A Blueprint to Address Research Gaps in the Development of Biomarkers for Pediatric Tuberculosis
A Blueprint to Address Research Gaps in the Development of Biomarkers for Pediatric Tuberculosis
Mark Patrick Nicol () 0
Devasena Gnanashanmugam 0
Renee Browning 0
Eleanor S. Click 0
Luis E. Cuevas 0
Anne Detjen 0
Steve M. Graham 0
Michael Levin 0
Mamodikoe Makhene 0
Payam Nahid 0
Carlos M. Perez-Velez 0
Klaus Reither 0
Rinn Song 0
Hans M. L. Spiegel 0
Carol Worrell 0
Heather J. Zar 0
Gerhard Walzl 0
0 Anzio Road, Observatory , Cape Town, 7925 South Africa
Childhood tuberculosis contributes significantly to the global tuberculosis disease burden but remains challenging to diagnose due to inadequate methods of pathogen detection in paucibacillary pediatric samples and lack of a child-specific host biomarker to identify disease. Accurately diagnosing tuberculosis in children is required to improve case detection, surveillance, healthcare delivery, and effective advocacy. In May 2014, the National Institutes of Health convened a workshop including researchers in the field to delineate priorities to address this research gap. This blueprint describes the consensus from the workshop, identifies critical research steps to advance this field, and aims to catalyze efforts toward harmonization and collaboration in this area.
tuberculosis; children; diagnosis; biomarker; blueprint.
Childhood tuberculosis is estimated to account for 6%
of the tuberculosis caseload globally, and for 4%–21%
of the caseload in the 22 high-incidence countries
that account for 80% of global tuberculosis cases .
Mathematical modeling suggests that only 35% of
tuberculosis cases in children are detected . Improving the
accuracy of tuberculosis diagnosis in children is required
to improve case detection and outcomes, surveillance,
efficiency of healthcare delivery, future research, and
However, attaining an accurate diagnosis in children
in tuberculosis-endemic settings remains challenging.
There is overlap of the clinical presentation of
tuberculosis with other common childhood diseases such as
pneumonia, human immunodeficiency virus (HIV)–
associated lung disease, and severe malnutrition .
Clinical and chest radiographic features are often
nonspecific and subject to variable interpretation .
Structured diagnostic scoring systems based on clinical and
radiological findings and tuberculin skin testing show high
variability in case yield, with poor agreement between
scoring systems . Microbiological confirmation is
possible in children of all ages, but is rarely attempted due to
perceived difficulties in obtaining respiratory specimens
and because both culture  and automated real-time nucleic
acid amplification tests are only positive in a proportion of
children who have been clinically diagnosed with tuberculosis [7–
9]. Current diagnostics that measure immunological responses
following infection with Mycobacterium tuberculosis have
uncertain sensitivity and are unable to distinguish active
tuberculosis from latent tuberculosis . The clinical distinction
between latent and active tuberculosis is unlikely to be
dichotomous, especially following recent infection—a common
scenario in children.
In 2014, the National Institutes of Health (NIH) of the United
States convened a group of panelists to develop a blueprint for the
process of discovery and implementation of new diagnostic
biomarkers for pediatric tuberculosis. In the 19th century, a
“blueprint” was a reproduction of a technical drawing through a
contact print process on light-sensitive sheets that allowed the
rapid and accurate reproduction of documents but was unable
to reproduce color or shades of gray. This article shares several
similarities with the original blueprint method, inasmuch as it
builds on existing efforts for pediatric diagnostic biomarker
discovery, qualification, validation, and implementation, but does
not dictate the exact approach in view of the rapidly changing
technological, regulatory, and health implementation realities.
The blueprint presented here outlines the issues facing the field
of pediatric tuberculosis biomarker development and is aligned
with the Stop TB Partnership and the World Health
Organization (WHO) International Roadmap for Tuberculosis Research
. The blueprint covers the following critical steps:
The target audience for this article includes researchers,
healthcare providers, funding agencies, and regulatory bodies,
in an effort to coordinate and streamline the challenging
process of pediatric tuberculosis diagnostic biomarker discovery,
validation, qualification, and implementation.
CONSENSUS STATEMENT PREPARATION
Wide consultation was sought in the development of this
blueprint with input from international experts from relevant clinical,
basic science, public health and regulatory fields, and other
stakeholders. Among the panelists specifically included were pediatric
tuberculosis clinicians, tuberculosis researchers, HIV research
network representatives, ethicists, representatives from research
funding agencies, and individuals from nongovernmental,
advocacy, and community research organizations. Panelists were
invited to a workshop entitled “Pediatric Tuberculosis: Addressing
Research Gaps in Diagnostic TB Biomarkers” organized by the
NIH in Bethesda, Maryland, in May 2014. Prior to the workshop,
conference calls were held to identify key questions for discussion
at the workshop. A table summarizing existing pediatric (and
major adult) specimen repositories was developed (Table 1).
During the workshop, there were timed discussions, including
break-out groups, with statement modification in real time.
The statements were reviewed in the subsequent plenary sessions.
Agreement was reached by consensus or by vote.
The consensus questions covered 3 key areas relating to
pediatric tuberculosis biomarker research:
1. What are the criteria for the optimal pediatric tuberculosis
2. What are the challenges and sustainability issues for a
pediatric tuberculosis specimen repository?
3. What are the custodianship, ownership, legal, regulatory,
and policy issues relating to such repositories?
This document, which captures the consensus from the
workshop, aims to generate further discussion about pediatric
tuberculosis biomarker research, and to catalyze efforts toward
harmonization and collaboration. An NIH-sponsored Pediatric
TB Biomarker Working Group has been established to assist in
moving this process forward.
THE CHARACTERISTICS OF AN “IDEAL”
BIOMARKER FOR TUBERCULOSIS IN CHILDREN
A biomarker (or set of biomarkers) that could be used to
develop an accurate test for tuberculosis in a child would ideally fulfill
the following requirements:
Measurable in a readily obtainable matrix such as blood
(eg, by fingerstick), urine, stool, saliva, buccal mucosal
transudates, or exhaled air given the challenges in obtaining
respiratory specimens from infants and young children (<5 years).
Identify M. tuberculosis with high sensitivity and
specificity, independent of age, nutritional status, or HIV status as the
cause of, or contributing factor to, the current illness in children
presenting clinically with pulmonary or extrapulmonary
Distinguish between children with latent tuberculosis
(including children with latent tuberculosis who have respiratory
symptoms due to another pathogen) and those with active
Suitable for incorporation into a diagnostic platform that
would provide a rapid, accurate result at, or close to, the point
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PRIORITIES FOR PEDIATRIC TUBERCULOSIS
search were identified:
ed) should be considered top priority.
should be given to young children aged 0–5 years who are
firmation of disease is most challenging.
nostic challenges highlighted above.
plification (NAAT) technologies include the development of
molecular detection of small fragments of tuberculosis-specific
transrenal DNA [17, 18] and fluorogenic enzymatic tests for the
specific detection of BlaC . The latter is a highly conserved
the potential for diagnostics based on miRNA profiles in
reported that specific miRNAs show the potential to
discriminate infected from healthy individuals, and active from latent
infection, and to be useful for monitoring response to treatment
. Similarly, pediatric studies have demonstrated that
genomewide host transcriptional RNA signatures in blood can
distinguish tuberculosis from other diseases and from latent
tuberculosis infection, and that risk scores based on gene
expression may be useful for ruling out tuberculosis . It has also
been recently shown that a T-cell activation marker present
on circulating M. tuberculosis–specific T cells can discriminate
active from latent infection in children . Given the rapid
emergence of new technologies, there is a need to better define
the strategy for biomarker selection and validation.
KEY DESIGN FEATURES FOR BIOMARKER
Diagnostic studies in children should be standardized with
regard to the key elements of study design such as eligibility
criteria, radiological and microbiological assessments, specimen
collection and storage, data collection and analysis, clinical
care, and clinical case definitions. The Pediatric TB Biomarker
Working Group is currently developing consensus guidelines in
this regard. Case definitions of disease should preferably follow
the NIH consensus definitions for diagnostic study categories
for intrathoracic tuberculosis , which have recently been
revised to define 3 categories of tuberculosis disease: (1)
confirmed tuberculosis (microbiologically confirmed); (2)
unconfirmed tuberculosis (formerly possible or probable
tuberculosis); (3) unlikely tuberculosis .
Ideally, prospective cohort studies with adequate follow-up to
monitor response to therapy should be undertaken to enable
accurate, standardized collection of data and specimens.
However, leveraging existing cohorts and biorepositories from
wellcharacterized cohorts (eg, Table 1) will also be of considerable
value. Children with a wide range of symptoms and clinical
manifestations should be included in studies to cover the full
spectrum of tuberculosis disease. Studies should be done in
different epidemiological settings that include a broad range of
frequent nontuberculosis conditions (eg, malnutrition,
HIVassociated infections, bacterial pneumonia, malaria) as
differential diagnoses. Detailed historical, clinical, laboratory, and
radiological characteristics should be collected to enable
standardized case definitions. A standardized data dictionary and
specimen collection template would be useful to enable
metaanalysis of data from different studies, cohorts, and repositories,
and should be developed by pediatric tuberculosis researchers.
Study design should include follow-up of all children, with
and without tuberculosis infection or disease, not only to
allow collection of sequential specimens, but also to strengthen
the case definitions for tuberculosis, by evaluating response to
therapy in children treated or not treated for tuberculosis.
The suggested follow-up times for children treated for
tuberculosis disease include visits at 2 weeks, 2 months, and 6 months
or treatment completion and a visit at 2 months for children not
treated for tuberculosis disease.
COLLECTION AND STORAGE OF SUITABLE
A key component of study design is the choice of specimen
type and collection method. Specimens should be collected
bearing in mind that both pathogen and host biomarker
detection strategies may be used, including, for example, proteomic,
metabolomic, and gene expression profiling. In children with
pulmonary tuberculosis, M. tuberculosis has been isolated
from a variety of respiratory and alimentary tract specimens,
and potential diagnostic biomarkers can also be detected in
blood products and urine [7, 8, 21, 25–28]. Selection of the
most suitable specimen types for a pediatric tuberculosis
diagnostic biomarker study should take into account the issues of
feasibility, acceptability, effectiveness, and cost of the collection
procedure under programmatic conditions. In general, this
would include blood (eg, serum, whole blood for RNA),
respiratory specimens (eg, induced or spontaneously expectorated
sputum, nasopharyngeal aspirate or swab, gastric aspirate/
lavage, string test), urine, and other extrapulmonary specimens
(when clinically indicated). To facilitate comparison of results
between biomarker studies and thereby increase their
usefulness, it is important to standardize specimen collection,
handling, processing, and storage across studies.
There are many considerations to take into account to assure
standardization. Collection of specimen types should be well
documented, including anatomical location (eg, nasopharyngeal/
oropharyngeal/laryngopharyngeal); specimen content (eg,
“pure” aspirate vs diluted lavage/wash); collection vehicle (eg,
swab tip vs suction catheter); swab type (eg, cotton vs synthetic
tip); additives used (eg, antimicrobial agent, nucleic acid
stabilizer, buffer); and volume accepted and processed, as volume
can significantly impact assay sensitivity. Handling, processing,
and storage of specimens vary by type, and considerations for
standardization include pH (eg, neutralization based on initial
pH); transport time, especially for nonsterile specimens;
centrifugation parameters; and refrigeration and storage temperature.
Ideally, standard operating procedures for the collection of
each specimen type should be collaboratively developed, piloted
and refined, and consistently implemented.
Stored specimens may be used not only for tuberculosis
biomarker discovery and detection, but, as improved diagnostics
for other respiratory pathogens become available, may also be
tested for such pathogens to improve the specificity of clinical
case definitions. Research protocols, reviewed by institutional
review boards, need to incorporate specific consent for storage
and future testing of samples.
DEVELOPING A SUSTAINABLE
The biomarker discovery and validation process would be
facilitated by the availability of biorepositories containing
wellcharacterized and appropriate pediatric specimens. Given the
substantial upfront effort and cost involved in carefully
characterizing a symptomatic cohort by clinical criteria and in
confirming a case of tuberculosis in a child, it is particularly
important to maximize the benefit associated with this initial
investment by establishing repositories for pediatric specimens.
At present, tuberculosis-specimen biorepositories are focused
on samples from adults, or are in the hands of individual
investigators (Table 1), with little coordination and standardization.
A key outcome of the workshop was an initiative to develop a
shared pediatric repository.
Key questions in designing a repository are: What is the
scientific objective of the work and what are the potential biomarker
targets of interest? Scientific objectives, for example, could
include discovery of new diagnostics for tuberculosis disease or of
markers of treatment response and will guide decisions about
whether to focus more on specimen collection at baseline vs
longitudinal specimen collection . Biomarker targets of interest
will guide decisions about which specimens to collect, how to
store specimens, and whether initial processing is required
prior to biobanking. Practical questions to be addressed in
repository planning are: (1) How many aliquots of each specimen to
biobank and in what volume, based on considerations of known
vs unknown potential applications, cost, and clinical limitations
(eg, blood volume)? (2) Which specimens should be prioritized
in case it is not possible to collect all specimens from every
participant? and (3) What are the time points for specimen
collection during longitudinal sampling?
The technical differences between methods used to collect,
transport, process, and store specimens in studies may be
contributing factors to variation in the published accuracy of
diagnostic markers . Data collection and process harmonization
are therefore critical for interpretability and comparability of
results. Detailed data on the specifics of specimen collection are
necessary to determine what potential targets are likely
preserved in a sample. Similarly, linkage to clinical metadata is
needed to identify the potential suitability of specific samples
for studies and the interpretation of study findings.
A large challenge to the integrity of repositories is the
requirement for meticulous record-keeping. Potential solutions
to record-keeping challenges include the use of barcode
labeling, electronic databases to indicate the location of each sample
and to link sample data to clinical metadata, and segregation of
samples by specimen type and aliquot number to facilitate easy
access at a later time.
As a part of first steps, it was determined at the workshop that
a Data Sharing Framework needed to be created with
agreedupon standard procedures and pediatric tuberculosis
nomenclature, and a Pediatric TB Biomarkers Working Group is
being organized by NIH, including the authors of this
manuscript, other tuberculosis experts, and microbiologists. This
group is currently developing standard nomenclature and
operating procedures for pediatric specimen collection.
The establishment, maintenance, and custodianship of a
biorepository will require dedicated funding. Funding of a
biorepository requires careful planning as costs for specimen collection,
handling, and processing for long-term storage, and for data
management and storage, are often not included in the initial
budget of research studies, and specific funding sources are
currently limited. Appropriate funders or partners will need to be
identified and key partnerships should be sought with existing
networks, such as the International Maternal, Pediatric,
Adolescent AIDS Clinical Trial Network (IMPAACT) and foundations
with experience in running biorepositories, such as the Foundation
for Innovative New Diagnostics (FIND), as well as potential
funders such as the NIH and the Bill & Melinda Gates Foundation.
Considering that prior experience has shown that the creation
and maintenance of such repositories is a large and expensive
undertaking, entities such as those listed above could pool resources
and each fund parts of the repository creation and maintenance.
Funders may be more amenable to provide resources when
existing specimens are pooled together in a single repository, as
opposed to generating de novo repositories or funding individual
repositories. A repository team would be needed to plan the
establishment of the repository in detail. This blueprint can be seen
as part of the lobbying exercise to facilitate such funding and may
stimulate the participation of other collaborators. Funding would
have to be for a period of at least 5 years, with clear milestones
and deliverables, culminating in a sample release phase during
which researchers could apply for sample release to facilitate
their test development work. A long-term plan for sustainability
should be developed, which may include contributions from
researchers wishing to access samples.
To encourage investigators to submit specimens to
biorepositories, funders could consider making submission of samples
a component of funding opportunities or incentivizing
submission through the provision of supplementary funding.
REGULATORY CHALLENGES FOR PEDIATRIC
Custodianship of a biorepository involves the provision of
careful oversight and management of samples from the point of
specimen collection all the way to the research use of biospecimens and
the linked data. A plan for selection of an appropriate custodian,
with explicit details on the responsibilities, should be developed
prior to the launch of a biobanking initiative. Policies need to
be in place to safeguard the quality of samples being entered
into the repository and their long-term storage. Additionally,
policies are needed for the appropriate use of biospecimens and data,
while assuring the privacy and confidentiality of participants and
their data. Research data standards, such as those provided by the
Clinical Data Interchange Standards Consortium , along with
a data-sharing framework and agreed-upon pediatric tuberculosis
nomenclature, should be established as part of the custodianship
plan. Local and national regulatory bodies may have restrictions
on types of specimens that may be collected as well as the
international exchange of samples. Such regulations vary by country,
by specimen type (eg, host DNA), and by target patient population
(eg, pediatric patients and pregnant women). A well-delineated
custodianship plan can help address some of these issues by
providing to the local and national regulatory bodies the agreed-upon
principles and protocols that govern the biorepository. The
National Cancer Institute Best Practices for Biospecimen Resources
 notes that the “custodian is the trusted intermediary and
caretaker of biospecimens and associated data, and the
custodian’s caretaking responsibilities should align with applicable
ethical and policy standards,” adding that the ideal custodian should
be someone other than research investigators or sponsor(s) of the
biospecimen resource (eg, a biospecimen resource manager) to
eliminate potential conflicts of interest.
As part of a governance plan for the biorepository, an
organizational structure should be delineated that includes a
biorepository team responsible for planning and managing the repository
as well as a sample access committee that governs access to
samples. These could be modeled on the framework established by
CTB2, a project of the TB Alliance, the CDC’s TB Trials
Consortium, and the AIDS Clinical Trials Group of the US National
Institute of Allergy and Infectious Diseases (NIAID), NIH. This
project is collecting high-quality patient specimens in late-stage
tuberculosis drug clinical trials where they are linked to detailed,
yet anonymized clinical documentation to enable discovery and
qualification of biomarkers for clinical development of improved
tuberculosis treatments for both drug-sensitive and
multidrugresistant tuberculosis (see http://www.tbbiorepository.org/). The
sample access committee should include scientists knowledgeable
about the research that may arise from the specimens,
experienced curators of biorepositories, and experts in epidemiology,
biostatistics, and informatics, among other technical consultants.
Patient advocates and research participants, where feasible,
should be included as members. The governance plan should
describe (1) the methods for safeguarding the integrity of the
samples and associated data, providing protocols used for
biospecimen collection and storage; (2) clear procedures for requesting
access to samples and data by investigators; (3) the review process
for sample and data requests, assuring that the composition of a
review committee that evaluates access requests is aligned with the
stated mission/goals of the biobank; (4) policies for the
dissemination of results from research that uses the samples; and (5)
responsible fiscal planning for the long-term storage of specimens
and data, as well as plans for securing future funds to maintain the
repository until samples are depleted. Early planning for the
custodianship, governance, and sample access mechanisms will help
mitigate potential legal, ethical, and regulatory complications that
can arise in the future operations of the biorepository.
Continued advocacy, collaboration, and communication are
needed to ensure the key elements of the blueprint are adopted
and implemented. Future pediatric tuberculosis biomarker efforts
will require focus on standardization in terms of case definitions,
specimen collection methods, and clinical data collection, as well
as the adaptability to evaluate new potential biomarkers and
technologies. Early inclusion of donors into the discussion
should emphasize the need of funding not only for diagnostic
development and pediatric cohorts, but also for building and
maintaining specimen repositories as part of a larger research
network. Developers of new tuberculosis diagnostics should be
involved at early stages to consider ways to integrate new
biomarkers into already existing, adapted, or new platforms.
Given the limited number of pediatric specimens in existing
centralized repositories, there is a clear need to continue to
enroll children in prospective cohort studies that collect
standardized high-quality data and samples and to optimize available
resources. Harmonization of specimen collection methods
and clinical data collection has begun with the formation of a
Pediatric TB Biomarker Working Group and a coordinating
committee from the attendees of this workshop, with the goal
of having unified standards for evaluating biomarkers from
existing and future repositories. Building on newly discovered
promising biomarkers and on new technologies with a focus
on the clinical challenges in children and the priority areas as
identified in this blueprint can serve as the initial stages of
tuberculosis diagnostics development. Successful identification of
a child-friendly tuberculosis diagnostic biomarker will require
input, collaboration, and coordination from many stakeholders,
from concept development of the study design to, ultimately,
the development of a point-of-care test appropriate for use in
those regions with the highest burden of pediatric tuberculosis.
Acknowledgments. We thank the following organizations: National
Institute of Allergy and Infectious Diseases (NIAID), National Institutes of
Health (NIH); Eunice Kennedy Shriver National Institute of Child Health
and Human Development (NICHD); Centers for Disease Control and
Prevention (CDC); Treatment Action Group, TB/HIV, New York,
New York. We thank the following individuals: Sheryl Zwerski, MSN,
CRNP, Peter Kim, MD: NIAID, Bethesda, Maryland; Patrick Jean-Philippe,
MD, Marco Schito, PhD, Arthur Stone: Henry M. Jackson Foundation–
Division of AIDS, Bethesda, Maryland; Lori Dodd, PhD: NIAID, Bethesda,
Maryland; Patrick Bossuyt, PhD: Academic Medical Center, University of
Amsterdam, Clinical Epidemiology, Biostatistics and Bioinformatics,
Amsterdam, Netherlands; Martina Casenghi, PhD: Médecins Sans
Frontières, Geneva, Switzerland; Maryline Bonnet, MD: Epicentre, Geneva,
Switzerland; David Murdoch, MD: University of Otago, Department of
Pathology, Christchurch, New Zealand; Lindsay McKenna, MPH;
Treatment Action Group, TB/HIV, New York, New York; Elizabeth Talbot,
MD: Dartmouth College, Infectious Diseases and International Health,
Dartmouth Hitchcock Medical Center, Lebanon, New Hampshire.
Disclaimer. The findings and conclusions in this article are those of the
authors and do not necessarily represent the official position of the CDC
Financial support. This work was supported by NIAID/NIH and the
Eunice Kennedy Shriver NICHD. This project has also been supported in
part with federal funds from the NIAID/NIH, US Department of Health
and Human Services (contract no. HHSN272200800014C).
Supplement sponsorship. This article appears as part of the supplement
“Advances inTuberculosis Research: A Blueprint for Opportunities.” This
article was sponsored by the Division of AIDS, National Institute of Allergy
and Infectious Diseases, National Institutes of Health.
Potential conflicts of interest. All authors: No potential conflicts of
All authors have submitted the ICMJE Form for Disclosure of Potential
Conflicts of Interest. Conflicts that the editors consider relevant to the
content of the manuscript have been disclosed.
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