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Biological and contextual determinants of early development in marginalized Roma communities: A research protocol of the RomaREACH study

International Journal for Equity in Health volume 23, Article number: 200 (2024) Cite this article

Abstract

Background

The period of early childhood bears significant importance from the lifespan perspective. Children from marginalized Roma communities face several risk factors that endanger their early development. Based on the gaps in available evidence, the aim of the RomaREACH research project (Research on Early Childhood in marginalized Roma communities) is, therefore, to explore the complex mechanisms influencing psychomotor development in the first 3 years of a child’s life in marginalized Roma communities, and to translate and adapt instruments for measuring development and parenting in marginalized Roma communities and assess their psychometric qualities and suitability

Methods

The project comprises two parts. The first part is a validation study of the translated Caregiver-Reported Early Development Instrument (CREDI) and the Comprehensive Early Childhood Parenting Questionnaire (CECPAQ), tools for the assessment of early development and of parenting strategies and practices. The second part is a longitudinal cohort study, in which the relationships of risk and protective factors with development are explored.

Discussion

The RomaREACH project is a multicomponent study of social determinants of health and development in early childhood that can provide new evidence on the relationship of risk and protective factors with early development. Such young children from difficult-to-reach marginalized Roma communities are rarely included in research, and information about the scope and the extent of inequities in health and development in the period of early childhood is scarce. The expected results of the RomaREACH project have the potential to influence policy and practice by providing validated tools and evidence-based insights that can help mitigate the developmental risks faced by children in marginalized Roma communities and contribute to improving developmental outcomes and equity.

Background

The period of early childhood bears great importance for a person’s quality of life throughout the lifespan, as the first years of a child’s life lay the foundation for further cognitive, socioemotional, behavioral, personal, motor and language development, with a later impact on academic and professional achievement [1]. In this period, a child is especially sensitive to risk and protective factors from the environment (Fig. 1, Shonkoff, 2010).

Fig. 1
figure 1

A biodevelopmental framework for understanding disparities in learning, behavior and health (Shonkoff, J. P. (2010). Building a New Biodevelopmental Framework to Guide the Future of Early Childhood Policy. Child Development, 81(1), 357–367.)

Full size image

Certain groups of children are particularly vulnerable, especially if there are multiple risk factors present in their environment. Children from marginalized Roma communities are a specific example of such a vulnerable group. Marginalized Roma communities (MRCs) regard separated or segregated groups within the Roma population who experience exclusion from mainstream social, economic, political, educational and cultural life [2]. According to the Atlas of Roma Communities published by the Slovak Government Plenipotentiary for Roma Communities and providing a detailed mapping and data on the distribution and conditions of Roma settlements across Slovakia, there are over a thousand smaller to bigger MRCs characterized by varying degrees of exclusion [3]. In Slovakia, as well as in other CEE countries, the health and development of children in marginalized Roma communities are influenced by severe poverty, lower quality living conditions and barriers to access to education, health care and the labor market [4,5,6,7]. Living in MRCs seriously damages health and health-related behaviors, regardless of socioeconomic differences within the community itself [4]. However, family affluence and the material conditions of families within such communities can still vary on the lower strata [8]. The use of health care is 36% lower among members of MRCs than in the nationwide population [6], which points to barriers in access to health care. The poorer health status of Roma from MRCs is also reflected in a shorter average life expectancy. Roma from MRCs who do not receive social support live on average 70.3 years; for social support recipients from MRCs this is only 67.7 years. This is an abysmal difference compared to the general Slovak life expectancy, which is 76 years [6].

Health inequalities between members of MRCs and the majority population in Slovakia have been shown to start as early as from pregnancy onward. Infant mortality is more than three-times higher in municipalities with a dominant Roma population compared to municipalities with low or no Roma population, and neonatal mortality is 2.5-times higher [9, 10]. Roma children are more often at risk of Sudden Infant Death Syndrome (SIDS), accounting for up to 24.2% of all SIDS deaths in Slovakia. Among the most significant factors contributing to the risk of SIDS in Roma children are maternal smoking during pregnancy and poor sanitation conditions [11]. Roma children are more likely to suffer from infectious diseases, injuries, poisonings and burns, respiratory diseases and chronic diseases. Approximately 60% of all hospitalized children are Roma; among infants, Roma make up to 80% [9] of hospitalized children, whereas the share of Roma children in the general population is estimated to be 13.3% [12]. According to stakeholders, promoting healthy early childhood development in MRCs needs to be targeted by coordinated efforts, focusing on living conditions, community interventions, quality and accessible health care and public resources. However, multiple barriers exist preventing the implementation of such coordinated efforts [13].

Extremely rapid brain development in the first years of life causes increased sensitivity to environmental conditions. In addition, the daily life of a young child takes place mainly in the home environment, so the quality of this environment in this period is crucial for further development [14]. Therefore, the effects of poverty experienced in early childhood may be significantly stronger than of poverty experienced later in life. Several biological and psychological mechanisms can potentially explain this relationship between poverty and outcomes in the early childhood of Roma.

Biological mechanisms include allostatic load as a consequence of psychosocial stressors, chronic inflammation, poor nutritional status and exposure to toxic substances. Allostatic load is the degree to which the exposure to chronic stress burdens the organism through disruption of the neuroendocrine, cardiovascular, metabolic and immune systems [15]. Chronic exposure to stress causes high allostatic load, which cumulates throughout life, with a negative impact on multiple biological systems throughout the lifespan [16, 17]. Chronic inflammation caused by frequent and numerous insults in the prenatal period and in early life may lead to impairments in neurodevelopment [18]. This effect can be observed in poorer outcomes in the motor and cognitive development of children with recurrent and chronic febrile, diarrheal and parasitic diseases [19,20,21,22,23]. Children from MRCs are disproportionately more at risk of parasitic diseases than their peers from better socioeconomic conditions [24,25,26,27]. Poverty and socioeconomic disadvantage also contribute to food insecurity, resulting in poor nutritional status, based on the deficiency of macronutrients and micronutrients essential for growth and neurodevelopment [28]. Another biological risk factor for children from disadvantaged settings is exposure to toxic substances, which can have major negative effects on brain function and neurodevelopment in early childhood [29,30,31,32,33,34]. The inhabitants of MRCs are at disproportionately higher risk of being exposed to toxic substances, as they often live near landfills [35] and industrial buildings, and because they heat with solid fuel, often including waste materials [36].

Psychological mechanisms, such as responsive parenting, can both mediate and moderate the negative influence of poverty on early health and development. Responsive parenting is considered to have a key role in shaping secure attachment [37], regardless of the social and cultural context [38, 39]. Secure attachment in early childhood becomes a basis for mental health in later life [39] and is a basic psychological need in early childhood [40]. Caring relationships are essential for all young children but are of particular importance to children living in poverty, who are at greater risk of health problems and developmental delays [41]. Responsive parenting acts as a buffer to protect a child from the long-term effects of stress [42] and reduces the detrimental immunological, epigenetic and metabolic effects of chronic stress [43]. However, it might be more difficult to put responsive parenting into practice in stressful situations [44], such as those related to living in poverty.

The links between exposures related to early life poverty and developmental outcomes have not yet been studied in the specific population of the children of marginalized Roma. In order to carry out research on early childhood development (ECD) in MRCs, valid, reliable and culturally sensitive tools are needed to measure ECD and the parenting strategies of Roma living in such communities. Slovakia lacks a valid ECD assessment tool that is easy to administer and culturally sensitive for use in both the non-Roma majority and the MRCs. The National Developmental Screening (S-PMV; [45]) that was introduced in 2019 [46] as a part of mandatory preventive pediatric visits has not been validated, and to date no data have been collected on its cultural sensitivity in Roma children. In addition, no data on ethnicity is collected during the administration of this screening questionnaire; therefore, no comparisons can be made between groups. However, direct testing of psychomotor development, for example with Bayley’s developmental assessment, requires intensive training of administrators, is time-consuming and costly, and is burdened by language barriers [47]. In addition, it is standardized for a different sociocultural environment and some items are not suitable for an environment of socioeconomic deprivation [47]. Therefore, using the questionnaire method in order to limit deviations in the performance based on a child’s stress and the bias caused by cultural differences between the researchers and the children is more feasible. Additionally, a questionnaire makes it possible to capture a child's more general abilities in different situations and times and is not subject to issues with motivation and cooperation, as might be the case in the direct assessment [48]. Parents are generally reliable reporters of their child’s skills and abilities, even when their child has a developmental disorder [49]. Based on this rationale, the Caregiver-Reported Early Development Instrument (CREDI) was chosen by the research team as the most fitting instrument. CREDI has been translated to 41 languages, has been used in 26 countries and is planned to be used in additional 18 countries with communities of different affluence [50,51,52,53,54,55].

The Comprehensive Early Childhood Parenting Questionnaire was chosen by the research team to assess parenting strategies and practices, as it is the only available instrument assessing 5 different domains of parenting central to the period of early childhood, with items focused specifically on the period between 1 and 4 years of age [56]. These characteristics allow the relationships between parenting practices specific for the period of early childhood and developmental outcomes of the children to be explored.

Based on the gaps in available evidence, the aim of the RomaREACH research project (Research on Early Childhood in marginalized Roma communities) is, therefore, to explore the complex mechanisms influencing psychomotor development in the first 3 years of a child’s life in marginalized Roma communities, and to translate and adapt instruments for measuring development and parenting in MRCs and assess their psychometric qualities and suitability.

Methods

In order to address the aims above, the RomaREACH project comprises two parts: (1) the validation study of the translated versions of the two questionnaires used in the second component of the research project, namely the Caregiver-Reported Early Development Instrument (CREDI) and The Comprehensive Early Childhood Parenting Questionnaire (CECPAQ); and (2) a longitudinal study exploring the mechanisms that influence the early development of children under the age of 3 in MRCs.

Part 1. Validation study

The aim of the validation study was to translate and adapt the Slovak version of the Caregiver-Reported Early Development Instrument (CREDI) and the Comprehensive Early Childhood Parenting Questionnaire (CECPAQ) and to assess their psychometric properties and the suitability of their use in both the general population and in MRCs in Slovakia.

Sample

In this study, 400 mother–child dyads will be included, i.e. 250 mothers from the Slovak majority population and 150 Roma mothers from segregated communities. Individual interviews (N = 30) will be carried out with selected mothers, and 10 observations of the children will be performed.

Procedure

For this part of the research project, the procedure consists of the translation and adaptation of the CREDI and CECPAQ instruments and the collection of data.

Translation and adaptation of the CREDI and CECPAQ

The standard forward translation–back translation procedure [57] will be used to translate the CREDI and CECPAQ from English into a Slovak version to be further used both in the Roma and non-Roma population. To ensure cultural sensitivity, understandability and appropriateness of the items in the sample of Roma mothers from the segregated communities, adaptations will be made, including translation of the CREDI Item Guide with item stem explanations into the Roma language and assisted administration of the questionnaires for women with low literacy. In order to confirm the appropriateness of use and to support the construct validity of the selected instrument in the marginalized Roma communities, the translation of the CREDI and CECPAQ will be discussed with professionals with relevant expertise, in accordance with the ITC guidelines for Translating and Adapting Tests [57].

Data collection

Participants (see Sample) will be recruited in cooperation with Roma health mediators and community centers, as well as online, using forums for parents. The questionnaire, including the CREDI, questions on socioeconomic status and demography, and the CECPAQ, will be then administered to mothers of children aged 0 – 3 years both on paper and online. In the case of Roma mothers with lower literacy, researchers will assist with the administration of the questionnaires. Individual interviews will be carried out with selected mothers, with questions focused on the clarity of the items and social desirability. Several randomly selected mother–child dyads will be observed in their home settings using structured observation and actively watching for behaviors targeted in the questionnaire in order to validate their answers. The questionnaire will be used to guide the behavioral observations. We will code responses using a coding scheme derived from CREDI questionnaire and inter-rater reliability calculated between the observation of the researcher and the report of the parent in CREDI.

Measures

CREDI

The Caregiver-Reported Early Development Instrument (CREDI), the long form, has been used to assess motor, cognitive, language and social-emotional development of children aged 0 – 36 months and consists of total 117 items (108 age-specific items for motor, cognitive, language and social-emotional development, and 9 items for the mental health for all children regardless of age). The items are divided into 6 age levels (0 – 5 months, 6 – 11 months, 12 – 17 months, 18 – 23 months, 24 – 29 months, 30 – 35 months) [50]. For the purpose of validation, the long form of the CREDI will be used, with the first item administered being the first item intended for the given age level. In case of a negative answer on the first item for the given age level, the first item administered will be the first item for the previous age level. The last item administered will be the fifth subsequent item with negative answer. If the child does not score negatively for five subsequent times, the instrument is administered in its entirety.

For the early development measurement, the CREDI was chosen out of all currently available development instruments because of its cultural sensitivity, language variability, parental literacy and time- and cost-effectiveness with regard to the set goals. The CREDI is a fast and easy-to-understand tool for detecting early development. Its good psychometric characteristics and international use make it possible to compare the results in our research groups with other groups of children, as it has been validated in 17 different low-, middle-, and high-income countries, is used in 26 countries and planned to be used in further 18 countries [50,51,52,53,54,55]. The average test–retest reliability for the final set of selected items was kappa = 0.62 (SD = 0.13, range = 0.41–0.86). The internal consistency (Cronbach’s alpha) of the CREDI was 0.89 for 0–5 mo, 0.86 for 6–11mo, 0.84 for 12–17 mo, 0.86 for 18–23 mo, 0.84 for 24–29 mo, and 0.80 for 30–35 mo [50]. To compute scores for overall development and various developmental domains, we will use the CREDI scoring application (credi.shinyapps.io/Scoring_App/), which will incorporate multidimensional domain loadings and allow individual items to inform multiple developmental domains. Subsequently, the raw scaled scores will be used as outcomes in the statistical analyses for hypothesis testing following the recommendations provided by the CREDI authors [58]. This instrument does not provide cut-off points to identify children at risk. Nevertheless, the z-scores delivered by the CREDI scoring application are norm-referenced standardized scores. These will be use to compare the scores of our sample to those from a CREDI reference group, which includes 4,652 children from advantageous home environments. A z-score of 0 will indicate that a child’s developmental status in that domain is comparable to the average developmental status of children of the same age in the CREDI reference sample [58].

CECPAQ

The CECPAQ was developed by researchers at Utrecht University to measure the parental behavior of parents of children aged one to five. The CECPAQ is composed of 54 items and focuses on 5 domains of parental behavior, which are of great importance for the optimal development of children at an early age. Specifically, it captures support (sensitivity, responsiveness, affection), stimulation (activities, exposures, toys), structuring (consistency, absence of negligence, and exaggerated reactions), positive discipline (leadership, positive reinforcement) and harsh discipline (physically, verbally and psychologically aggressive punishments). A validation study on a sample of Dutch parents showed the good psychometric characteristics of the instrument. Model fit indices were only acceptable for the 5-factor solution. All lower-order factor loadings were statistically significant (p < 0.001), with standardized loadings ranging from β = 0.43 to β = 0.92. In addition, the factor loadings regarding the higher order factors were all statistically significant (p < 0.001) and ranged from 0.66 to 0.96. The correlations between the parenting domains were all statistically significant (p < 0.001) and ranged from r = − 0.14 to r = − 0.71. Cronbach’s alpha’s for mothers/fathers were respectively: 0.88/0.88 for Support, 0.82/0.86 for Stimulation, 0.75/0.77 for Structure, 0.79/0.79 for Harsh Discipline, and 0.76/0.77 for Positive Discipline [56]. Similar results were confirmed by the validation study of the Chinese version of the tool [59]. The tool is currently translated and used in Chinese, Spanish, Persian, Turkish, Hungarian, Italian and Slovenian.

Other variables

Gold standards for measuring early development will not be used, as these have not been previously standardized for use in Roma. We further collected standard demographic data, data on the socioeconomic situation of the family (employment, education, household equipment, household indebtedness, problems with monthly payments, number of household members, overcrowding), and data on environmental stimuli (toy presence, book presence, coloring books, visits to preschools, children’s groups) as background characteristics within the questionnaires reported by mothers.

Analysis

To assess the internal consistency of the CREDI, Kuder-Richardson 20 coefficients will be computed to confirm the internal consistency for each of the five factors. In order to confirm the structure equivalence, confirmatory factor analysis will be performed, and the factor structures of the original version, the Slovak version used in non-Roma mothers and the Slovak version used in Roma mothers, will be compared. In order to assess criterion validity, the CREDI scores will be correlated with the CECPAQ scores and socioeconomic status using Pearson correlation. Item Response Theory analysis will be performed to compare the differential item functioning (DIF) between participants from the MRCs and the majority population and to confirm the item-level validity. Interviews will be analysed using a combination of conventional and directed content analysis as a combination of inductive and deductive research method [60]. The questionnaire will be used as a structure for behavioral observation and inter-rater reliability calculated between the observation of the researcher and the report of the parent in CREDI.

Part 2. A longitudinal study of early development

Sample

For the longitudinal study, children aged 15–18 months at baseline will be recruited (T1). The sample groups will consist of children from MRCs (N = 150), from the middle-class majority (N = 150) and from the low-SES majority (N = 150) in the Košice and Prešov regions. The participants will be recruited during mandatory preventive examinations through their pediatricians. The same participants will be contacted at 30–36 months (T2) again.

Measures and procedures

At T1, data will be collected using questionnaires on background information on the family’s socioeconomic situation (employment, education, household equipment, household indebtedness, billing problems, number of household members, overcrowding), environmental stimuli (toy presence, book presence, coloring books, visits to preschools, children’s groups), on the child’s psychomotor development (CREDI), on parenting, upbringing and nutrition (CECPAQ, breastfeeding, feeding, quality of food), quality of relationships and social support in the family and community, health-related behavior of parents, including maternal behavior during pregnancy and breastfeeding (smoking, alcohol and other psychoactive substance use), and familial stressors (adverse events, SES-related stressors, COVID-related stress).

Additionally, anthropometric, anamnestic and clinical data from the extended preventive examination will be collected. Anthropometric measurements regard body growth and can be considered as indicators of nutrition. These include weight, height and head circumference. Data extracted from clients’ medical records include gestational age, APGAR score, birth length and weight, parity, complications during pregnancy and childbirth, former hospitalizations, former illnesses (febrile, respiratory, diarrhea, antibiotic use, eczema, chronic illnesses, allergies) and previous psychomotor development. Clinical data include parameters assessed in blood samples of the child, hair cortisol and the presence of parasites in stool samples (cysts, oocysts of protozoan parasites including detection of Cryptosporidium spp., eggs of helminths). In blood samples, indicators of immune system activation in acute and chronic inflammatory processes, the markers of parasites and allergies (high-sensitive CRP, IL-6, α1-acid glycoprotein—AGP, eosinophils, and IgE antibodies) and indicators of nutritional status (iron, zinc, iodine, vitamins A and D) will be assessed. The procedures and kits used for assessing the parameters in blood, stool and hair samples are described in more detail in the Appendix.

Statistical analysis

Descriptive statistical methods will be used to describe the research sample. To explore the complex mechanisms influencing psychomotor development in the first 3 years of a child’s life in the MRCs, correlation coefficients, univariable and multiple regression, and mediation and moderation analyses will be applied to assess the relationships of the anthropometric, anamnestic and clinical variables with the developmental outcomes. Specifically, we will use linear regression models to explore differences in development (as measured with the CREDI) between groups, adjusting for mediators and moderators such as socioeconomic status, parenting behavior, maternal stress, nutritional status, and markers of inflammation. Significant relationships will be further examined to identify potential mediation pathways and moderating effects. Given the longitudinal nature of the data, the relationships between the variables will be examined using structural equation modelling. Statistical analyses will be performed using IBM SPSS, PROCESS Macro and the programming language R.

Ethics and dissemination of all parts

Each of the components of the research project has obtained separate approval from the ethics committees. The validation study has obtained approval from the Ethics Committee of the Faculty of Medicine at Pavol Jozef Šafárik University in Košice. The longitudinal cohort study was approved by the Ethics Committee of the Faculty of Medicine at Pavol Jozef Šafárik University in Košice and the Ethics Committee at the Košice Self-governing Region.

The study investigators have significant previous experience with the dissemination of scientific findings among the scientific, professional and general public. The team of investigators strives to publish the findings of this research in scientific journals and as a monograph, to share them at scientific conferences and also to share them with the general public in an accessible and understandable way. Previous relationships with policymakers and practitioners were established and are further developed in order to ensure knowledge translation.

Hypotheses

Based on the current state of knowledge, we formulated the following hypotheses:

  1. (1)

    Children from MRCs will have a delay in all of the domains of psychomotor development compared to their peers from the majority control group due to exposure to health-damaging factors resulting from living in conditions of socio-cultural disadvantage at the age of 15–18 months and at age 30 – 36 months.

  2. (2)

    Differences in psychomotor development between children from MRCs and the control group increase between 15 – 18 and 30 – 36 months.

  3. (3)

    Psychomotor development is negatively influenced by all examined risk factors, including maternal stress, socioeconomic status, parasites, insufficient nutrition, and inflammation, endangering the health and healthy development of children.

  4. (4)

    Nutritional profile, inflammation, and presence of parasites will be associated with delays in psychomotor development and worse health outcomes in T1 and predicting further delays in psychomotor development in T2 in children from MRCs compared to the majority control group.

  5. (5)

    Responsive parenting will serve as a buffer of the impact of toxic stress on psychomotor development in early childhood.

Discussion

The RomaREACH project focuses on ECD in MRCs in Slovakia and consists of two parts: a validation study of the used measures and a longitudinal prospective cohort study. This study is innovative because it focuses on some previously unexplored relationships. While many studies have previously explored the relationships between socioeconomic status, prenatal exposures, immune system activation, parasitic disease, nutrition, early life stress, parenting strategies and ECD, these relationships have not yet been explored in the context of MRCs to this extent. Moreover, previous studies were more narrowly focused on assessing separate factors influencing health and development in early childhood, rather than trying to explore more complex mechanisms.

The use of culturally sensitive and validated measures, a longitudinal design and a wide set of relevant indicators will enable us to explore the long-term effects of socioeconomic disadvantages, environmental exposures, health risks and parental practices on early development, while accounting for potential bias caused by different ethnic and socioeconomic groups being included in the study. An important strength of the RomaREACH project is its multidisciplinary approach by a team of investigators consisting of public health researchers, psychologists, epidemiologists, physicians, biochemists and parasitologists. The different backgrounds of the investigators mean that the topic can be explored from different perspectives, allowing for a richer interpretation and application of the knowledge gained into policymaking and practice. Another strength of this project stems from its multimethod nature, which enables examining the topic from several perspectives and integrating knowledge from different sources. Finally, a strength of this research project lays in the variability of the sample, with focus on the inclusion of participants of different socioeconomic status, from both urban and rural settlements of various sizes, allowing for greater generalizability.

The biggest limitation of the RomaREACH project is that the sample is only recruited from one self-governing region of Slovakia, which may affect the generalizability of the findings to all the MRCs in Slovakia or elsewhere. Given the heterogeneity of MRCs, future results should be generalized with caution. The research team will try to address this limitation by recruiting participants from different types of settlements in order to account for the variability. Another limitation of the project stems from the cultural distance between the participants from an ethnic minority and researchers of a different cultural background, which can result in bias. The research team will try to address this problem by consulting the members of the target group on all the steps of the project.

The RomaREACH project is a unique project focusing on difficult-to-reach children from MRCs, that are rarely included in research. The project aims to adapt valid, reliable and suitable instruments for measuring development and parenting, which can be used for both the general population and segregated Roma in Slovakia, and to explore the complex mechanisms influencing the early development of children in MRCs.

Availability of data and materials

Not applicable, as participant recruitment and data collection is still ongoing.

Abbreviations

RomaREACH:

Research on Early Childhood in Marginalized Roma Communities

MRCs:

Marginalized Roma Communities

CREDI:

Caregiver-Reported Early Development Instrument

CECPAQ:

Comprehensive Early Childhood Parenting Questionnaire

CEE:

Central European countries

SIDS:

Sudden infant death syndrome

ECD:

Early childhood development

ITC:

International Test Commission

DIF:

Differential item functioning

SES:

Socioeconomic status

COVID:

Corona virus disease

CRP:

C-reactive protein

IgE:

Immunoglobulin E

AGP:

α1-Acid glycoprotein

IBM SPSS:

IBM Statistical Package for Social Sciences

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Acknowledgements

Not applicable.

Funding

This work was supported by the Slovak Research and Development Agency under Contract no. APVV-19–0493 and by the Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences, Reg. No. 1/0593/21. These agencies are public and state-funded organizations, they provide funding based on assessment of the research protocol and the research team reports the results and financial management to the agencies.

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Authors and Affiliations

  1. Graduate School Kosice Institute for Society and Health, PJ Safarik University, Trieda SNP 1, Kosice, 040 11, Slovak Republic

    Shoshana Chovan, Daniela Fiľakovská Bobáková & Andrea Madarasová Gecková

  2. Department of Health Psychology and Research Methodology, Medical Faculty, PJ Safarik University, Trieda SNP 1, Kosice, 040 01, Slovak Republic

    Shoshana Chovan, Daniela Fiľakovská Bobáková & Andrea Madarasová Gecková

  3. Olomouc University Social Health Institute, Palacky University in Olomouc, Univerzitni 22, Olomouc, 771 11, Czech Republic

    Daniela Fiľakovská Bobáková

  4. Faculty of Social and Economic Sciences, Institute of Applied Psychology, Comenius Universityin Bratislava, Mlynske Luhy 4, Bratislava, 821 05, Slovakia

    Andrea Madarasová Gecková

  5. Department of Medical and Clinical Biochemistry, PJ Safarik University, Trieda SNP 1, Kosice, 040 11, Slovak Republic

    Beáta Hubková

  6. Department of Epizootiology, Parasitology and Protection of One Health, University of Veterinary Medicine and Pharmacy in Kosice, Komenskeho 73, Kosice, 040 01, Slovak Republic

    Gabriela Štrkolcová

  7. Department of Community & Occupational Medicine, University Medical Center Groningen, University of Groningen, Antonius Deusinglaan 1, Groningen, 9713 AV, the Netherlands

    Sijmen A. Reijneveld & Marlou L. A. de Kroon

  8. Department of Environment and Health, Youth Health Care, University of Leuven, KU Leuven, Kapucijnenvoer 35, Louven, 3000, Belgium

    Marlou L. A. de Kroon

Authors
  1. Shoshana Chovan
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  2. Daniela Fiľakovská Bobáková
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  3. Andrea Madarasová Gecková
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  5. Gabriela Štrkolcová
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  7. Marlou L. A. de Kroon
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Contributions

SC, BH and GŠ was responsible for the study design and manuscript writing. DFB, AMG, SAR and MLAdK were responsible for the study design and manuscript review.

Corresponding author

Correspondence to Shoshana Chovan.

Ethics declarations

Ethics approval and consent to participate

This study was approved by the Ethical Committee at the Faculty of Medicine, University of Pavol Jozef Šafárik in Košice on 24th February 2020 under the No. 8N/2020 and on 18th October 2021, under the No. 16N/2021. The study was also approved by the Ethical Committee of Košice Self-governing Region on 26th August 2021 under “RomaREACH” and by the Ethical Committee of Prešov Self-governing Region on 2nd June 2022 under the No. 03682/2022/OZ-20. The study was also approved by the Ethics Committee of Children’s Hospital in Kosice on 27th June 2022.

Informed consent was obtained by the legal guardians of the participants in written form, one for the participation of the child and one for the participation of the parent. Specific consent forms were formulated for each of the phases of the project and for each of the data collection scenarios.

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Not applicable.

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The authors declare no competing interests.

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Appendix

Appendix

Blood samples will be collected under standard conditions from venous blood after overnight fasting in pyrogen/endotoxin-free serum separator tubes. After clot formation, the samples will be centrifuged at 3000 rpm for 15 min and serum will be collected. To avoid repeated freeze–thaw cycles, undiluted serum samples will be stored at -70 °C in aliquots. The following blood parameters will be analyzed in the collected serum samples using the kits: human thyroid stimulating hormone (RCD028R – Human Thyroid Stimulating Hormone ELISA kit, BioVendor), vitamin A (MBS729269 – Human Vitamin A ELISA kit, MyBioSource), 25-hydroxy-vitamin D (RIS020R – 25OH Vitamin D Total ELISA kit, BioVendor), interleukin-6 (RD194015200R – Interleukin-6 Human ELISA, BioVendor), alpha-1 acid glycoprotein (PRB-5044 – Human Alpha 1 Acid Glycoprotein ELIOSA Kit, Cell Biolabs), C-reactive protein (ab260058 – Human CRP SimpleStep ELISA kit, Abcam), ferritin (ab108698 – Ferritin Human ELISA kit, Abcam), zinc (ab 102,507 – Zinc Quantification Kit, convenient colorimetric assay, Abcam), hemoglobin (ab234046 – Hemoglobin Assay Kit, colorimetric assay, Abcam), total iron-binding capacity (ab239715 – Total Iron-Binding Capacity and Serum Iron Assay Kit, colorimetric assay, Abcam), transferrin (ab187391 – Human Transferrin SimpleStep Elisa Kit, Abcam).

Hair cortisol will be assessed in the hair samples of the mothers. A 3 cm segment of hair with a minimum weight of 50 mg will be cut with fine scissors as close as possible to the scalp from a posterior vertex position. A minimum of 50 mg of hair will be put into a 15 ml Falcon tube and 2.5 ml of isopropanol will be added. Subsequently, the tube will be gently mixed on an overhead shaker (Polymax 1040, Heidolph) for 3 min. After decanting, the wash cycle will be repeated two more times. After the wash, the hair samples will dry for at least 12 h. The washed and dried hair samples will be powdered using a Fritsch ball mill for 5 min at 30 Hz. Individual powdered hair samples will be placed in glass vials and weighed. Then 1.5 ml of pure methanol will be added per 50 mg of powdered hair in vials and the vials will be slowly rotated over 24 h for steroid extraction. Samples will spin in a centrifuge (Z 326 K, Hermle) at 10,000 rpm for 2 min and 1 ml of the clear supernatant will be transferred into a new 2 ml cryovial. The methanol will evaporate at 40 °C under a constant stream of nitrogen for 30 min or until the samples are completely dried. The dry residues will be re-suspended in 0.4 ml of distilled water and the tubes will be vortexed for 15 s, until completely dissolved. The samples will be stored at -70 °C until the hair cortisol measurement is carried out by the ELISA method (EIAHCOR – Cortisol Competitive Human ELISA kit, Invitrogen).

Parasites will be assessed in the stool samples of children. All of the children’s samples will be examined by applying the flotation concentration technique for the presence of oocysts, cysts of protozoan parasites and eggs of helminths, as well as for the qualitative evidence of coproantigen of Cryptosporidium spp. carried out by the ELISA method. The flotation concentration method is based on the principle of differences between the specific gravity of propagative stage of parasites (oocysts, cysts and eggs) and the specific gravity of a flotation solution. The propagative forms have lower specific gravity and therefore float to the top of the solution. The samples collected from the children will be processed by applying the flotation method with the use of two flotation solutions; in particular, zinc sulphate (specific gravity of 1.18 g.cm–3) for the diagnostics of the cysts of Giardia duodenalis, and a flotation solution with a specific gravity of 1.24 g.cm–3 for the diagnostics of oocysts of protozoan parasites and eggs of helminths. Subsequently, the samples will be microscopically examined (Garcia and Bruckner 1997). Detection of the presence of coproantigen of Cryptosporidium spp. will be carried out by the ELISA method with the use of the CRYPTOSPORIDIUM (FECAL) commercial assay by Diagnostic Automation, INC, Calabasas, USA, which is used for the qualitative detection of cryptosporidium antigen in the feces of animals and humans. The assay sensitivity is 93% and the specificity is 98%. The sandwich ELISA is based on the principle of a highly specific antigen–antibody reaction, wherein one of the two binds to an enzyme (most frequently a peroxidase or an alkaline phosphatase). The antigen is bound to an insoluble carrier of the micro-titration plate by covalent bonds. The enzyme catalyzes chemical transformation of the substrate added to the reaction mixture into a colored product. The plates will be washed using the DYNAWASH AM60, Multi-Reagent Washer (DYNEX Technologies Inc., USA). The reaction will be assessed by measuring the absorbance using the ELISA reader (OPSYS MR Thermo Labsystems) at a wavelength of 450/630 nm. The samples that exhibit the absorbance of 0.15 OD units and more will be regarded as positive.

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Chovan, S., Fiľakovská Bobáková, D., Madarasová Gecková, A. et al. Biological and contextual determinants of early development in marginalized Roma communities: A research protocol of the RomaREACH study. Int J Equity Health 23, 200 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12939-024-02287-0

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International Journal for Equity in Health

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