Clinico immunological profile of immune-mediated disorders in South Indian children- A prospective observational study

ABSTRACT

Introduction:

Immune-mediated diseases can affect any part of the body of genetically susceptible individuals. Few diseases predominantly affect a single organ, while others present with multisystemic manifestations.

Methods:

We collected data on the clinical, demographic, and immunological details of children with immune-mediated diseases presenting over a period of three years from January 2020 to December 2022 retrospectively from the children enrolled in Pediatric immunology clinic of a tertiary care hospital in South India.

Results:

The prevalence of immune-mediated diseases among children visiting the hospital for various illnesses was 5 per 1000 children, and the percentage of positive autoantibodies in children with connective tissue disorders and single-organ autoimmune disorders was 41.3% and 40.7%, respectively. The most common connective tissue disorder in our study was juvenile idiopathic arthritis (21 patients), among which 9 / 21 (42.8%) were autoantibody positive. ANA was the most common antibody in both groups (47.5%). Only 3.4% had rheumatoid factor positivity in the connective tissue group. The most common single-organ autoimmune disorders in children are autoimmune thyroid disorders (25), followed by autoimmune hematological disorders (18), while autoantibodies were more common in patients with thyroid disorders (18 / 25) and autoimmune hepatitis (7 / 8).

Conclusion:

Early clinical suspicion and prompt diagnosis in children with prolonged illness or atypical presentation is essential to ensure remission and prevent complications, morbidity and mortality. Further research is needed to identify early biomarkers in immune-mediated pediatric disorders that will help to confirm the diagnosis and guide physicians to start immunosuppressive therapy at the right time.

Introduction

Pediatric immunology and rheumatology cover a wide spectrum of diseases, the basic underlying pathology of which is the body’s immune system.[1] Although the spectrum of diseases, such as rheumatological disorders, vasculitides, primary immunodeficiency diseases (PIDs), and autoimmune disorders, fall under clinical immunology, various institutions worldwide have different clinical programs for this specialty. Clinical Immunology is a clinical and laboratory field focused on conditions stemming from dysfunctional immune mechanisms, as well as situations where immune interventions play a crucial role in therapy or prevention. Children may be affected by long-term systemic autoimmune diseases (i.e., involving two or more target organs, such as SLE) or single organ-specific diseases, such as autoimmune thyroiditis or hepatitis. Some diseases can be one-time events with acute presentation, such as Henoch–Schonlein purpura, Kawasaki disease, or acute disseminated encephalomyelitis, which occur only once in their lifetime but require long-term follow-up. Few Indian studies regarding the clinical profile of rheumatologic diseases in children have shown different patterns of diseases in various parts of India[2,3] and very few immunologic markers are available to confirm these diseases. Clinico-immunologic profile of rheumatologic diseases like Juvenile idiopathic arthritis varies in the western world and Asian countries (4). There is a growing need for novel biomarkers in autoimmune diseases to predict clinical phenotypes, disease activity and severity, clinical remission and relapse, treatment response, and the progression of disease over time (5).

We collected data on the clinical, demographic, and immunological details of children with immune-mediated diseases presenting for three years from January 2020 to December 2022. This study aimed to assess the prevalence of immune-mediated diseases among children visiting the hospital for various illnesses and the percentage of positive autoantibodies for each immune-mediated pediatric disease.

Materials and Methods

This observational study was conducted at the Department of Pediatrics, Velammal Medical College Hospital, Madurai, between January 2020 and December 2022. Children aged 1–15 years who presented to the outpatient department with musculoskeletal complaints or systemic symptoms suggestive of immune-mediated disease were registered at the pediatric immunology clinic. Patients who were diagnosed with arthritis due to infections such as tuberculosis or HIV and those who were postinfectious, such as rheumatic arthritis, were excluded from the study. Post-COVID-19 immune-mediated multisystem syndrome (MIS-C), Allergy disorders, such as asthma, allergic rhinitis, and atopic dermatitis, were not included in this study. Patients with autoimmune disorders involving the eyes and skin were also excluded from the study because they were referred to the concerned departments at their first contact on an outpatient basis. Type 1 diabetes mellitus and primary and secondary immunodeficiency disorders were excluded from this study as they had different spectrum of clinical manifestation and treatment

All other children confirmed to have immune-related diseases were admitted to the Pediatric Immunology Clinic. We analyzed the symptomatology at presentation, investigations performed, and spectrum of diagnosis and management details of those who were regularly followed up at our institute during the study period. Details of the children including complete workup done to confirm the underlying diagnosis were collected from their clinic records. Laboratory data regarding autoantibody workup and their reports were collected, and the parameters were recorded and missing data were collected by contacting the family over phone. The data was entered into a Microsoft Excel sheet, and data analysis was performed manually. For continuous data, the mean was calculated, and for discrete data, such as symptoms and clinical signs, the frequency was calculated and expressed as a percentage. The proposal was approved by the Institute Ethics Committee (IEC) of Velammal Medical College Hospital IEC no. VMCIEC/28/2022.

Results

Of the 205 children who were initially registered in the clinic, forty-four children were excluded from the study due to alternative diagnoses, such as leukemia (8), tubercular arthritis (5), rheumatic arthritis (13), post-streptococcal reactive arthritis (PSRA-5), chikungunya arthritis (3), and hemophiliac arthritis (2).

We divided the 161 children included in the study into two broad groups comprising pediatric rheumatology problems (58), which included connective tissue disorders, arthritis, vasculitis, and other autoimmune disorders (103), predominantly affecting a single system or organ [Figure 1].

Figure 1.

Flowchart of study methodology

Out of the 31414 pediatric patients seen in the hospital (both inpatients and outpatients) during the study period from January 2020 to Dec 2022, five out of 1000 pediatric patients visiting the hospital were diagnosed with immune-mediated disorders.

The median age of the children in the connective tissue group [Table 1] was 13 years, whereas that of the children in the Juvenile Idiopathic arthritis (JIA) and autoimmune groups was 11 years. The median age of presentation of Kawasaki disease and Henoch Schonlein purpura was 5 and 8 years respectively.

Table 1.

Clinical presentation of Pediatric rheumatologic disorders

Disease	Total no	Median age (IQR)	M: F ratio	Symptoms	Autoantibody positive cases
Systemic lupus erythematosus	8 (13.8%)	14 (5–15)	0:8	fever (98%), fatigue (100%), malar rash (25%) Myalgia (75%) Arthralgia (75%) Acute cutaneous lupus (3/8) Renal (5/8) CNS (3/8) Hematology (5/8)	8 (100%)
Mixed Connective tissue disorder (MCTD)	4 (6.9%)	8 (7–14)	1:3	fever (100%), fatigue (100%), malar rash (25%) Myalgia (50%) Arthralgia (75%) Arthritis (75%) Peripheral gangrene (50%)	4 (100%)
Systemic sclerosis (SSc)	1 (1.7%)	8	0:1	Fatigue Poor weight gain Arthralgia	1 (100%)
Juvenile dermatomyositis (JDM)	2 (3.4%)	12 (12)	0:2	Muscle weakness Skin rash	0
Systemic onset JIA (SoJIA)	3 (5.2%)	4.5 (2–5)	2:1	Fever Skin rash Joint swelling	2
ANCA associated vasculitis (AAV)	2 (3.4%)	13.5	0:2	Gangrene (50%) Breathlessness (50%) Hematuria (50%)	2 ANCA
Takayasu arteritis	3 (5.2%)	12 (7–14)	2:1	Hypertension (66.6%) Myalgia (100%)	0
Kawasaki disease	10 (17.2%)	5 (4–11)	6:4	Fever (75%) Skin rash Mucosal changes	0
Henoch Schoenlein purpura (HSP)	7 (12%)	8 (5–12)	4:3	Skin rash (100%) Hematuria (4/7) 57.1%	0
Pauci/Poly articular JIA	14 (24.1%)	11 (3–14)	10:4	Joint pain (100%) Joint swelling (100%) Movement restriction (85.7%)	5ANA, RF2
Enthesitis related arthritis (ERA)	4 (6.9%)	12.5 (12–14)	3:1	Back pain (100%) Joint swelling (100%)	0
Total	58				24

Among pediatric rheumatologic disorders, the most common were juvenile idiopathic arthritis (29.3%), Kawasaki disease (17.2%), and systemic lupus erythematosus (13.8%).

All eight patients with confirmed SLE based on the SLICC criteria were female. The median age at the time of diagnosis was 14 years. The clinical features of the patients were nonspecific, and included fever (98%), fatigue (100%), and malar rash (25%). Five patients had predominant renal manifestations, while three had acute cutaneous lupus erythematosus manifestations.

Mixed connective tissue disorder (MCTD) was diagnosed in four children with a median age of 8 years. The patients were initially treated for systemic lupus erythematosus; however, at follow-up, they developed clinical findings of systemic sclerosis, dermatomyositis, or JIA along with anti-U1RNP antibody positivity. Two children with typical cutaneous symptoms were diagnosed with juvenile dermatomyositis based on the EULAR Against Rheumatism criteria and skin biopsy or MR. The patient did not have any autoantibody positivity, and one of them experienced rapid progression of muscle weakness involving the respiratory muscles, leading to mortality. The most common autoantibody in both multiorgan and single organ groups was the Antinuclear antibody (ANA) [Figure 2].

Figure 2.

Pediatric immune disorders with ANA positivity

Seven children with SLE and MCTD had hypercoagulable states associated with APLA positivity, and three had peripheral gangrene. Two teenage girls who were diagnosed in later stages of MCTD required limb amputation because they did not respond to medical management for thrombosis and superimposed infection. The central nervous system was involved in 3 children with SLE who presented with recurrent episodes of depression, psychosis, and seizures.

Among the 17 patients confirmed to have juvenile idiopathic arthritis [Figure 3], eight with pauciarticular JIA, six with polyarticular JIA, three with systemic-onset JIA, and four with enthesitis-related arthritis were enrolled in the study. Thirteen patients were males, and five were females. The most common presentation was joint swelling and pain (100%), with movement restriction (85.7%). Pauciarticular JIA was more prevalent (8), of which 5 children had ANA positivity with a good response to intra-articular steroid joint injections. The median age at presentation was 11 years with predominant knee involvement. Among children with polyarticular JIA (6), two had RA factor positivity and a more severe form with small joint involvement along with severe restriction. Three children who developed uveitis on follow-up and required immunosuppression stepping-up were included in the study.

Figure 3.

Juvenile Idiopathic Arthritis categories and autoantibody positivity

Among childhood vasculitis cases, Kawasaki disease (10) and Henoch–Schonlein purpura (7) are more common. The median age at which the patients presented with Kawasaki disease was 5 years, with an equal sex distribution. However, males had more severe disease. All children who were diagnosed with Kawasaki disease based on the clinical criteria had a skin rash, strawberry tongue (100%), and high-grade fever (75%). Five of the 10 children had coronary involvement, requiring long-term aspirin therapy. The acute episodes were treated with intravenous immunoglobulin and high-dose aspirin.

ANCA-associated vasculitis was diagnosed in two adolescent girls, both of whom had severe renal and lung involvement and required intensive care treatment; one of them succumbed to the illness. Takayasu arteritis was diagnosed in 2 children based on CT angiography, with 1 adolescent boy presenting with hypertensive encephalopathy who was later confirmed to have renal artery stenosis that required renal artery stenting in addition to steroids and cyclophosphamide.

Single system involvement:

Among pediatric patients with a single-organ autoimmune disorders [Table 2] like, those with nervous system involvement (39), thyroid (25) hematologic disorders (18), or gastrointestinal disorders (15) were included in this study. ANA was the most common autoantibody identified in both groups (47.5%).

Table 2.

Clinical presentation of Single-system Autoimmune disorders

System	Disease	Total no	Median age at presentation (IQR)	M: F	Antibody positivity
Hematology (18)	ITP	12 (11.6%)	9 (6–15)	3:9	ANA 6 (50%)
	Autoimmune hemolytic anemia	6 (5.8%)		4:2
Thyroid (25)	Autoimmune thyroiditis	23 (22.3%)	9 (5–12)	16:7	Anti TPO 12, anti TG 4,
	Graves	2 (1.9%)		1:1	anti-TSH 2
CNS (39)	Autoimmune encephalitis	17 (16.5%)	5 (3–6)		Anti-NMDA 6
	ADEM	6 (5.8%)	7 (5–9)	2:4
	GBS	16 (15.5%)	4 (3–9)	13:3
GIT (15)	IBD	7 (6.8%)	8 (4–12)	3:4
	Autoimmune hepatitis	8 (7.8%)	6 (4–9)	2:6	Anti-SMA – 4, ANA – 3, anti-LKM 3
Renal (6)	Hemolytic uremic syndrome	6 (5.8%)	12 (5–14)	1:1	2- Anti-factor H
	Total	103			42

Thirty-nine children had neurological presentations, such as refractory seizures or abnormal movements, and 17 of them were diagnosed with Autoimmune Encephalitis, with a median age of five years at presentation. Most patients presented with seizures (50%), muscle weakness, and abnormal motor movement (27%). Anti-NMDA antibody was positive in 6/17 children diagnosed clinically as autoimmune encephalitis. All patients were treated with corticosteroids, Intravenous Immunoglobulin (IvIg), or rituximab if they did not respond to steroids. Sixteen patients were confirmed to have Gullian-Barré syndrome based on neuroimaging and CSF analysis. Six children were diagnosed with acute disseminated encephalomyelitis (ADEM) based on clinical and neuroimaging findings, of which 2 were male and 4 were female. The median age of the patients at diagnosis was 7 years. The main clinical presentations of these patients were fever (66%) and an altered sensorium (33%). No specific antibodies were detected in the study group. Patients were treated with intravenous steroids and immunosuppressants.

Of the 16 patients diagnosed with Gullian–Barré syndrome (GBS), 13 were male and 3 were female. The median age at diagnosis was four years. Patients presented with ascending proximal weakness in the lower limbs (60%), respiratory paralysis (40%), and visual disturbances (12.5%). Among them, only five who were affording underwent antibody testing for anti-NMO and antiganglioside antibodies, but the results were negative. All patients were treated with intravenous immunoglobulin, and six of them required oral immunosuppressants.

Twenty-five children, with a median age of 9 years, were confirmed to have autoimmune thyroid involvement. It had a female predominance (17 females, 8 males) and mostly had hypothyroid presentations, such as lethargy, constipation, and irregular menstruation, while two had hyperthyroid presentations, and they were confirmed to have Graves’ disease with the presence of anti-TSH antibodies. Anti-TPO antibody was positive in 12 patients, and anti-thyroglobulin antibody was positive in 4 patients who were diagnosed with Hashimoto’s thyroiditis. All patients received regular treatment and were followed up on an outpatient basis.

Children with hematologic disorders such as autoimmune hemolytic anemia (AIHA) (6) and chronic immune thrombocytopenic purpura (ITP) (12) were included in the study. Of the 18 patients, 7 were male and 11 were female with a median age of 9 years at diagnosis. In the AIHA group, the Direct Coombs test result was positive in all patients. The common clinical presentations of chronic ITP are erythematous rash (44%), bleeding gums/nostrils (33%), and heavy menstrual flow (23%). ANA was positive in 50% of the adolescent children with chronic ITP.

Among gastrointestinal hepatobiliary disorders, eight children with autoimmune hepatitis (AIH) and seven children with inflammatory bowel disorders (IBD) were included in the study. The median age of the patients at presentation was 6 years. Of the eight patients who were diagnosed with autoimmune hepatitis, six were female and autoantibodies were positive in 7 of them. Three of them were both ANA- and anti-SMA-positive, one child was positive for anti-SMA alone, and three children were positive for only anti-LKM. None of the children with IBD had any identifiable autoantibody. The common gastrointestinal presentations were jaundice (85%), abdominal pain (28%), and bowel disturbances (26.6%).

Discussion

The incidence of autoimmune disorders, although rarely suspected, is slowly increasing in pediatric patients. Many autoimmune diseases are likely to be initiated by infectious agents involving pathogenic antigen mimics in genetically susceptible individuals.[4] Self-reactive T cells cross-recognize microbial antigens and assist bystander autoimmune B cells. The early identification of these pathogenic factors and proper treatment can prevent the progression of immune disorders to more severe forms.

The most commonly performed screening tests for suspected immune disorders are CRP level and ESR, which are useful in the initial evaluation and follow-up testing for disease progression. Although these methods are inexpensive and easily available, we can neither confirm any autoimmune disorders nor rule out underlying infectious etiologies. Autoantibody testing is an important method to confirm immune-mediated illnesses. However, it only identified B cell-mediated immune disorders. In our study, the rates of autoantibody positivity [Figure 4] in children with connective tissue (24/58)and single-organ autoimmune disorders (42/103) were 41.3% and 40.7%, respectively, with ANA being the most predominant antibody (47.5%).

Figure 4.

Types of autoantibody positivity

Among the 58 children in the connective tissue disorders group, antibodies were present in 24 children (41.3%). The most common childhood vasculitis cases, such as Henoch–Schönlein purpura and Kawasaki disease, have no specific antibodies identified to confirm their diagnoses. The common symptom in most of the connective tissue disorders was fever but it is difficult to differentiate whether the fever is due to an infectious or immune-mediated etiology. However, most of the children were presenting the tertiary care after antibiotic therapy or after 2–4 weeks of illness.

The most common connective tissue disorder in our study was juvenile idiopathic arthritis (21 patients), among which 9/21 (42.8%) were autoantibody (ANA)-positive, and only 3.4% had rheumatoid factor positivity. Pauciarticular JIA was the most common, followed by polyarticular JIA. Few studies from Eastern and Northern India have reported polyarthritic JIA as the most common subtype in Indian children,[5,6,7] in contrast to Western countries,[8] where the oligoarthritic subtype is more common. A study by Hegde et al. in northern India[9] revealed more cases of enthesitis-related arthritis. While ANA was positive in 5/8 (62.5%) pauciarticular JIA, the Rheumatoid factor was positive in only 2/6 (33.3%) Polyarticular JIA children. The incidence of rheumatoid factor positivity is lower in children compared to adults in other studies.[10] Juvenile idiopathic arthritis (JIA) is the most common childhood rheumatic disease worldwide[11,12] and is mostly diagnosed clinically through a combination of presenting signs and symptoms, blood tests, and, if necessary, imaging. Several biomarkers, such as RF, ANA, anti-CCP, S100, and HLA-B27, are being studied to assist in the diagnosis and predict the course of the disease, response to treatment, and relapse. In a study by Tebo et al.,[12] anti-CCP antibodies were identified in RF-positive polyarticular JIA (73%) compared with other categories (13% in SoJIA and 19% in extended oligoarticular JIA). Anti-CCP antibodies are included in the new PRINTO criteria for JIA.[13] Disease-specific genetic markers are useful in the diagnosis of certain diseases, such as HLA-B27, which is present in >90% of patients with AS but is not specific. However, HLA-B27 is also present in >50% of patients with reactive or psoriatic arthritis. As India is a country with diverse ethnicities, socioeconomic backgrounds, and climatic conditions, more multicenter studies from different regions are needed to determine the epidemiological pattern of JIA in Indian children.

In our country, rheumatic arthritis must be ruled out before starting treatment as a JIA. ANAs are most frequently found in 65–85% of young girls with oligoarthritis and uveitis, representing a unique condition in JIA.[13,14] PRINTO proposed a new diagnostic criterion called early onset ANA-positive JIA that was not addressed in the ILAR criteria.[15,16] Studies have also shown that S100 proteins and IL-18 are useful markers for predicting treatment response and relapse in patients with systemic JIA and for diagnosing the JIA subtype.[12]

Recent clinical studies[17,18] have shown that estimations of NT-proBNP and IL-17 levels may be useful for differentiating Kawasaki disease from other confounding infectious diseases in children. Many markers, such as tumor necrosis factor (TNF), vascular endothelial growth factor (VEGF), and endocan, have been studied to identify vascular endothelium damage biomarkers for HSP.[19] A study by Berthelot et al.[20] in an adult prospective cohort identified urine IgA as a specific marker of IgA vasculitis nephropathy. In our study, IgA deposits were shown in all skin biopsy specimens. Renal biopsy was done in only 2 children.

In our study, the most common single-organ autoimmune disorder in children was autoimmune thyroid disorder (25), followed by autoimmune hematological disorder (18). The autoantibody positivity rate was 42/103 (40.7%), which was more common in patients with thyroid disorders (18/25) and autoimmune hepatitis (AIH-7/8). ANA positivity was detected in only 6/18 autoimmune hematological patients. In autoimmune hemolytic anemia, peripheral smear findings of hemolysis, elevated reticulocyte count, elevated LDH, and unconjugated bilirubin are common findings, as is a positive direct Coombs agglutination test.[21,22] Currently, the direct agglutination test is the gold standard for AIHA detection. In a pediatric study by Arora et al.[23] Ten patients were positive for polymerase-specific direct coombs (DCTs). Upon further evaluation, 2/10 polyspecific DCT-positive patients were positive for immunoglobulin G (IgG) and C3d, whereas the remaining eight patients were positive for IgG only. It may coexist with immune thrombocytopenia (ITP), which is termed Evans syndrome, and accounts for up to 30% of childhood AIHA cases. One child was diagnosed with Evans syndrome with autoimmune anemia and thrombocytopenia and developed blindness in both eyes as a rare complication during follow-up, as described in a few case reports.[24]

A large cohort study[25] by Khandelwal et al. of 138 Indian children demonstrated an association between anti-CFH autoantibodies and atypical HU. The recent discovery of the anti-complement factor H (CFH) antibody has revolutionized the understanding of atypical hemolytic uremic syndrome (aHUS) and also led to newer treatment options like Eculizumab. With the rising incidence of immune dysfunction in many chronic diseases, there is need to identify newer biomarkers or autoantibodies which will help in early diagnosis of other disorders also.

Autoimmune encephalitis which is increasingly diagnosed worldwide in the past few years, with the identification of specific autoantibodies like anti-NMDAR, anti-CASPR2, anti-GABABR, and anti-LGI1 that are as common as viral causes in many parts of the world.[26,27] Other neural antibodies, such as anti-MOG and leucine-rich glioma inactivated protein 1 (LGI1), have been used to diagnose various other immune-mediated conditions, such as multiple sclerosis and ADEM. Treatment often involves corticosteroids, intravenous immunoglobulins, and/or plasma exchange[28] in most of this immune mediated neurologic damage. In our study, only autoimmune encephalitis had positive autoantibody (anti NMDA 35.3%) in the currently available serological and CSF tests. We couldn’t identify any autoantibody in ADEM or GBS among the children included in this study.

There is a need to identify simpler screening biomarkers for pediatric autoimmune disorders, so that clinical suspicion can be correlated with objective laboratory evidence. Many recent research studies[29] have shown that Th22 and Th9 cell levels and the levels of cytokines, such as IL-22 and IL-9, are elevated in patients with SLE, RA, ITP, autoimmune hepatitis (AIH), autoimmune thyroid disease (AITD), inflammatory bowel disease (IBD), immune-related pancytopenia, or thrombocytopenia. However, none of these markers are specific to a particular disease, and they can also be elevated in post-infectious conditions. With the emergence of new viruses and increased incidence of autoimmune diseases, the breach in immune tolerance should be identified earlier by serology or biomarkers.[30] Our study revealed that only approximately 40% of children can be diagnosed with autoimmune and connective tissue disorders with currently available antibody testing. Most predominantly T cell-mediated autoimmune disorders and vasculitis require a high degree of clinical suspicion and criteria-based diagnosis. Further research is needed to identify early biomarkers in immune-mediated pediatric disorders that will help to confirm the diagnosis and guide physicians to start immunosuppressive therapy at the right time.

The strengths of this study include a wide clinical spectrum of immunologic diseases along with the data about the prevalence of these diseases in South India and the need for autoantibody testing in children presenting with prolonged illness including fever without focus. Our study had a few limitations, as the duration of the study overlapped with that of the COVID-19 pandemic and patients with milder manifestations of immune disorders did not seek medical help. Because our study was performed at a tertiary medical college, which has a referral bias, the prevalence of immune-mediated disorders in children may be estimated falsely higher than the community prevalence. However, being a second-tier city, the non-availability of certain tests and the poor affordability of patients prevented them from undergoing newer autoantibody or cytokine testing.

We conclude that early clinical suspicion and prompt diagnosis in children with prolonged illness or atypical presentation is essential to ensure remission and prevent complications, morbidity and mortality. The currently available antibody test results may not be positive in approximately 60% of childhood connective tissue disorders and autoimmune conditions. This can lead to delayed diagnosis, resulting in poor prognosis and complications. Newer biomarkers and easily available simple diagnostic tests are needed to confirm autoimmune diseases in peripheral healthcare settings.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.