A Cross-Sectional Study to Compare Differences on Clinical and Laboratory Findings in Children with Seropositive and Seronegative Lupus

Background: Lupus is an inammatory and autoimmune disease that involves various tissues and organs of the body. Identication of diagnostic elements to rapid identication of seronegative lupus cases is very important in order to prevent morbidity and progression of disease. This study aimed to compare clinical and laboratory ndings of seropositive cases with seronegative lupus patients. Methods: This cross-sectional analytic study was performed on 43 children (17 seronegative and 26 seropositive) with lupus who were admitted to Ali Asghar Hospital during 2007-2017. Seropositive patients had anti-nuclear antibody (ANA) titration >1/80, while seronegative patients had ANA titration <1/80 (at the time of disease diagnosis). Clinical and laboratory ndings were compared between two groups. Results: Serositis in patients with ANA - was signicantly higher than ANA + (41.17% vs. 23.07%; p = 0.042). ANA - group had higher autoimmune disease history than ANA + group (42.85% vs. 15.0%; p = 0.041). The family history of the disease in the ANA - group was greater than ANA + group (50% vs. 23.52%). The percentage of hypertensive patients in ANA - group was higher than ANA + group (52.94% vs. 26.92%; p = 0.037). Neurologic symptoms in ANA + and ANA - groups were 38.46% and 17.64%, respectively (p = 0.043). The frequency of patients with thrombocytopenia in ANA + group was signicantly greater than ANA - group (32% vs. 12.5%; p=0.041). There was no signicant difference in other clinical and laboratory ndings between two groups. Conclusion: Seronegative lupus patients musculoskeletal symptoms, autoimmune familial disease, hypertension, while neurological and thrombocytopenia in (13-45%), weight loss (-32%). 21%), mucocutaneous (60-90%), musculoskeletal (60-90%), nephritis (48-78%), NPSLE (15-95%), gastrointestinal problems (24-40%), hematological disorders (50%-100%), heart problems (25-60%), and pulmonary problems (18-81%).


Introduction
Lupus is a chronic in ammatory disease in which the autoimmune system attacks various tissues and organs of the body (e.g. joints, skin, kidneys, blood cells, brain, heart and lungs) and causes serious damage [1]. Lupus is usually divided through several categories, including systemic lupus erythematous (SLE), skin involvement lupus, drug-induced lupus erythematous, neonatal lupus erythematous and pediatric lupus [2]. It is usually accompanied by symptoms such as fatigue, fever and arthritis [3]. Lupus in children is usually somewhat similar to adult lupus; however, children are often involved in the disease for a longer period of time before a de nitive diagnosis [4,5]. Since children are sick for a longer period of time before lupus diagnosis, their internal organs are more likely to be involved and hurt at the time of diagnosis [5].The risk of kidney involvement in children with lupus is approximately twice more than adult patients [4]. Therefore, children suspected of be involved with lupus need serious care strategies than adults [6]. Symptoms in pediatric lupus are highly various with more abnormal clinical presentation compared to adults [7]. In Some cases the disease is presented with typical symptoms such as fever, rash (40 to 60% of cases) and renal dysfunction [8]. Others may be presented with fatigue and pain. Some children look good, but their urine sample may have blood or other invisible problems that can help a physician to diagnose it [9]. The other clinical symptoms are light sensitivity (35 to 50% of cases), alopecia (15 to 30% of cases), oral ulcers (20 to 30% of cases), arthritis (60 to 70% of cases), central nervous involvement (20 to 45% of cases), respiratory problems (15 to 40% of cases), and pericarditis (10 to 15%) [7,10]. The laboratory ndings of pediatrics lupus usually contain hyper-gammaglobulinemia, anemia, leukopenia or lymphopenia, thrombocytopenia, hypo-albuminemia, and increased erythrocyte deposition [7,11]. The presence of anti-dsDNA (in 50 to 85% of cases) is commonly used to con rm the diagnosis diagnosis. Although the positive result of anti-nuclear antibodies (ANA) test is needed for de nitive con rmation of the diagnosis in more than 90% of cases, there are also many ANA positive children without lupus [12]. On the other hand, there are children with lupus who may have negative ANA test result. These children with seronegative lupus usually comprise 1-5% of the lupus patients [13].
Despite these clinical and laboratory tests, there are still patients with negative serological test results which make the precise diagnosis di cult for clinicians. Therefore, evaluating of clinical and laboratory test results in children with seronegative lupus is very helpful to nd out valuable ndings (s) for rapid diagnosis of the disease. In this study, we aimed to consider clinical and laboratory ndings of children with lupus (including seronegative and seropositive cases) who referred to Ali Asghar Hospital from the year of 2007 to 2017 to nd out appropriate elements to identify seronegative patients.

Materials And Methods
In this cross-sectional analytic study, all seronegative and seropositive Systemic Lupus Erythmatosis patients aged less than 16 years who were admitted to Ali Asghar Hospital (Tehran, Iran) from the year of 2007 to 2017 were included. The study was approved by the Ethics Committee of Iran University of Medical Sciences (IR.IUMS.FMD.REC.1397.256). At the beginning of the study, a checklist was provided in which information on demographic and basic clinical data of all patients such as age, sex, BMI, systolic (SBP) and diastolic blood (DBP) pressures, family history of diseases, history of drug consumption, and family history of autoimmune diseases were recorded. Patients with incomplete clinical and laboratory documents were excluded from the study. Eventually, 43 patients (17 seropositive and 26 seronegative) were selected for further evaluations. Patients were divided into two groups of ANA positive (titration >1/80) and ANA negative (titration <1/80) at the time of the disease diagnosis. Patients in both groups were considered phenotypically for general, cutaneous and musculoskeletal symptoms, as well as for neuropsychological, gastrointestinal, pulmonary and renal symptoms, and renal biopsy test results.
Furthermore, the laboratory and immunological test results of patients in each group, including CBC, proteinuria, hypergammaglobulinemia, hematuria, direct coombs test, complements levels, and serum antibodies (e.g. Anti-dsDNA, Anti-phospholipid antibodies, p-ANCA and c-ANCA) were compared.

Statistical analysis
All quantitative data were analyzed using the descriptive program and presented as Mean ± SD.
Crosstabs and Chi-Square tests were used to compare the percentage or frequency of ndings between two groups. The comparison of the mean of quantitative data between two groups was analyzed using independent sample-t test. In this study, p< 0.05 was considered statistically signi cant. The SPSS software (IBM, version 19) was applied for data analysis.

Results
A total of 43 cases with mean age of 10.81 ± 3.16 years (ranged from 1 to 15 years old) and BMI of 18.25 ± 2.63 kg/m 2 (ranged from 13.36 kg/m 2 to 24.49 kg/m 2 ) were included into the study. Overall, 13 patients (30.2%) were boys and 30 cases (69.8%) were girls.
Comparison of the basic demographic and clinical ndings of cases between ANA + and ANA − is summarized in Table 1. Totally, 26 patients (60.46%) were ANA + and 17 cases (39.53%) were ANA − . There was no signi cant in the mean of age (p=0.42), BMI (p=0.3) and sex distribution (p=0.55) between ANA + and ANA − groups. The percentage of girls in both groups was signi cantly higher than boys (p<0.05). Twenty-ve (58.13%) out of 43 patients had at least one of the general symptoms such as fatigue, anorexia, weight loss, fever and lymphadenopathy. The prevalence of general symptoms in ANA + and ANA − groups was 53.84% and 64.7%, respectively (p=0.39). Overall, cutaneous examination of all patients showed that 29 patients (67.44%) had at least one of the mucocutaneous manifestations, including malar rash, photosynthesis, oral ulcer, nasal ulcer and maculopapular rash. The frequency of cutaneous manifestation in ANA + and ANA − groups was 69.23% and 64.7%, respectively (p=0.79). Thirteen patients (30.23%) had at least one of the neuropsychological manifestations, including convulsion, psychosis, headache, depression, and anxiety. The incidence of neuropsychological symptoms in patients in the ANA + group was approximately twice that of the ANA − group (38.46% vs. 17.64%; p=0.043). Seventeen patients (39.53%) had at least one of the gastrointestinal symptoms, including hepatosplenomegaly, intestinal vasculitis, pancreatitis, abdominal pain, and rectal surgery. The distribution of gastrointestinal symptoms in ANA + and ANA − groups was 38.46% and 41.17%, respectively (p=0.62). Twenty-three patients (53.48%) had at least one musculoskeletal manifestations such as arthritis, synovitis, arthralgia and myalgia. The frequency of musculoskeletal symptoms in ANA − group was slightly greater than ANA + group (64.7% vs. 46.15%; p=0.09). Seventeen patients (39.53%) had at least one of the renal symptoms such as edema, nausea, vomiting, ascites and oliguria. The distribution of renal complaints in ANA + and ANA − groups was 38.46% and 41.17%, respectively (p=0.68).
The information of the history of autoimmune disease (such as hypothyroidism, vitiligo ....) was available for 27 patients (20 in ANA + and 7 in ANA − group). Patients in the ANA − had signi cantly higher percentage of autoimmune disease history compared to those in the ANA + group (42.85% vs. 15.0%; p=0.041). The information of family history of the autoimmune disease was available for 21 patients (17 in ANA + and 4 in ANA − group). The family history of the autoimmune disease in ANA − group was relatively higher than that in ANA + group (50% vs. 23.52%; p=0.29). The prevalence of patients with hypertension in ANA − group was signi cantly higher than ANA + group (52.94% vs. 26.92%; p=0.037).
Thirteen patients (30.23%) had serositis (7 patients in ANA + and 6 patients in ANA − group). The prevalence of patients with serositis in ANA − group was signi cantly higher than ANA + group (41.17% vs. 23.07%; p=0.042). The frequency of patients with leukopenia or lymphopenia in ANA + and ANA − group was 40.0% and 31.25%, respectively (p=0.68). The frequency of patients with thrombocytopenia in ANA + group was signi cantly greater than ANA − group (32% vs. 12.5%; p=0.041). Although there was no signi cant difference in the frequency of positive and negative results of direct Coombs between two groups, the direct positive Coombs in ANA + group was relatively higher than ANA group (43.75% vs. 20%; p=0.34). There was no signi cant difference in the frequency of dsDNA test result between two groups; however, patients in ANA + had slightly higher percentage of dsDNA positive compared to those with ANA − (80% vs. 66.66%; p=0.34). Anti-phospholipid antibody test result didn't show a signi cant difference between two groups (p=0.21). Most patients in both groups (88.88% in ANA + and 100% in ANA − ) had negative anti-phospholipid antibody test result. There was no signi cant difference in the p-ANCA and c-ANCA test results between two groups.

Discussion
In this study, 26 patients (60.46%) were seropositive (ANA + ) and 17 patients (39.53%) were seronegative (ANA − ). The frequency of seronegative patients in our study was somewhat more than other previous studies because this research was conducted in long period of time at the Ali Asghar Pediatric Hospital, a referral center in Iran in which many patients even from neighboring countries are referred for diagnostic and treatment procedures. Furthermore, we compared the clinical and laboratory ndings of patients at the time of diagnosis and we didn't intend ANA − patients who had been changed to ANA + in the course of the disease because the purpose of this study was to identify the underlying factors for the diagnose of the disease and seroconversion was not the purpose of this study. We also found that the proportion of female patients in both groups was nearly twice as high as that of boys, indicating a higher incidence of Lupus in girls than boys. Many studies reported that the incidence of lupus in girls is higher than boys [6, 12,14]. For example, Murashima et al., [15], showed that the incidence of ANA + was signi cantly higher in girls than boys; and 9 out of 10 patients who had ANA + at the second visit were girl. In another study, Moradinejad et al.,[16] reported that the ratio of female to male with SLE was 8 to 1 and aged between o 3 to 16 years(sample size=45). In another study, Hiraki et al., [17]examined 256 children with SLE and revealed that the ratio of girls to boys with SLE was 4.7, which is somewhat consistent with the results of our study. Salah et al., [18] showed that the ration of girls to boys with SLE was 2.7 which is in line with our results. In our study, although the percentage of girls with SLE was higher than boys, there was no signi cant difference in sex distribution between ANA + and ANA − groups.
So far, very few studies have compared the results of clinical and laboratory ndings between patients with lupus, especially between seropositive and seropositive children. The results of our study didn't show a signi cant difference in general symptoms such as fatigue, anorexia, weight loss, fever and lymphadenopathy between the ANA + and ANA − groups. The frequency of general symptoms in ANA + and ANA − patients was 53.84% and 64.7%, respectively. Our study also did not show a signi cant difference in the frequency of skin symptoms between the two groups. Interestingly, the results of our study showed a signi cant difference in the frequency of neuropsychiatric symptoms, including convulsion, psychosis, headache, depression, anxiety, and between the two groups. The incidence of neurological symptoms in ANA + group was approximately twice that of the ANA − group. The frequency of neurological symptoms in patients in ANA + and ANA − groups was 38.46% and 17.64%, respectively. Thus, the results of this study suggest that neuropsychiatric manifestations can be one of the diagnostic tools used in the differentiation of seropositive and seronegative SLE patients.
The results of our study did not show a signi cant difference in the frequency of gastrointestinal involvement, including HSM, intestinal vasculitis, pancreatitis, abdominal pain. The frequency of gastrointestinal presentations in ANA + and ANA − groups was 38.46% and 41.17%, respectively. Although there was no signi cant difference in the frequency of musculoskeletal symptoms between two groups, its prevalence was somewhat higher in ANA − patients (64.7%) than ANA + (46.15%). Therefore, it seems that musculoskeletal symptoms may also be a useful diagnostic tool for the differentiation of seropositive and seropositive patients; however, further studies with larger sample sizes are needed to con rm it. The frequency of renal complaints, including edema, nausea, vomiting, ascites, and oliguria in ANA + and ANA − groups was 38.46% and 41.17%, respectively, and didn't show a signi cant difference.
Another nding of our study was related to the history of autoimmune disease between two groups.
Patients in ANA − group had higher autoimmune disease history compared to ANA + group (42.85% vs 15.0%). It is clinically valuable and can be helpful for the prognosis and distinguish of seronegative from seropositive patients. However, further studies with larger sample size should be undertaken. In our study, although there was no signi cant difference in the family history of the disease between two groups, patients in ANA − group had somewhat higher family history of the disease (50% compared to 23.52%).
Interestingly, the results of our study showed a signi cant difference in the frequency of hypertension between two groups. The frequency of hypertensive patients in ANA − group (52.94%) was signi cantly higher than those in ANA + group (26.92%). This data suggests that a history of hypertension can be considered as one of the risk factors for the possibility of lupus seronegative. Along with hypertension, our results showed that the prevalence of serositis ANA − group (41.17%) was signi cantly higher than patients in ANA + group (23.07%). We didn't nd signi cant difference in other parameters such as CBC, complements levels, direct coombs, renal biopsy result, prevalence of proteinuria and hematuria, as well as prevalence of leukopenia and lymphopenia between two groups; however, the prevalence of patients with thrombocytopenia in ANA + group was signi cantly higher than those in ANA − group (32% vs. 12.5%). There was no signi cant difference in the serum antibodies levels including anti-dsDNA, antiphospholipid antibodies, p-ANCA and c-ANCA between two groups. Similarly, Murashima et al., [15]found nonsigni cant difference in the prevalence of anti-DNA and anti-phospholipid antibodies in children with SLE and the control group. Therefore, it seems that investigating several risk factors such as autoimmune and family history of the disease together, hypertension, serositis and musculoskeletal symptoms can be considered for the diagnosis seronegative patients from seropositive patients.

Conclusion
According to the results of this study, seronegative lupus patients compared with seropositive patients have higher percentage of musculoskeletal symptoms, autoimmune disease history, familial history of disease, and hypertension, whereas neurological and thrombocytopenia symptoms were higher in seropositive patients compared to seronegative cases. Therefore, evaluation of these factors can be helpful to diagnose and differentiate of seronegative from seropositive patients. Since one of the limitations of this study was related to small sample size, it is worthwhile to conduct another comprehensive study with larger sample size to con rm the diagnostic role of these factors in seronegative lupus children.

Declarations
Funding: No funds, grants, or other support was received.
Con icts of interest/Competing interests: The author declare they have no con lict of interest Availability of data and material: Not applicable Code availability: Not applicable Authors' contributions: SSH, the acquisition, analysis and interpretation of data for the article.. RS, contributed the data and designed analysis. RH, Approved the version to be published MSH, collected the data.