This was an unmatched case-control study conducted among adolescent schoolchildren (61 with depression and 61 healthy controls) at government schools in the locality of Almatamah, northern Sudan, from August to September 2022. Almatamah is located in the River Nile State in northern Sudan, approximately 100 kilometers from Khartoum, the capital of Sudan.

Is there a difference in serum ferritin levels and iron deficiency between adolescents with depression and healthy controls in northern Sudan?

In this study, the strengthening of the reporting of observational studies in epidemiology (STROBE) guidelines were strictly followed (23). Adolescents were considered depressed when their Patient Health Questionnaire (PHQ-9) scores were more than 4; any adolescent with a PHQ-9 score of four or less was considered a control. More details are provided in our previously published work regarding the PHQ-9 scores for depression (24).

In Almatamah locality, there are three districts, among which is Wad Hamid district. In Wad Hamid district, there are 16 government schools, each serving male or female students. Six schools (three for each sex) were randomly selected using simple random sampling via a lottery method. Sixty-one students, the desired sample size, were chosen from each arm of the designated schools. In each school, the assigned sample size was selected using a simple random technique (lottery method) from the list of students.

The inclusion criterion was adolescents aged 10–19 years. These age cutoff points were chosen based on the definition of adolescents by the World Health Organization (WHO) (25). Students with a younger age (<10 years) or older age (>19 years); who did not give consent to participate in the study; who had chronic diseases such as thyroid, hemolytic disorder; who had previously been diagnosed with a mental disorder, such as depression or bipolar disorder according to DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition) (26); who received mental health disorder medications; who were currently on iron supplements; or who were pregnant or lactating girls (have different physiological parameters for anemia and serum ferritin) were excluded from participating in this study.

A sample size of 61 participants in each study group was computed with an equal number of cases and controls (ratio of 1:1). The sample size was calculated assuming a mean difference in serum ferritin (8.0 μg/l) between depressed and nondepressed adolescents. This assumption was based on a study showing a mean serum ferritin level of 8.0 μg/l among adolescents in the same area as this study (27). The sample size (61 participants in each group) was determined to detect a 5% difference at an α-value of 0.05 with 80% power.

A questionnaire was developed based on previous studies (18, 21, 28). This questionnaire included data on sociodemographic characteristics, such as age in years, gender (male or female), parental educational levels (<secondary or ≥secondary), mother’s occupational status (housewife or employed), father’s occupational status (skilled worker or laborer), tobacco use (yes or no), and clinical data, such as family history of mental illness. Two medical officers, one male and one female, were trained by the investigators for data collection.

After the participants and their guardians agreed to participate and signed informed consent forms, the selected students were approached, and the questionnaires were completed through face-to-face interviews (by the medical officers). The selected students were informed about the study’s aims. They provided all necessary information, including the voluntary nature of their participation in the research and their right to withdraw at any time without giving a reason. All preventive measures were taken to ensure the privacy and confidentiality of the participants, including the exclusion of personal identifiers during data collection. Depression, weight, height, and serum ferritin were measured using the standard procedures detailed below.

The students’ weights were measured in kilograms (kg) using standard procedures, which included well-calibrated scales and adjustments to zero before each measurement. Weight was measured to the nearest 100 grams (g). The students stood with minimal movement, with their hands by their sides. Furthermore, shoes and excess clothing were removed. Height was measured to the nearest 0.1 centimeter (cm), with each student standing straight against a wall with their feet together. Finally, the body mass index (BMI) for the age Z-score was determined based on the WHO’s standards (29).

From each student, 3 ml of blood was taken under aseptic conditions for serum ferritin and C-reactive protein (CRP) analyses, using the laboratory methods described in our previously published work (30, 31). The collected blood samples were allowed to clot and then centrifuged and stored at −20°C until analysis. Serum ferritin was measured using an immunofluorescent assay on the IMMULITE 1000 (SIEMENS, CA, USA), following the manufacturer’s instructions. Ferritin concentration is a promising biomarker of iron stores and should be used to diagnose iron deficiency in otherwise apparently healthy individuals (32, 33). Therefore, according to the WHO cutoff point, iron deficiency was defined as serum ferritin less than 12 μg/l with normal CRP (32).

Data were entered into a computer using the IBM Statistical Package for the Social Sciences (SPSS^®) for Windows, version 22.0 (SPSS Inc., Armonk, New York, United States). The Shapiro–Wilk test was used to evaluate the normality of continuous data, such as age, and serum ferritin, none of which was found to be normally distributed; hence, values are expressed as median (interquartile range [IQR]) and were compared between the two groups by a nonparametric test (Mann–Whitney U test). A chi-square test was used to compare the categorized variables. Spearman’s correlation was performed between the depression scale and serum ferritin levels. Univariate analysis was performed with depression as the dependent variable. In contrast, the independent variables (covariates) included sociodemographic characteristics (age, gender, and parental educational level), family history of mental illness, BMI, and serum ferritin levels. Furthermore, a multivariable model was utilized using variables from the univariate analysis with a p-value of less than 0.20, with backward elimination to adjust for covariates. Adjusted odds ratios (AORs), 95% confidence intervals (CIs), coefficients, and standard errors were calculated as needed. A two-sided p-value of less than 0.05 was considered statistically significant.

These results have significant implications for public health interventions, underscoring the need for targeted nutritional programs, particularly for adolescent girls, to address iron deficiency and its associated mental health consequences. Furthermore, this study advocates for integrating mental health screening in schools, mainly focusing on at-risk demographics, such as older adolescents and females. Future research should focus on longitudinal studies to unravel the causal pathways between iron status and mental health and explore whether supplementation can mitigate depressive symptoms. It is very important to consider the ongoing war and its devastating impact on food security and mental health, which might worsen children and adolescents’ nutritional status and mental health (46–48).

To the best of the authors’ knowledge, this is the first study to investigate the association between serum ferritin and iron deficiency and depression in Sudan, especially in an understudied area. It is important to mention that iron deficiency can be prevented with adequate nutritional intake and even intravenous iron infusions, if required (12, 33, 38, 39), and, as a consequence, decreasing depression among adolescents. However, this study has some limitations that need to be mentioned. Sample size and generalizability: The school-based and localized nature of the study, specifically in northern Sudan, may have limited the sample size, thereby affecting the generalizability of the findings. The findings may not be representative of other demographics, including different age groups, cultural backgrounds, or regions within Sudan. Study design: Because this was a case-control study design (fails to establish causality, making it difficult to determine whether low serum ferritin contributes to depression or vice versa) investigated the association between the studied variables at one point in time. A longitudinal study is recommended since the bidirectional association between nutrition and depression remains unclear (15). Confounding variables: There may be other confounding variables that the study did not fully account for that could affect the relationship between ferritin levels, iron deficiency, and depression in adolescents. These factors may include socioeconomic status, nutritional intake, genetic predispositions, or other health conditions that can impact both iron levels and mental health. The study uses the PHQ-9 questionnaire, which is a validated screening tool but not a diagnostic tool or a severity assessment tool. Due to the sample size limitation, the cutoff of >4 for depression may include mild cases. These limitations should be taken into account in future research.