Initial manifestations in Patients with Inborn Errors of Immunity Based on Onset Age: a Study from a Nationwide Survey in Japan

Patients with inborn errors of immunity (IEI) manifest various initial symptoms; however, those that are critical for the early diagnosis of IEI have not been identified. Also, the significance of the ten warning signs of primary immunodeficiency (PID) among infants has not been established. This study aimed to conduct a nationwide survey of IEI in Japan and investigated the initial manifestations based on onset age. Among 1298 patients, data regarding the initial manifestation were available from 505 patients. Patients with autoinflammatory diseases, complement deficiency, and phenocopies of IEI were excluded. The ten warning signs were positive in 67.3% of the cases. The positivity rate was low (20.5%) in patients with immune dysregulation. Although the positivity rate was low (36.6%) in patients aged less than 3 months, they were highly positive for family history of IEI (26.8%). Infectious symptoms were the most commonly observed in all age groups and in all disease categories. Symptoms of “immune dysregulation” were present in approximately 15% of the patients. Regarding the anatomical category, almost all initial symptoms were “systemic” infections in patients with X-linked severe combined immunodeficiency. Moreover, “respiratory” symptoms were the most common in patients with IEI aged ≥ 1 year and accounted for more than 50% in all age groups in patients with common variable immunodeficiency. These results highlight the significance of the 10 warning signs and may serve as clinical indicators for early diagnosis, considering the initial presentation of IEI.


Introduction
Inborn errors of immunity (IEI) are a group of diseases, occurring only in approximately 2-10 per 100,000 people, and it includes more than 450 diseases with varying clinical characteristics [1][2][3][4][5]. Early diagnosis enables prompt therapeutic intervention, thus improving the prognosis of patients [6]. It also decreases the incidence of infection or inappropriate vaccination [7,8]. However, vaccination programs in children vary among different countries or areas depending on the epidemic status of infectious diseases. Recently, the "10 warning signs of primary immunodeficiency" presented by the Jeffery and Model Foundation have been used to allow physicians and patients' families to remain vigilant for the clinical signs of IEI, which are expected to play a role in the early diagnosis of IEI [9]. We used the modified 10 warning signs (Supplementary Table 1) [10] in Japan, which includes signs of infectious disease caused by Bacillus Calmette-Guérin (BCG) since 2010. Although these warning signs have been tested and verified for their ability to identify patients with IEI [9,11,12], their significance has not been established in countries like Japan, where the newborn screening system has not been widely implemented, and routine BCG vaccination is in use [8,13].
Although frequent or severe respiratory tract infections (including pneumonia, otitis media, and sinusitis) is emphasized in the 10 warning signs, infections at sites other than the respiratory tract, such as the gastrointestinal tract, should also be acknowledged [14]. In addition, patients with IEI manifest various symptoms initially, with 18-26% of them displaying symptoms of immune dysregulation [15,16]. Currently, the symptoms that are critical for the early diagnosis of IEI have not been identified. Recently, we conducted the first nationwide survey of patients with IEI in Japan over 10 years [8]. The purpose of this study was to characterize the initial manifestation of IEI in patients and to elucidate the performance of the modified 10 warning signs using nationwide study data, which may help in the early diagnosis of IEI.

Study Participants
This study was conducted according to the nationwide epidemiological survey manual for patients with intractable diseases (3rd edition, 2017) [17], similar to a previous survey [4]. Details of the survey results are presented in our previous report [8]. We sent questionnaires to approximately 20% of all 12,517 departments (pediatrics, internal medicine, hematology, rheumatology, and dermatology) in Japan using stratified random sampling. Departments treated at least 1 IEI patient during 2018 were asked the respective patient details. Diseases were classified according to the International Union of Immunological Societies 2017 classification [18].
Based on the secondary survey results, patients with autoinflammatory diseases and complement deficiency were excluded because of an insufficient description of the initial manifestation in many cases. Patients with insufficient data for age either, at disease onset or initial symptoms, were also excluded from the analysis

Initial Presenting Symptoms and the Disease Category
In the secondary survey, physicians were questioned about the patients' age, sex, disease, initial symptoms, and age at onset. As initial symptoms, they were asked about the presence or absence of each of the 10 warning signs (Japanese version) [10]. The presence of other symptoms, if any, was noted in the free text field (free description). All initial symptoms, including the 10 warning signs and those written in the free-text field, were categorized into pathological and anatomical categories (Table 1). In line with previous reports, the pathological categories were "infectious," "immune dysregulation," "malignancy," "syndromic," "inflammatory," and "no symptoms" [16]. Anatomical categories were newly established by the authors (Table 1) and were defined as "respiratory" (e.g., pneumonia, bronchitis, otitis, sinusitis), "mucocutaneus" (atopic dermatitis, eczema, subcutaneous abscess, lymphadenopathy), "gastrointestinal" (vomiting, diarrhea, bloody stool including inflammatory bowel disease), "hematological" (hemophagocytic syndrome, cytopenia, hyper/hypo gammaglobulinemia), "deep-seated" (bacterial infections or abscesses in deep-seated organs), "systemic," and "not specified". For example, an airway infection was pathologically classified as "infectious," and anatomically classified as "respiratory."

Data Analysis
To evaluate the performance of the 10 warning signs, focusing on the symptoms at the time of initial onset, the positivity rate for each of the 10 warning signs and other symptoms in each disease category was analyzed. We also calculated the positivity rate for the 10 warning signs excluding family history (nine warning signs), since family history may indicate early diagnosis before symptom onset.
The difference between disease category or age groups were tested using the chi-square test, Fisher's exact test, or Kruskal-Wallis test as appropriate. All tests were two-tailed, and a p-value of <.05 was considered statistically significant. Statistical analyses were performed using STATA version 15 (StataCorp, Texas, USA).

Declarations
The survey was conducted in accordance with the Epidemiological Surveillance of Intractable Diseases Manual [17] of the Ministry of Health, Labour and Welfare, with due consideration to maintain confidentiality of personal information. This study was conducted in accordance with the principles of the Declaration of Helsinki. Informed consent was obtained from parents or their legal guardians in the form of an opt-out on the website. The study protocol was approved by the ethics committee of the University of Tsukuba Hospital (H30-208) and was funded by Health and Labor Sciences Research Grants for Research on Intractable Diseases of Japan (20FC1053).

Background of the Patients
Questionnaires were sent to 2506 departments from a total of 12,517 departments nationwide, and detailed information was obtained from 89 departments for 750 patients. The most common IEI was autoinflammatory disease (n=191, 26%), followed by antibody deficiency (n=183, 24%), and congenital defects in phagocyte number or function (n=121, 16%). The sex ratio was approximately 1.5:1 (males:females). The details of this questionnaire study have been presented in a previous report [8].
The number of patients in each disease category, sex, and median age at disease onset is shown in Table 2. The distribution of age and sex in each disease group was significantly different. The median age at first onset for autoinflammatory diseases, complement deficiency, and phenocopies of IEI was higher than those of the other disease categories. After excluding these patients and those whose age at disease onset was not described, 505 patients with IEI were included in the study. The median age at initial presentation was less than 3 years (Fig. 1).

Positivity Rate of the 10 Warning Signs at First Onset Based on Disease Category
The positivity rates for each disease category are shown in Table 3. The 10 warning signs were positive in 67.3% (340/505) of the cases. Defects in the intrinsic and innate immunity had the highest positivity rate (83.3%, 20/24). In contrast, immune dysregulation had the lowest rate at 20.5% (9/44). These variations by disease group were statistically significant (p<0.01). Among individual manifestations, The patients were divided into the following four age groups according to disease onset: (1) less than 0.25 years (3 months of age), (2) 0.25 to 1 year, (3) 1 to 6 years, and (4) > 6 years. Positivity rates for the ten warning signs in each age group are shown in Table 4. The percentage of positive 10 warning signs exceeded 60% in all age groups, with no statistically significant variation. Compared to the other age groups, the positivity rate of family history was high (26.8%) and that for the remaining nine warning signs was low (36.6%) in patients aged less than 0.25 years. Of all the patients, 10.3% were asymptomatic, had no family history of the disease, and were incidentally diagnosed through abnormal blood test results.

Category of Symptoms by patients' Age at Onset
The initial symptoms of patients with IEI were classified into pathological and anatomical categories (Fig. 2). In the pathological category (Fig. 2b), the frequency of "infectious" symptoms was the highest in all age groups, accounting for > 75% of the patients. Similarly, immune dysregulation symptoms were present in approximately 15% of the patients in all age groups. Regarding the anatomical category, "respiratory" symptoms were the most common in patients aged ≥ 1 year, and the frequency decreased in the younger age groups.

Initial Symptoms in Common IEI
Four common IEI were selected to analyze the prevalence of the initial symptoms (Fig. 3). The age of onset was low in patients with X-linked severe combined immunodeficiency (XSCID) and chronic granulomatous disease (CGD) but was relatively high in patients with common variable immunodeficiency (CVID). Moreover, the number of patients with hyper-IgE syndrome (STAT3 deficiency) was not significantly different among the four age groups. Infectious symptoms were common in all disease categories. Almost all initial symptoms were "systemic" infections in patients with XSCID. In patients with CVID, "respiratory" symptoms accounted for > 50% of all age groups.

Discussion
We conducted a nationwide survey of patients with IEI in Japan and analyzed their initial symptoms. The Japanese version of the 10 warning signs was positive in 67.3% of   the patients (Table 3). In patients with combined immunodeficiency, the positivity rate was as high as 80.4%; however, it was low in patients with immune dysregulation (20.5%). Although infection was the major manifestation of IEI, symptoms associated with inflammation, malignancy, syndromic features, and immune dysregulation were observed (Fig. 2). In addition, the significant rate of mucocutaneous, gastrointestinal, and systemic symptoms suggested that they should also be considered as markers for early diagnosis (Fig. 2). Because IEI encompasses a wide  Deep seated Systemic variety of diseases, this symptom distribution IEI does not imply a pathophysiology of IEI. However, it may be useful to inform non-specialized health care professionals, such as primary care physicians, about IEI, as well as to inform them about the 10 warning signs. The positivity rate of the 10 warning signs was low in patients aged less than 3 months, and they showed an increased tendency to have a family history of IEI (Table 4). Notably, the ten warning signs were not very effective in detecting IEI in patients younger than 3 months old because only 36.6% of them were positive for warning signs other than family history. As previously reported, the 10 warning signs is a list of symptoms that are more frequently seen during the course of the disease, rather than at the onset [9,10,16]. They focus more on susceptibility to infectious diseases rather than other symptoms, such as autoimmune diseases, malignancies, and skin and mucocutaneous manifestations [19,20]. Therefore, these 10 warning signs must be referred to considering the knowledge of these findings. Recent advances in the treatment of IEI have increased the significance of the early diagnosis of IEI, which can reduce the risk of various complications and increase the success rate of therapies, including hematopoietic stem cell transplantation, while maintaining high levels of quality of life in patients. The usefulness of neonatal screening for primary immunodeficiency diseases, such as SCID and X-linked agammaglobulinemia, using TREC/KREC testing has been reported [21] and has already been adopted as a universal screening tool in some regions [22][23][24]. However, this contributes to only a small proportion of the total IEI. Newborn screening is not conducted in many countries. There is still a great need for awareness of the primary manifestations of IEI in families and primary care physicians. It is occasionally difficult to avoid serious adverse events of vaccination in patients with IEI because they may receive vaccination before they are accurately diagnosed with IEI [8]. Under these circumstances, we should suspect the possibility of IEI based on some key manifestations of IEI at the earliest. In this regard, gastrointestinal symptoms and symptoms based on immune dysregulation should be considered [16]. In this study, we observed similar findings, although the positivity rate for gastrointestinal manifestations was not very high. It is noteworthy that "mucocutaneus" manifestation which  Fig. 3 Initial symptoms of anatomical/pathological category in patients with four common inborn errors of immunity by age of onset. Duplication is allowed for cases with more than one initial symptom in a patient. XSCID: X-linked severe combined immunodeficiency, CGD: chronic granulomatous disease, CVID: common variable immune deficiency, BTK: Bruton's tyrosine kinase deficiency is the body surface findings such as lymphadenopathy and lesions of the skin and oral mucosa was frequently observed even in early infancy, similar to the rate at which airway symptoms were detected. This finding was consistent with a recent report [15] and may be important for early diagnosis. To our knowledge, this is the first study analyzing initial symptoms of IEI in Asian population. In addition, this study identifies some other important markers for the early diagnosis of some common IEI. Many patients with CGD manifest deep-seated infections, mucocutaneous lesions, and systemic manifestations, irrespective of the age of onset. Patients with XSCID often develop systemic viral infection. Patients with hyper-IgE syndrome (STAT3 deficiency) manifest bacterial infectious diseases in several organs, including the airways, and manifest mucocutaneous lesions.
Moreover, many asymptomatic cases (10.3% in all disease categories) are incidentally detected during blood tests. Indeed, previous reports showed that lymphocyte abnormalities alone are indicative, with 86% sensitivity and 94% specificity for SCID [25]. In contrast, lymphocytopenia may be the most commonly overlooked laboratory abnormality in IEI because its normal range varies greatly with age [25]. The situation was similar for the immunoglobulin levels. A study of severely infected patients in a pediatric intensive care unit (PICU) revealed that 70% of patients with IEI could be diagnosed using immunoglobulin screening alone [26].
The present study, being a questionnaire survey conducted by clinicians, had several limitations. First, the response rate was not very high, which may not reflect truly comprehensive data of patients with IEI and may affect the accuracy to a certain extent. However, this was a nationwide study and we obtained a relatively high response rate from the pediatric department, particularly from university hospitals and other institutions equipped with specialized staff for IEI [8]. We believe that we were able to present at least the current situation of Japanese patients with IEI. Second, there may be incorrect clinical records or physician memories. In recent years, registration systems for patients with IEI have been established in many regions [27], where all patients are registered from the time of initial presentation and records are shared. In Japan, patient registration has been established and the number of registered patients is increasing. The analysis of such a database should be promoted and may help us obtain more accurate IEI data. Third, although the pathological categories in this study were used in line with a previous study [16], it may be difficult to confirm their suitability for such an analysis. The same was true for the original anatomical categories. To minimize bias in the analysis, the authors (HT=immunologist, KI=gastroenterologist, TK=PICU physician) discussed and agreed on the classification into such categories.

Conclusion
Based on the results of a nationwide survey on IEI in Japan, we examined the initial symptoms of patients with IEI. Infections, respiratory infections and family history were the leading warning signs of IEI at symptom onset. However, the ten established warning signs may be insufficient for early diagnosis, particularly in early infancy. Widespread awareness of mucocutaneous symptoms and other signs of IEI and systematically ensuring that the laboratory abnormalities are not overlooked may be useful for early diagnosis.