Parasitic infections as potential risk factors for attention deficit hyperactivity disorder (ADHD) in children

Abstract

Attention deficit hyperactivity disorder (ADHD) represents a mysterious neuropsychiatric alarming concern due to indefinite etiopathogenesis among children. Notably, the studies which investigated the correlation between ADHD and parasitic infections are insufficient. Therefore, this research aimed to assess the correlation between ADHD and some tissue dwelling and intestinal parasitic infections in children. The study was conducted on 200 children, including 100 children suffering from ADHD (Group I) and 100 healthy children as a control group (Group II). All caregivers fulfilled predesigned sociodemographic form and Conners parent rating scale (CPRS-48) questionnaire. Blood samples were collected to determine hemoglobin level as well as relative eosinophilic count. The presence of anti-Toxoplasma IgG and anti-Toxocara IgG in serum by Enzyme-Linked Immunosorbent Assay (ELISA) was further investigated. Also, micronutrients as zinc, iron, and copper levels were measured. Schistosoma antigen was investigated in urine samples. Stool samples were subjected to direct wet smear, concentration technique and modified Ziehl–Neelsen (MZN) staining for coccidian parasites detection. Cryptosporidium parvum, Giardia lamblia and Entamoeba histolytica antigens were investigated in stool samples. Group I expressed more liability to sociodemographic risk factors, decreased levels of Hb, iron, zinc, and copper with statistically significant difference (P < 0.001). Comparison between Group I and Group II regarding the detected parasitic infections exhibited statistically significant difference except Schistosoma antigen positivity which expressed no statistical significance. The present study concluded that the parasitic infections with their consequences are potential risk factors in children with ADHD indicating that their early diagnosis and treatment may help in ADHD prevention.

Introduction

Attention deficit hyperactivity disorder (ADHD) is one of the most imperative neuropsychiatric developmental complaints with worldwide prevalence affecting about 3–12% of children before 12 years. This behavioral disorder may persist from childhood to adulthood in more than 70% of cases with more affection of males than females (Nayeri et al. 2020). It usually impairs social performance especially in the family life and interpersonal interaction in the form of repeated suicidal attacks, poor confidence, criminal behavior, and increased incidence of injuries or accidents. It also affects the school performance resulting in significant educational delay and learning deficits (Nourredine et al. 2021). Although many details of ADHD’s pathophysiology are mysterious, it was found that impairment of the dopamine neurotransmitter level plays an important role in hyperactivity, dyskinesia, tics, attention disorders, and behavioral problems (Demontis et al. 2021).

Recently, Toxoplasma gondii (T. gondii) infection was highlighted as a chief risk factor for neuropsychiatric disorders as it targets neurons and becomes latent causing cerebral toxoplasmosis, as well as performing functional impairments in dopaminergic systems and neurotransmitters (Lam et al. 2020). However, the studies that have surveyed the association of T. gondii infection with ADHD are still insufficient with many discrepancies in the obtained results (Nayeri et al. 2020).

It was noted that nearly all children are physiologically vulnerable to parasitic infections due to poor hygiene, nutritional deficiencies and immunosuppression especially Cryptosporidium parvum (C. parvum), Giardia lamblia (G. lamblia) and Entamoeba histolytica (E. histolytica) infections (Sucheta et al. 2021). Although associations of ADHD with multiple infections have been confirmed in few studies, little is known about the relation between parasitic infections and ADHD (Heikkilä et al. 2021). Parasitic infections have long been implicated in various neurological symptoms. For example, the larvae of Toxocara species can reach the central nervous system (CNS) causing a variety of neurological disorders (neurotoxocariasis) (Nicoletti 2020). On the other hand, long term diarrhea and intestinal dysbiosis caused by parasitic infections deprive the infected children from vitamins, minerals, and amino acids hence affecting children’s physical and cognitive development (Allain and Buret 2020). Scarce studies have been conducted to explore the relationships of early childhood intestinal dysbiosis with subsequent affection of neuroglial cells and neurocognitive impairment (Koszewiczz et al. 2020). Regarding Egypt as a Schistosoma endemic area, this would have a salutary impact on a range of child-health outcomes including anemia, malnutrition, and neurocognitive deficits (Ezeamama et al. 2018).

Regarding previous knowledge, it seems that the role of parasitic infections is neglected in the research era of ADHD etiopathogenesis. Therefore, the aim of this study was to identify the correlation between some intestinal and tissue dwelling parasitic infections and ADHD in children in the Nile Delta, Egypt. Filling this knowledge gap can offer holistic insights about the etiology of ADHD hence paving the way for successful prevention and treatment of such disorder.

Subjects and methods

Type of the study and ethical standards

This cross-sectional study was conducted in two major university hospitals in Egypt after its approval by the ethical committee of the Faculty of Medicine, Tanta University (Approval code: 34477). All the terms of Helsinki Declaration were followed, and all the personal information was saved anonymously. All caregivers of children gave signed written informed consents after clear explanation of the study aims and techniques.

Study area

All cases were selected by random sampling technique representing both urban and rural communities of Nile Delta.

Inclusion criteria and exclusion criteria

Children of both sexes aged 3–15 years and diagnosed with ADHD by rating scale (CPRS-48) were included in the study group. Whereas, children aged above 15 or below 3 years, children suffering from any other neuro-psychiatric or chronic illness, and children who had received mineral-vitamin supplements during the last 6 months were excluded from the study.

Study design

This study was conducted on 200 children of both genders aged 3–15 years old. Two groups were included in the present study; Group I (G1): 100 children referred to Pediatric Neurology Unit of Kafr Elshiekh and Tanta University Hospitals diagnosed with ADHD (ADHD Group). Group II (G2): 100 healthy children (Control group). The study was performed in collaboration with Medical Parasitology Physiology and Clinical Pathology Departments, Faculty of Medicine, Tanta University, Egypt.

Tools of the study

Predesigned questionnaire

Parents and legal caregivers were asked to fill a predesigned questionnaire to assess personal demographic data; risk factors for parasitic infections as contact with animals, contact with soil, and consumption of undercooked meat or unfiltered water. History of the present illness including onset, progress of the disease, investigations and received treatment was also recorded. Children suffering from ADHD were diagnosed by Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-V) criteria (Nourredine et al. 2021) and score was given by (CPRS-48) (Catale et al. 2014). All patients were applied to CPRS-48 which is a revised 48 item version created from the 93-item version. This modified rating form provides a qualitative and quantitative image of children’s emotions and behavior (Hemamy et al. 2020).

Blood sampling technique

Blood samples of 6 ml were collected by venipuncture under sterile conditions. Two ml of blood were collected in EDTA tube to be used for determination of hemoglobin level as well as total and differential leucocytic count for calculation of relative eosinophilic count (Blomme et al. 2020). Other 2 ml of blood were centrifuged at 2000 rpm for 15 min; the separated sera were kept in sterile Eppendorfs and labeled with the child's name and number at − 20 °C for immunological analysis. Hemolyzed samples were discarded to prevent any potential bias in the results. The last 2 ml were examined for the levels of micronutrients in serum (zinc (Zn), iron, and copper (Cu) levels) by using the Spectrophotometer (ICP-OES Optima 2X00/7000 Series DV, PerkinElmer, Waltham, Massachusetts, USA). Results were expressed as μg/dl (Lu et al. 2021).

Immunological assays

Anti-Toxoplasma IgG antibodies levels were investigated using the Enzyme-Linked Immunosorbent Assay (ELISA) technique. Screening of the samples for anti-Toxoplasma gondii IgG antibodies was done by T. gondii IgG (Toxo IgG) ELISA Kit (AccuDiag™, Cat # 1101-1, Diagnostic Automation/Cortez Diagnostics, Inc., California, USA) according to the manufacturer’s instructions. The anti-Toxocara IgG antibodies were detected by commercial ELISA kit (AccuDiag™ Toxocara IgG ELISA Kit Cat # 8206–35, Diagnostic Automation/Cortez Diagnostics, Inc., California, USA) according to the manufacturer’s instructions.

Urine samples were collected under sterile conditions then the circulating cathodic antigen (CCA) specific to Schistosoma mansoni (S. mansoni), and S. haematobium was investigated for the qualitative presumptive detection of active schistosomiasis. The CCA was investigated in the samples by a rapid immunological diagnostic test called CCA cassette test obtained from Rapid Medical Diagnostics (Pretoria, South Africa) according to the manufacturer’s instructions (Colley et al. 2020).

Detection of parasites in stool

Typical steps were used in collecting fresh stool specimens in a 100-mL clean leak proof stool cups. All the data of participating children including name, serial number, group number, and date of sample collection were recorded obviously on the stool cups. All samples were transported within 30 min to the Medical Parasitology Department, Faculty of Medicine, Tanta University. Stool consistency was reported. Direct microscopic examination of wet mount preparations of stool specimens was done to screen for parasitic stages. Each stool sample was well mixed and stored in10% formalin at 4 °C, using suitable containers labeled with the same information. Afterwards, specimens were concentrated using formalin-ethyl acetate sedimentation method to identify cysts. The smears from each stool sample were stained using a modified Ziehl–Neelsen stain technique specific for coccidia detection and examined carefully by the light microscope using 10 × and 40 × objective lenses (Robinson and Chalmers 2020).

Cryptosporidium parvum, G. lamblia and E. histolytica infections were diagnosed by detection of their antigens (Ag) in stool samples using Crypto-Giardia-Entamoeba Rapid Test (Catalog Number: DTS700, CD Creative diagnostics, USA) which is an immunochromatographic rapid assay for the qualitative determination of these infections. As E. histolytica is morphologically indistinguishable from the noninvasive and more prevalent Entamoeba dispar (E. dispar), detection of E. histolytica antigen in stool was required (Samie et al. 2020).

All blood, urine, and stool remnants of samples were discarded in the general incinerator according to the research and safe disposal rules of the Faculty of Medicine, Tanta University. The children who were positive for any of the examined parasitic infections were referred to Tanta university Hospital to receive free treatment.

Statistical analysis

Data were collected, tabulated, statistically analyzed using Statistical Package of Social Science (SPSS) version 22. Qualitative data were expressed as number and percent and tested by the chi-squared test. Quantitative data were expressed as mean and standard deviation (Mean ± SD) and tested by the t-test. P value < 0.05 was set to be significant. Logistic regression and Spearman correlation analysis were also applied.

Results

Regarding the children gender, ADHD was more prevalent in male than female children but with no statistically significant difference between the two groups (P > 0.05) (Table 1). Meanwhile, children suffering from ADHD (G1) expressed more liability to be exposed to demographic risk factors in the form of contact with animals, contact with soil, consumption of undercooked meat and unfiltered water with statistically significant differences with G 2 (P < 0.001) (Table 1 and Fig. 1). On the other hand, the mean ± SD of the levels of Hb, iron, zinc, copper, and eosinophils percentages showed statistically significant differences between G1 and G2 (P < 0.001) (Table 2).

Table 1.

Comparison between ADHD (G1) and control group (G2) regarding the demographic risk factors

Demographic risk factors	G 1 (n = 100)	G 2 (n = 100)	X2	P-value
Sex
Male
N	72	59	3.739	0.053
%	72.0%	59.0%
Female
N	28	41
%	28.0%	41.0%
Contact with animals
Yes
N	28	9	11.971	0.001*
%	28.0%	9.0%
No
N	72	91
%	72.0%	91.0%
Contact with soil
Yes
N	43	14	20.636	0.001*
%	43.0%	14.0%
No
N	57	86
%	57.0%	86.0%
Consumption of undercooked meat
Yes
N	39	16	13.266	0.001*
%	39.0%	16.0%
No
N	61	84
%	61.0%	84.0%
Consumption of unfiltered water
Yes
N	71	43	15.993	0.001*
%	71.0%	43.0%
No
N	29	57
%	29.0%	57.0%

Data were expressed by using P-value for comparing between G1 and G2

^*: Statistically significant at P ≤ 0.05

Fig. 1.

Illustration of the prevalence of demographic risk factors among the study groups

Table 2.

Comparison between ADHD (G1) and control group (G2) regarding the Mean ± SD of Hb levels, Eosinophils % and micronutrients levels (n = 100 for each group)

Blood variables	Group	Range	Mean ± S.D	t. test	P value
Hb level (g/dl)	G 1	9.8–13	11.05 ± 0.89	11.267	0.001*
	G 2	11.3–13.3	12.27 ± 0.62
Eosinophils %	G 1	0.5–11	3.55 ± 2.33	4.865	0.001*
	G 2	0.4–4	2.34 ± 0.90
Iron level (μg/dl)	G 1	20–35	29.14 ± 4.13	11.645	0.001*
	G 2	32–37	34.31 ± 1.62
Zn level (μg/dl)	G 1	40–80	56.94 ± 11.03	19.833	0.001*
	G 2	75–88	80.03 ± 3.73
Cu level (μg/dl)	G 1	70–99	80.00 ± 6.89	13.132	0.001*
	G 2	85–100	91.15 ± 4.97

Data were expressed by using Range, Mean ± SD, and P-value for comparing between G1 and G2

^*: Statistically significant at P ≤ 0.05

Abbreviations; Hb: hemoglobin, Zn: zinc Cu: copper

Regarding the prevalence of the examined parasitic infections, there were statistically significant differences between G1 and G2 (P < 0.001) concerning the positivity of anti-Toxoplasma gondii IgG, anti-Toxocara IgG, E. histolytica Ag, G. lamblia Ag and C. parvum Ag (Table 3 and Fig. 2). Conversely, Schistosoma CCA positivity was found in 5% of G1 and only one case (1%) of G2 with no statistically significant difference (P > 0.05) (Table 3 and Fig. 2).Direct stool examination using wet mount preparations, formalin-ethyl acetate sedimentation technique revealed E. histolytica cysts in 33% of G1 and in 10% of G2 in addition to G. lamblia cysts in 29% of G1 and in 13% of G2. However, helminthic ova were not detected in both groups. Modified Ziehl–Neelsen stain revealed C. Parvum oocysts in 24% of G1 and in 11% of G2 with statistically significant difference (P < 0.001) (Table 4 and Figs. 3, 4).

Table 3.

Prevalence of the examined parasitic infections in ADHD group (G1) and control group (G2)

Parasitic infections	G 1 (n = 100)	G 2 (n = 100)	X2	P-value
Serum anti-Toxoplasma gondii IgG
+ ve
N	36	19	7.248	0.007*
%	36.0%	19.0%
− ve
N	64	81
%	64.0%	81.0%
Serum anti-Toxocara IgG
+ ve
N	18	4	10.010	0.002*
%	18.0%	4.0%
− ve
N	82	96
%	82.0%	96.0%
Urine Schistosoma CCA
+ ve
N	5	1	2.749	0.097
%	5.0%	1.0%
− ve
N	95	99
%	95.0%	99.0%
Stool E. histolytica Ag
+ ve
N	49	28	9.313	0.002*
%	49.0%	28.0%
− ve
N	51	72
%	51.0%	72.0%
Stool G. lamblia Ag
+ ve
N	45	20	14.245	0.001*
%	45.0%	20.0%
− ve
N	55	80
%	55.0%	80.0%
Stool C. parvum Ag
+ ve
N	58	25	22.428	0.001*
%	58.0%	25.0%
− ve
N	42	75
%	42.0%	75.0%

Data were expressed by using P-value for comparing between G1 and G2

^*: Statistically significant at P ≤ 0.05

Abbreviations; IgG: immunoglobulin G, CCA: circulating cathodic antigen, E. histolytica: Entamoeba histolytica, G. lamblia: Giardia lamblia, and C. parvum: Cryptosporidium parvum. Ag: antigen

Fig. 2.

Prevalence of the parasitic infections in ADHD group (G1) and control group (G2)

Table 4.

Prevalence of the intestinal protozoa by direct stool examination in ADHD group (G1) and control group (G2)

Intestinal protozoa	G 1 (n = 100)	G 2 (n = 100)	X2	P-value
E. histolytica cysts
+ ve
N	33	10	15.669	0.001*
%	33.0%	10.0%
− ve
N	67	90
%	67.0%	90.0%
G. lamblia cysts
+ ve
N	29	13	7.719	0.005*
%	29.0%	13.0%
− ve
N	71	87
%	71.0%	87.0%
C. parvum oocysts
+ ve
N	24	11	5.849	0.016*
%	24.0%	11.0%
− ve
N	76	89
%	76.0%	89.0%

Data were expressed by using P-value for comparing between G1 and G2

^*: Statistically significant at P ≤ 0.05

Abbreviations; E. histolytica: Entamoeba histolytica, G. lamblia: Giardia lamblia, and C. parvum: Cryptosporidium parvum

Fig. 3.

Prevalence of intestinal protozoa by direct stool examination in study groups

Fig. 4.

A stool smear stained with modified Ziehl–Neelsen, after formol-ether sedimentation, showing Cryptosporidium oocysts (× 1000)

Notably, the logistic regression analysis of the risk factors for ADHD in G1 highlighted the risk of contact with soil, consumption of unfiltered water as well as decreased Hb, iron, and zinc levels. Moreover, the positivity of anti-Toxoplasma IgG, E. histolytica Ag, G. lamblia Ag and C. parvum Ag indicated their highly eminent role in predisposing ADHD (P < 0.05). On the other hand, children who showed contact with animals, consumption of undercooked meat, reduced Cu level, and anti-Toxocara IgG positivity had reduced risk of ADHD with P > 0.05 (Table 5).

Table 5.

Logistic regression model of risk factors for ADHD conditioned on most relevant significant risk factors. (Odds ratio (OR) and 95% confidence interval (CI))

Independent variables	OR (95% CI)	P-value
Contact with animals	1.748(0.591–5.169)	0.313
Contact with soil	3.790(1.527–9.411)	0.004*
Consumption of undercooked meat	1.391(0.585–3.309)	0.215
Consumption of unfiltered water	2.266(1.117–4.597)	0.023*
Hb level (g/dl)	1.659(1.097–7.251)	0.021*
Iron level (μg/dl)	1.634(1.110–2.406)	0.013*
Zn level(μg/dl)	2.292(1.305–4.024)	0.004*
Cu level (μg/dl)	1.212(0.8.5–1.427)	0.102
Anti-Toxoplasma gondii IgG	2.867(1.181–6.958)	0.020*
Anti-Toxocara IgG	1.730(0.430–7.435)	0.632
E. histolytica Ag	2.049(1.138–4.341)	0.043*
G. lamblia Ag	3.644(1.640–8.097)	0.002*
C. parvum Ag	4.059(1.945–8.623)	0.001*

Data were expressed by using Logistic regression (Odds ratio (OR) and 95% confidence interval (CI)), P-value for comparing between G1 and G2

^*: Statistically significant at P ≤ 0.05

Abbreviations; Hb: hemoglobin, Zn: zinc Cu: copper. IgG: immunoglobulin G, E. histolytica: Entamoeba histolytica, G. lamblia: Giardia lamblia, and C. parvum: Cryptosporidium parvum. Ag: antigen

As regards the correlation between the detected parasites and Hb levels, eosinophils percentages, and micronutrients levels in ADHD group, it was found that there was a correlation between toxocariasis and eosinophils percentages. Amoebiasis was correlated to low iron level, low Hb level and eosinophils percentages. Furthermore, there was a correlation between cryptosporidiosis and low Zn and Cu levels. It was found that there was a correlation between giardiasis and low Zn level as well (Table 6).

Table 6.

Correlation between the detected Toxoplasma IgG, Toxocara IgG, E. histolytica Ag, G. lamblia Ag and C. parvum Ag with Hb levels, Eosinophils %, and micronutrients levels of ADHD group (G1) (n = 100)

Independent variables	Hb level		Eosinophils %		Iron level		Zn level		Cu level
	r	P	r	P	r	P	r	P	r	P
Anti-Toxoplasma IgG	− 0.016	0.875	− 0.121	0.229	0.096	0.342	− 0.069	0.498	− 0.126	0.212
Anti-Toxocara IgG	− 0.161	0.109	0.583	0.001*	− 0.180	0.074	0.123	0.222	− 0.124	0.219
E. histolytica Ag	− 0.408	0.001*	0.232	0.020*	− 0.718	0.001*	0.081	0.422	− 0.104	0.305
G. lamblia Ag	0.046	0.649	− 0.083	0.412	− 0.043	0.671	− 0.564	0.001*	− 0.187	0.063
C. parvum Ag	0.030	0.765	0.040	0.692	0.086	0.393	− 0.396	0.001*	− 0.525	0.001*

Data were expressed by using r: Spearman correlation and P-value

^*: Statistically significant at P ≤ 0.05

Abbreviations; Hb: hemoglobin, Zn: zinc Cu: copper. IgG: immunoglobulin G, E. histolytica: Entamoeba histolytica, G. lamblia: Giardia lamblia, and C. parvum: Cryptosporidium parvum. Ag: antigen

Discussion

Globally, ADHD worth to be greatly considered in children as it can touch every aspect of the child's life activities and school presentation with subsequent educational and academic discrepancies (Nourredine et al. 2021). Based on the previous knowledge, this research is the foremost to investigate the causal association of ADHD with tissue dwelling and intestinal parasites along with various micronutrients and blood indices. We postulated that childhood parasitic infections may impact the neuropsychiatric development with special concern to gut-brain axis.

Compared with G2, children with ADHD significantly had higher odds of exposure to demographic risk factors for parasitic infections as contact with animals, contact with soil, and consumption of undercooked meat or unfiltered water. This result indicates that ADHD children carry higher potential to acquire parasitic infections which may either directly cause or even augment ADHD by its sequels (El-Beshbishi et al. 2018). The role of demographic risk factors in the development and spread of parasitic infection was well reported by Geneidy (2019). Furthermore, the levels of Hb, iron, zinc and copper showed statistically significant decrease in G1. These results are very logic coinciding with those of Marques et al. (2020) who reported that Amazonian children were suffering from anemia and poor nutritional status as a chronic sequel of parasitic infections. The results of the present work are also confirmed by those obtained in Egyptian children by Mahmoud et al. (2017) who indicated the causal relationship between intestinal parasitic infections and iron deficiency anemia. Therefore, several studies highlighted the parasitic infections as dangerous health problem owing to their impact on nutritional status and micronutrients level that leads to learning, physical, and mental disorders (Shalaby et al. 2017). On the other hand, parasitic infections significantly impact the brain activities by altering brainwave amplitudes triggering mental sluggishness, learning deficits, and ADHD but with unidentified mechanisms (Davoudi et al. 2020).

The statistically significant levels of seropositivity of Toxoplasma IgG in ADHD children in the present work can be interpreted by numerous behavioral studies which proved that latent toxoplasmosis may result in behavioral changes (Noori et al. 2020). At the same time, the behavioral changes of T. gondii-infected rodents such as rapid loss of concentration and elevated activity levels were clinical shown to bear resemblance to those manifested in ADHD patients (Nayeri et al. 2020). Moreover, latent toxoplasmosis has been associated with lethargy, impaired concentration, discrepancies in motor functions, and increased risk of injury which all described in patients with ADHD (Lam et al. 2020).

The mechanistic potentials by which T. gondii can manipulate brain functions include direct interactions with brain neurons which are the main target of Toxoplasma (Cabral et al. 2016). Experimental T. gondii infection resulted in neuro-inflammation and loss of brain parenchyma with subsequent neuro-psychiatric abnormalities (Hermes et al. 2008). Moreover, T. gondii was reported to interact with host genomics and proteomics throughout its life cycle, including human ADHD predisposing genes with a specific emphasis on the dopaminergic signaling and neurotransmitter pathways (Carter 2013). This can be interpreted by its direct enhancing impact on dopamine neurotransmitter release from neurons by emphasizing genes encoding tyrosine hydroxylase enzyme which is dopamine biosynthesis inhibitor (Volkow et al. 2009). In order to explain the hypothesis of the current work, the high prevalence of Toxoplasma IgG in ADHD children was interpreted by the finding that T. gondii cysts were mainly concentrated in the amygdala, nucleus accumbens, and hypothalamus which are dopamine-containing brain areas that control fear and movements with great emphasis on ADHD (Volkow et al. 2009; Lam et al. 2020). Furthermore, the antipsychotic dopamine receptor agonists, the mood stabilizers, or the selective dopamine reuptake inhibitor were reported to inhibit T. gondii multiplication as well as the associated behavioral disorder (Webster et al. 2006; Skallova et al. 2006). Similarly, L-tyrosine supplements, a crucial precursor for dopamine and norepinephrine, recorded positive results in ADHD patients (Wood et al. 1985).

In the present work, there was statistically significant level of seropositivity of anti-Toxocara IgG in ADHD group. The results herein coincide with experimental research which indicated that neurotoxocariasis leads to variable behavioral changes in infected animals (Othman et al. 2010). In addition, cohort studies indicated that neurotoxocariasis is one of the major causes of academic shortages as well as other psychological discrepancies depending on the intensity of parasite-induced pathology (Nicoletti 2020). Increased prevalence of anti-Toxocara IgG in ADHD group can be explained by Maiga et al. (2007) and Lompo et al. (2012) who revealed that Toxocara larvae release toxins that can cause direct neuronal damage. Furthermore, chronic Toxocara infection was found to augment expression of inflammatory cytokines such as TNF-mRNA with associated TNF-disturbances leading to sleep and cognitive impairment (Turrin and Plata-Salamán 2000). The role of toxocariasis induced inflammatory mediators cannot be neglected in the affected brain circuits in experimental animals (Othman et al. 2010). This fact was also confirmed by Tonelli et al. (2009) who indicated the role of inflammatory cytokines in prompting the behavioral and emotional neuroimmune mechanistic potentials that may activate the hypothalamus–pituitary–adrenal axis. Furthermore, inflammatory cytokines were found to amend the metabolism of norepinephrine and dopamine which were identified to be involved in ADHD pathogenesis (Chen et al. 2019). Taken together, the mechanisms by which toxocariasis can impact the pathogenesis of ADHD may involve inflammatory cytokines in parallel with brain neurotransmitters in experimental animals and perhaps in humans as well accounting for the behavioral and cognitive deficits (Othman et al. 2010).

In the current work, no helminthic eggs were detected in direct stool examination though; Schistosoma CCA was detected in G1 but with no statistical significance. Regarding schistosomiasis as the second most common parasitic infection in the world and one of the most imperative causes of anemia (Mnkugwe et al. 2020), it was logic to interpret the existence of schistosomiasis in ADHD group. Urinary and gastrointestinal schistosomiasis can predispose to iron deficiency anemia either by direct blood loss in urine and stool or by associated schistosomal hypersplenism (Adam et al. 2021). On the other hand, Schistosoma infection was reported to be highly associated with learning and memory deficits due to cognitive dysfunction by different mechanisms either directly through eggs deposition in the brain, or indirectly, via infection associated iron-deficiency and malnutrition (Ezeamama et al. 2018). On the other hand, the role of other helminthic infections in triggering neurotransmitters changes was highlighted in several researches (Brahem et al. 2006; Ismail et al. 2007). Therefore, according to the previously mentioned mechanisms, future research is needed to explore the cross talk between helminthic infections and neurodevelopmental disorders including ADHD.

There is no doubt that intestinal parasitic infections among school children can significantly trigger iron deficiency anemia, physical and mental retardation (Marques et al. 2020). This systemic impact may be linked to significant reduction of Hb levels in children as parasites require iron, carbohydrates, lipids, minerals and vitamins to gain energy for their life cycles, growth and multiplication (Teja et al. 2020). According to Mahmoud et al. (2017), G. lamblia and C. parvum associated diarrhea as well as E. histolytica associated dysentery was recorded to predispose to iron deficiency anemia among Egyptian school children. The mechanical damage of the intestinal epithelium by the ventral suckers of G. lamblia, toxic injury with subsequent increase of the intestinal permeability, and malabsorption are the main causes of anemia in giardiasis (Hussein et al. 2016). The results of the current study agree with Shalaby et al. (2017) who reported the suppressor impact of G. lamblia, C. parvum and E. histolytica infection on Zn, iron, and Cu serum levels due to their malabsorption. In the present study, G. lamblia was highly correlated to decreased Zn levels. This result can be interpreted by the supposition that the mucosal lesions triggered by the sucking discs of G. lamblia may significantly impair the intestinal Zn absorption. Moreover, these parasitic infections shift zinc to the liver with significant decrease of its level in serum (Mahmoud et al. 2017).

Protozoal diarrhea is one of the foremost causes of anemia and dehydration as well as loss of minerals. Furthermore, their treatment with anti-protozoal drugs had amended the micronutrient serum levels (Wiser 2021). On the other hand, Zn serum level can be easily dropped especially in children because of their higher growth demands as it cannot be stored in the body (Skalny et al. 2020). Zinc is well-thought to be crucial in the metabolism of nucleic acids and the protein synthesis, therefore its deficiency delay the growth process in children (Zhu et al. 2021). Evidence of mineral deficiencies in children suffering from ADHD such as zinc, iron, calcium, magnesium, and selenium was proved by Viktorinova et al. (2009) as decreased Zn and iron levels might be allied with substantial dampening of dopaminergic transmission exacerbating anxiety and behavioral disorders (Oner et al. 2010; Ajsuvakova et al. 2020). On the other hand, zinc supplementation in children reported reduced ADHD manifestations (Noorazar et al. 2020).

Regarding the results of the present work, the mean ± SD of the levels of Hb, iron, zinc, copper, and eosinophils percentages showed statistically significant differences between G1 and G2 which highlight various ADHD associated comorbidities. Decreased levels of Hb and iron in the present study in ADHD group highly coincides with (Rodríguez et al. 2021) who correlated low levels of serum iron in children as a risk for cognitive, behavioral, and learning problems. Likewise, iron deficiency may interfere with oxygen supply of the brain altering the cognitive ability hence the development of ADHD (Robberecht et al. 2020). As iron deficiency anemia is the most dominant form of anemia it was proved to be a potential risk factor for hyperactivity (Oner et al. 2012) and neurological discrepancies including ADHD (Bener et al. 2014). Moreover, decreased iron stores were also supposed to dampen the psychostimulants efficacy in children with ADHD (Cortese et al. 2012). Regarding iron as a cofactor of tyrosine hydroxylase and tryptophan hydroxylase biosynthesis, its depletion can alter neurotransmitters such as dopamine (Erikson et al. 2000).

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by AMS, DAE and AMS. The first draft of the manuscript was written by DAE and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.”

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Ethics approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Tanta University (Approval code: 34477).

Consent to participate

Written informed consent was obtained from the parents

Consent to publish

The authors affirm that human research participants provided informed consent for publication.