1.0 Page 7 of 16

Version 1.0 Page 8 of 16

5.2 Inclusion and Exclusion Criteria

Version 1.0 Page 9 of 16

6 Study Procedures and Evaluations

Quality Assurance and Management

6.2 Data Management

Questionnaires will be administered via a tablet. Responses will be uploaded to a local

Version 1.0 Page 10 of 16

7.2 Sample Size Considerations

7.3 Participant Enrollment and Follow-Up

7.4 Analysis Plan

Version 1.0 Page 11 of 16

Version 1.0 Page 12 of 16

8.1 Future Use of Stored Specimens

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9 Informed Consent Process

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2. World Health Organization | WHO vaccine-preventable diseases: monitoring system.

3. World Health Organization India | Measles.

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Supplements and Appendices

Version 1.0 Page 16 of 16

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