Prospective cohort study methods and procedures

The following section describes the study objectives, study population and study procedures as described in the study protocol and manual of procedures (MOP).

Study objectives

The primary objective of this prospective natural history cohort study was to characterize the acute and post-acute clinical, neurological and neurodevelopmental outcomes of postnatally-acquired, laboratory-confirmed ZIKV infection in newborn infants and their mothers (infant-mother pairs), as well as older children ages 1.5–3.5 years old who may have been postnatally exposed to ZIKV during the 2015–2017 outbreak. The study also aimed to describe the clinical characteristics, viral load and kinetics of humoral immune responses during acute laboratory-confirmed ZIKV infection in infants and young children, including the potential for antibody-dependent enhancement (ADE) during acute ZIKV infection in those previously exposed to or with maternally-derived antibodies against DENV.

Study population

Children and mothers were enrolled from 22 communities, living within a 200 km² area in the southwest Trifinio (SWT) region of Guatemala, a rural lowland area that borders Chiapas, Mexico and is hyperendemic for vector-borne infections [8] A community needs assessment conducted in 2012 identified high levels of food insecurity, poverty, poor access to healthcare, and high maternal-child morbidity and mortality in this area [5]. In 2013, as a response to the needs assessment, CHD, an academic-private global health partnership (CU and AgroAmerica), began implementing clinical services and community maternal-child programs to register and follow pregnant women and their children under 3 years of age to improve child growth and development [9,10]

Eligibility criteria

Infant-mother pairs (cohorts B and M, respectively) were eligible if the child was 0–2.9 months of age, and the mother was ≥16 years of age. Informed consent was signed by the mother if ≥18 years old. If the mother was 16–17 years of age a grandparent signed the informed consent and the mother provided assent (as per local ethics committee requirements). Older children 1.5–3.5 years of age at enrollment were eligible if they either participated in a prior 2015–2016 dengue acute febrile illness (AFI) surveillance study (cohort A) or were a sibling of an infant enrolled in cohort B (cohort C), and consent for participation in this study was signed by one of the parents.

Ethics statement

The study was granted approval by the Institutional Review Boards (IRBs) of Baylor College of Medicine (BCM), University of Colorado (CU) Multiple IRB (COMIRB), and the Ministry of Public Health and Social Welfare (MSPAS) of Guatemala in May of 2017. All study participants provided guardian informed consent prior to initiating study activities.

Participant enrollment

Infant-mother pairs were screened and enrolled from the CHD maternal and child health program and from referrals of pregnant women who were close to delivery or had delivered an infant in the previous two months by community health workers. Children 1.5–3.5 years of age who previously participated in the dengue AFI surveillance study were contacted for enrollment in this study.

Baseline and follow-up visits

At baseline, we recorded demographic information, including each child’s date of birth, sex, birth weight (if available), gestational age (reported by mother), household location and household characteristics. We also documented medical history and information about initiation of breastfeeding and history of vaccinations. The child’s length, weight, and head circumference were measured using WHO’s recommended procedures [11].Baseline physical, neurological, neurodevelopmental, ophthalmologic visual acuity, anterior segment and fundoscopy, and hearing otoacoustic emissions (OAE) evaluations were conducted in all infants and children. For mothers, prenatal and delivery history along with past medical history were obtained, as well as measurements of mother’s height and weight. Biological samples (blood, urine, and saliva) were collected from all children, infants and their mothers. Breastmilk was also collected from mothers if available.

Protocol-required household visits at various intervals were conducted by trained study staff at the subjects’ homes or at the study clinic in order to collect health data and to perform anthropometric measurements, clinical and neurological exams, neurodevelopmental tests, and ophthalmologic and hearing evaluations. Blood, urine and breast milk samples were also collected during household visits (Table 1).

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Table 1. Study schedule of procedures for each study cohort.

https://doi.org/10.1371/journal.pntd.0010480.t001

ZIKV and DENV case ascertainment

Both ZIKV and DENV infections can be asymptomatic or subclinical in 70–80% of the infected population, or present as an acute illness with or without fever [12,13]. Therefore, we conducted active illness surveillance using a case definition of flavivirus-like illness (FLI) which included two or more of the following signs or symptoms: fever >38.0° C for ≥ 2 days, rash, conjunctivitis (non-purulent/hyperemic), arthralgia, myalgia, or peri-articular edema for ≥ 1 day. Surveillance was conducted by weekly home visits with collection of saliva samples every other week, regardless of the presence of symptoms (Fig 1). Caregivers responded to a standardized questionnaire designed to document the presence of FLI and other Integrated Management of Childhood Illness (IMCI) danger signs [14]. If a child or mother met defined FLI criteria, blood, urine and saliva samples were collected within 72 hours of illness report and a second time at the 2^nd, 3^rd or 4^th week after the first visit to evaluate seroconversion. The timing of the second visit was selected at random for each study subject.

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Fig 1. Organization of competency-based teams.

https://doi.org/10.1371/journal.pntd.0010480.g001

Challenges and lessons learned during the operationalization of the study protocol

After providing an overview of the study procedures, the following section describes the challenges, solutions and lessons learned as our team operationalized the study protocol. To identify challenges and solutions, our team of investigators (EA, FM, DO, MC) and study coordinators (AP, NR, EB, DB) held meetings after completing the enrollment phase to develop a comprehensive list of the most salient challenges faced during the study operationalization, which were summarized and iteratively reviewed by the study team until no other challenges were identified. For each of the challenges, we describe at least one solution that was implemented by our team. To accurately describe such solutions, the first author (AP) revised the manuals of procedures, minutes from team meetings held since the start of the study, administrative documents, and emails that documented key decisions on study operationalization made by the study team. We grouped challenges and solutions into the following categories: neurodevelopmental assessments, human resources, bioethical procedures, community engagement, data collection and quality control (See Table 2). Through a process of team discussions and reflections based on our experience conducting the present study and multiple other studies in Guatemala and other LMICs, we identified overarching lessons learned and recommendations that may guide other researchers and clinicians conducting prospective multi-disciplinary studies in other low-resource settings in response to emerging infections (See Table 3).

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Table 2. Challenges of operationalizing the study protocol and implemented solutions.

https://doi.org/10.1371/journal.pntd.0010480.t002

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Table 3. Lessons learned / Recommendations.

https://doi.org/10.1371/journal.pntd.0010480.t003

Adapting instruments for neurodevelopment assessments

While many options for neurodevelopmental assessment exist in higher-income countries, few tests have been standardized and validated for use in low resource areas, and none for the Guatemalan population and our study population age range [15,16].As this study aimed to evaluate children while the ZIKV epidemic was ongoing, the development of a new neurodevelopmental measure, which can take several years, was deemed impractical [17–19]. The process of the selection, translation, cultural adaptation, and use of neurodevelopmental measures for this study has been previously described in detail [20]. In brief, a multi-disciplinary team composed of psychologists with content, cultural, and local expertise, along with neuropsychologists from the UC, selected the most appropriate test from existing tools, considering the relevance of these developmental domains in the local community and the developmental domains targeted by the study. We selected the Mullen Scales of Early Learning (MSEL) as our primary performance-based developmental assessment measure. We also used two parent report measures: the Pediatric Quality of Life Questionnaire (PedsQL), which is a measure of health-related quality of life available in Mexican Spanish, and the Ages and Stages Questionnaire-Third Edition (ASQ3), a developmental screening tool available in Spanish. The MSEL was translated to Spanish and culturally adapted, while the PedsQL and ASQ3 were culturally adapted. Visual Likert scales were developed to aid parents’ understanding of questions and responses. Questionnaires were administered verbally to all parents to account for differences in levels of literacy and limited familiarity with neurodevelopmental assessments. Psychologists were trained in how to maintain standardization conducting assessments in a variety of settings, including our clinic, a converted bus, satellite clinics, and in the home. Data analysis demonstrated no significant differences in child performance between testing spaces. We conducted assessments of interrater reliability, internal consistency, and construct validity of the adapted and translated version of the MSEL among a subsample of 84 children 0–5 years old, which have been previously reported [21,22]. Our assessments showed strong psychometric properties suggesting that this test may be used validly and reliably within our population of children.

Training and managing human resources

Upon contract approval by NIH in January 2017, and due to the urgency to enroll participants in the study given the ongoing ZIKV epidemic, we proceeded to hire and train a physician who served as the study coordinator, three psychologists who adapted and conducted the neurodevelopmental evaluations, and one physician who conducted physical and neurological evaluations, focusing on personnel with expressed interest in research and public health. Two experienced research coordinators who held graduate degrees in nursing and were already hired by CHD became the field coordinators. We rapidly expanded our team from 4 to 20 study nurses who held graduate and technical-level degrees in nursing) and three new data managers, who were graduates from business administration or accounting due to the scarcity of trained data managers at the study site. Forty community health workers who were mothers recognized as leaders in the community, were financially compensated to serve as the bridge between study nurses and community members, helping to disseminate information about the study, identifying potential participants, sending study visits reminders, and communicating community concerns to the study team. All personnel, including community health workers, were trained in NIH human subjects protection (HSP), Good Clinical Practices (GCP) and principles of clinical research following the NIH regulatory training, the Special Programme for Research and Training in Tropical Diseases (TDR) Global Competency Framework for Clinical Research, and the WHO Ethics in epidemics, emergencies and disasters: Research, surveillance and patient care manual [23,24].

The study manual of procedures (MOP) and various standard operation procedures (SOPs) were developed, reviewed and refined as needed during the study operationalization. Study coordinators developed checklists summarizing visits and key procedures (e.g., enrollment visit, informed consent process, specimen collection and transport, data collection and electronic data entry) to train, supervise and provide feedback to the research staff on an ongoing basis. To ensure the implementation of multiple and complex procedures as required by the study protocol, we created competency-based sub-teams responsible for certain procedure clusters: recruitment and enrollment visits, symptomatic surveillance, FLI visits, and follow-up visits. Each sub-team included 2–6 study nurses and one field coordinator (Fig 1). Weekly and daily specific and measurable goals were established and monitored by each field coordinator to ensure that enrollment, retention and study procedures goals were met.

Psychologists received on-site training from a CU neuropsychologist at the start of the study and then periodically throughout. Weekly videoconferencing, video reviews of assessments, and quality checks then occurred to ensure high quality neurodevelopmental assessments. As the number of study visits and procedures increased, we trained one group of nurses on physical examination and another group to conduct ASQ3 and PedsQL assessments with supervision by physicians and psychologists. This division of labor allowed us to complete procedures in a rapidly growing number of study subjects (Fig 2) within the required visit timeframes, while psychologist and physicians focused on more complex procedures and complicated cases.

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Fig 2. Enrollment and follow-up visits by study week.

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A mentorship and training network with PIs, co-PIs and coordinators was established, to promote interest in research and public health among study staff. A strong culture of community service, continued learning and improvement, and team-based work was promoted throughout the study. This shared spirit ensured that study personnel stayed motivated to collaborate among sub-teams, and meet demanding study goals in a challenging environment.

Engaging community members

The community-based nature of this cohort study and the need to include villages in the study that were not familiar with CHD activities, required strong community engagement. During the 5-month preparatory phase, study coordinators and research nurses met with members of the local CABR and leaders of each of the 22 participating communities to explain the purpose and procedures of the study and to obtain feedback for its deployment. CHD established the CABR in 2015 to engage the local communities in all research and programmatic activities. The board is composed of community and religious leaders, teachers, and public health-post nurses who were trained by a consultant from the Colorado Multiple Institutional Review Board (COMIRB) on how to review research studies and advocate for their communities. Research nurses from the participating communities provided their local expertise and helped enhance the study buy-in. Local leaders, midwives and nurses identified and referred potential study participants and maintained communication with families enrolled in the study. Community collaborators were trained on the essentials of GCP and Human Subjects Protection by an anthropologist from CU.

Adapting bioethical procedures to local context

Communities in SWT are characterized by involving community leaders and families in decision making. For this reason, it was important to socialize the study informed consent to communities and families, before individuals’ consenting. Prior to initiating research activities, lead study staff obtained community buy-in by presenting and discussing the study aims, procedures, and potential risks and benefits with community leaders. A simple and culturally-appropriate visual informed consent tool (available on request) was used to explain the study objectives and procedures in Spanish (the local language) to potential participants, highlighting the differences between research and healthcare services to families with low literacy levels and limited or no previous exposure to medical research. Informed participants older than 18 years old provided informed consent prior to study procedures. If participating mothers were 16 to 17 years old, informed consent was obtained from one of their parents and assent was provided by the mother. If the participant providing consent was illiterate, a third party witnessed the consent process and signed the informed consent form, while the participant provided thumbprints. Our approach to community engagement and bioethical procedures facilitated the enrollment of 500 infants along with nearly 400 children 1.5–3.5 years of age with a high percentage of enrolled-to-screened participants and a low percentage of lost to follow-up participants. Results will be published in a future manuscript.

Dissemination of final results (aggregated and individual results to participants) was planned in collaboration with the Community Advisory Board for Research (CABR), community leaders and local public health facilities.

Collecting data and ensuring quality control

The study catchment area covered a large geographic zone, with deteriorated roads and limited transportation. For this reason, reducing participant mobilization and finding alternative sites, other than CHD clinic, to conduct study visits was needed. Visits were therefore conducted at multiple settings, including satellite clinics, a converted bus and participant’s homes. In order to collect large amounts of data in a location with limited access to internet, an electronic, smartphone-based CommCare app (Dimagi, Cambridge, MA, USA) was developed. Study staff collected data online or offline and uploaded it to an electronic server in real-time or as soon as internet was available, at least once daily. Research nurses completed most questionnaires (e.g., sociodemographic, medical history, illness visits, etc.), while trained psychologists and physicians documented neurodevelopmental and medical evaluations into paper-based source documents and later entered in the CommCare mobile application. In addition, information related to clinical visits and hospitalizations since the previous study visit was collected and entered into a secure local electronic REDCap database (Vanderbilt University, TN, USA) if participants consented.

To ensure adherence to GCP standards, a quality control plan was developed with a priority list of indicators for quality assurance. The quality control coordinator reviewed all the informed consent forms and 10–20% of the individual study records, conducting a comparison audit of the paper-based forms and the electronic databases. We aimed for 100% compliance with informed consent indicators. As data were entered in real-time, all completed data collection forms were reviewed daily by study coordinators. In addition, The Emmes Company, LLC, a data management group, updated online process quality reports daily or weekly allowing local data managers to address data issues (e.g., missing or duplicated forms, missing or extreme values, unexpected specimen samples) on an ongoing basis. Site specific report logs were maintained in a secure file sharing Website that provided real-time, site-specific feedback to identify errors and/or omissions in data entry and to facilitate corrections with minimal time delay.

Study coordinators conducted staff refresher trainings every two months as well as monthly field supervision of study staff procedures and data collection, while study investigators conducted random spot-checks of field work and home visits. Feedback through on-the-spot discussion of supervisors’ findings, weekly staff meetings, and monthly written reports to all staff and external collaborators was done with the aim of ensuring high quality performance of study procedures.

During weekly meetings, study progress, challenges and solutions were discussed among leaders from the field team, CU, Baylor University, Emmes, and NIH representatives. In addition, principal investigators conducted monthly on-site supervisions.

Building laboratory capacity for biological sample collection and processing

Trained research nurses collected blood, saliva, urine and breast milk samples at the participants’ households or at the satellite clinics per study protocol (Table 1). Given the high ambient temperature (25–38°C), long transit times and harsh environment at SWT, a strict protocol for ensuring quality collection of samples and maintaining cold chain in the field was developed, allowing for 95% of samples to meet with temperature requirements. Whole blood was collected in EDTA tubes for ZIKV and DENV serologic and molecular testing and processed to obtain plasma for ZIKV serologic and molecular testing, and other congenital infections. Saliva was collected using a CultureSwab Collection and Transport System by brushing the participants’ cheek and soaking it with saliva for at least 20 seconds, and the sample was then mixed with 200 uL of Invitrogen (Thermo Fisher Scientific, Carlsbad, CA) nucleic acid stabilizer buffer. Urine and breast milk samples for ZIKV molecular testing were collected in sterile containers. All samples were transported in temperature-controlled portable coolers and delivered to the study laboratory for storage at 2–8°C until processing. Samples were stored at -80°C from processing until international shipment. Retained blood samples included whole blood and plasma aliquots. Samples were shipped periodically to the NIH sample repository (Fisher Bioservices, Maryland, USA) preserving the cold chain and following international guidelines for shipping biologic materials. Specimen analyses included IgM, IgM, neutralizing antibodies and PCR.

In 2015, the CHD began enhancing its research capacity by building a 500 square foot (sqf) research laboratory and 1500 sqf research operations area for personnel, data management, secure document storage and meetings, with the support of the CU Strategic Initiative for Research. While the CHD has a connection to the power grid and a high-capacity generator, the stability of energy supply was reinforced by two additional generators to safeguard samples stored at the laboratory. During the conduct of this study, a solar-based energy system was installed. The laboratory had the capacity to process and store approximately 10,000 samples or aliquots, stored at -20°C or -80°C.