Variable indicator matrix for primary, secondary and other outcomes
| Variables | Indicator | Method |
| Primary outcomes | ||
| Nutritional status | BMI for age z-score weight | Anthropometric measurement (analysed with WHO AnthroPlus) |
| COVID-19 antibody response | IgG titre concentration against SARS-CoV-2 | Blood sampling electrochemiluminescence immunoassay (ECLIA) |
| Secondary outcomes | ||
| COVID-19 antibody response | sIgA | Saliva sampling Competitive ELISA |
| Immunity affinity (Baseline and endline) | Antibody affinity (monoclonal antibody affinity against SARS-CoV-2 spike protein) | Blood sampling Surface plasmon resonance (SPR) determined antibody binding or antibody affinity to prefusion SARS-CoV-2 spike protein |
| Gut microbiota profiling (baseline and endline subsample) | α and β-gut microbiota diversity | Faecal sampling 16S rRNA, NGS α-diversity: (1) Chao1, (2) phylogenetic diversity and (3) the Shannon index β-diversity: as the variability in community composition (the identity of taxa observed) among samples |
| Dietary intake (baseline, monitoring and endline) | Nutrient intake, dietary quality: Healthy Eating Index | 24 hours recall and Food Frequency Questionnaire |
| Other outcomes | ||
| SARS-CoV-2 infection | Proportion of symptomatic COVID-19 and positive PCR | Reported swab PCR (secondary data from healthcare centres) |
| History of COVID-19 vaccination | Time of first and second dose of COVID-19 vaccine Time of COVID-19 vaccine booster Type of vaccine booster | Interview with questionnaire (proven by vaccine certificate) Interview with questionnaire Interview with questionnaire |
| Nutritional status | Height for age z-score, waist circumference, MUAC, WHR, WHtR | Anthropometric measurement (analysed with WHO AnthroPlus) |
| Body composition | Weight, body fat, muscle mass, visceral fat level (analysed by BIA) | |
| COVID-19 antibody response | IgM Total neutralising antibody titre | Blood sampling, ECLIA Blood sampling, surrogate virus Neutralisation test using commercial kit |
| Total neutralising antibody titre | Antibody agglutination | |
| Gut integrity | Level of zonulin | Blood sampling ELISA |
| Gut microbiota profiling | Type of gut microbiota, firmicutes to bacteroides ratio | Faecal sampling, NGS |
| Short-chain fatty acids (SCFAs) | The concentrations of SCFAs in human faeces | Human stool samples will be analysed to determine SCFA concentration using gas chromatography mass spectrometry and metabolomic approach (combining bioscience, analytical chemistry and bioinformatics)61 |
| Gastrointestinal tract inflammation | Faecal calprotectin concentration43 44 | Faecal samples, ELISA |
| Gastrointestinal tract permeability | Faecal zonulin level62–64 | Faecal samples, ELISA |
| Faecal occludin level65 | Faecal samples, ELISA | |
| Sociodemographic | Ethnicity, urban/rural, family determinants (parents’ education, occupation, income, family structure), gender39 | Interview with questionnaire |
| Physical activity | International Physical Activity Questionnaire for Adolescent | Interview with questionnaire |
| Puberty stage | Self-reported according to the five stages described by Tanner, based on the assessment of two components: pubic hair growth for boys and girls, plus breast development in girls and genital development in boys. A 5-point maturity rating will be used where stage 1 corresponds to the prepubertal state and stage 5 to mature state. | Interview with questionnaire |
| Parenting support | Parental support questionnaire40 | Interview with questionnaire |
| Peer support | Peer support questionnaire40 | Interview with questionnaire |
| Behaviour/mental disorder and self-esteem | Body image (BSQ-34), peer pressure, substance use, depression (PHQ-A), self-esteem (Rosenberg Self-Esteem scale) | Interview with questionnaire |
| Quality of life | Youth Quality of Life for adolescents-Research version | Interview with questionnaire |
| Morbidity and mortality | History of illness, including COVID-19, gastrointestinal symptoms, medication history | Interview with questionnaire |
| Inflammation | Potential markers for gut and systemic inflammation (hs-CRP) | Blood and faecal sample |
| Cognitive function | Raven’s test, arithmetic test, brain-derived neurotrophic factor | Interview with questionnaire Blood sample |
| Routine blood measurement | Haemoglobin, erythrocyte, leucocyte and thrombocyte count, blood sedimentation rate | Blood sample |
BIA, bioelectrical impedance analysis; BMI, body mass index; MUAC, mid-upper arm circumference; NGS, next-generation sequencing; WHR, waist-hip ratio; WHtR, waist-to-height ratio.