Systemic lupus erythematosus (SLE) is an autoimmune disorder that manifests with inflammation and destruction of multiple organs [1]. Juvenile-onset SLE (jSLE) is clinically similar to adult-onset SLE; however, it is often associated with a more severe form of the disease, with a prevalence estimated to be between 1.89 and 34.1 cases per 100,000 children [1,2]. It can manifest with a range of non-specific signs and symptoms, including oral ulceration, fever, arthralgia, headaches, and weight loss, which can occur in children for various underlying causes [1].

The diagnosis and classification of SLE have proven to be a challenging task for clinicians due to its complex nature, which can resemble other autoimmune, infectious, or hematologic conditions [3]. A thorough evaluation of both clinical and laboratory findings is required, following the exclusion of alternative diagnoses. Therefore, various classification criteria have been developed, including the American College of Rheumatology (ACR), Systemic Lupus International Collaborating Clinics (SLICC), and the 2019 European Alliance of Associations for Rheumatology (EULAR)/ACR criteria.

Antinuclear antibodies (ANA) are the immunological hallmark of SLE, and its high sensitivity makes it an important tool in diagnosing, as 98% of patients with SLE were found to have positive ANA [4]. However, its limited specificity poses challenges, as positive ANA can be found in various conditions, including autoimmune diseases and infections. Seronegative SLE is a condition where a patient's clinical features and laboratory values are consistent with SLE, but their serology test for ANA is negative. It appears that ANA-negative SLE is a subtype of SLE that is also called lupus-like, that has been understudied [5]. Some diagnostic criteria require positive serology, while others only depend on clinical features and laboratory values, which makes individuals with negative ANA (more common in jSLE) may go unnoticed, leading to increased morbidity and mortality due to delayed diagnosis and treatment [1,5]. Moreover, monogenic lupus had been also found to have variable results of ANA titers and need to be excluded in such patients [1].

In this article, we present two rare cases of childhood lupus nephritis (cLN) with negative ANA. The first case presented as lupus cerebritis and LN class IV and V which showed improvement with treatment. The other one was also diagnosed as LN class IV but showed poor outcomes even with aggressive immunosuppressive treatment.

Case 1

A 13-year-old previously healthy female presented to the Emergency Department (ED) with a complaint of headache for two days, accompanied by generalized body swelling and decreased urine output. One month prior, the patient started to experience lower limb and periorbital swelling that progressed to the abdomen. The patient's recent headache was accompanied by nausea and vomiting, but there were no symptoms of skin rash or any known allergies. The patient has a familial history of hypertension but no history of autoimmune, rheumatologic, or renal disorders.

On examination, the patient was conscious but severely hypertensive and tachycardic with a blood pressure (BP) of 162/130 mmHg, a pulse rate of 120 beats per minute, a respiratory rate of 22 breaths, a temperature of 37°C and an oxygen saturation of 100% on room air. The patient appeared unwell with a puffy face, bilateral periorbital edema, level 3 and 4 bilateral lower limb pitting edema reaching the thighs, abdominal swelling with shifting dullness, sacral and genital swelling. Four days later, the patient experienced the same headache with vomiting and had new-onset of two generalized seizure attacks with a high blood pressure of 186/90 mmHg which was controlled at the ED.

Upon laboratory evaluation, the patient was found to have normocytic anemia, hypoalbuminemia (20 g/L), and an elevated microalbumin/creatinine ratio (810~813 mg/g). Additionally, urinalysis revealed hematuria. Immunological tests, including ANA and other antibodies related to SLE, were negative. Liver and renal function tests were within normal limits, and chest X-ray and electrocardiography revealed no abnormalities. Ophthalmologic examinations were also normal, and the Coombs test was negative (Table 1).

Table 1 . Laboratory evaluations for the two cases at diagnosis.

Investigation	Case 1	Case 2	Unit	Reference range
White blood cells	5.02	5.1	109/L	4.5~13.5
Hemoglobin	11.0	7.5	g/dL	12~14
Neutrophils	1.77	4	109/L	1.8~8
Lymphocytes	2.38	1	109/L	1.2~5.2
Platelet	315	302	109/L	150~350
ESR	5	35	mm/H	0~15
Blood Urea	3.3	32	mmol/L	2.1~7.1
Creatinine	28	482	µmol/L	44~88
eGFR	161	11	mL/min/1.73m	90
CRP	3.22	0.5	mg/L	0~5
C3	0.5	1.11	g/L	0.83~1.77
C4	0.05	0.34	g/L	0.15~0.45
Serum Albumin	20	28	g/L	36~52
Sodium	142	133	mmol/L	135~147
Potassium	3.6	4.9	mmol/L	3.4~4.7
Calcium	1.94	1.88	mmol/L	2.1~2.7
Chloride	112	97	mmol/L	97~107
Urine protein/creatinine ratio	1,700	821	Mg/mmoL	0~30
Urine analysis	Normal	>10 RBC	HPF	<5
ANA	Negative	Negative
APLA	Negative	Negative
ANCA	Negative	Negative
WES	Negative	Negative

ESR: erythrocyte sedimentation rate, CRP: C-reactive protein, C3: complement component 3, C4: complement component 4, ANA: antinuclear antibody, APLA: antiphospholipid antibodies, ANCA: anti-neutrophil cytoplasmic antibodies, WES: whole exome sequencing, eGFR: estimated glomerular filtration rate, RBC: red blood cell, HPF: high power field, g/dL: gram/deciliter, mm/H: millimeters per hour, mmol/L: millimoles per liter, µmol/L: micromoles per liter, mg/L: milligrams per liter, g/L: gram per liter..

Renal biopsy was consistent with the diagnosis of SLE, featuring combined diffuse LN and membranous LN of class IV and V, according to the International Society of Nephrology and the Renal Pathology Society (ISNRPS). Abnormal brain magnetic resonance imaging (MRI) showed multifocal cortical and subcortical signal intensities, suggesting small infarct regions, as well as a few white matter and brainstem high T2 fluid attenuated inversion recovery (FLAIR) signal intensities, which may be due to small vessel disease. Leptomeningitis was also observed.

The patient was diagnosed with LN with cerebritis and started on prednisolone, mycophenolate mofetil (MMF), and antihypertensive medications. Additionally, the patient received two infusions of rituximab, which led to improvement of her clinical condition and albuminuria. However, typical serological tests associated with SLE remained negative through her follow-ups for 12 months. To exclude monogenic cause, a whole exome sequencing (WES) had been performed and came back negative.

Case 2

An 11-year-old girl presented with a change in urine color, headache, decreased urine output, and vomiting for 5 days. She was found to have acute kidney injury, nephrotic range proteinuria, and hematuria along with hypertension (BP 140/77 mmHg) and fluid overload (lower limb pitting edema and ascites). Her renal function deteriorated quickly, presenting a picture of rapidly progressive glomerulonephritis (serum creatinine from 482 to a peak of 870 µmol/L). She underwent a renal biopsy and required supportive management, which included dialysis, diuretics, antihypertensive medication, and high-dose pulse methylprednisolone.

The renal biopsy revealed full-house, immune-mediated glomerulonephritis, resembling LN class IV, with significant damage (50% global sclerosis and moderate tubular atrophy). No extra-renal manifestations of lupus were observed, and an extensive workup for lupus and vasculitis was negative, including normal complement 3 and 4 levels and absence of autoantibodies (antinuclear antibody, anti-double-stranded DNA [anti-dsDNA], extractable nuclear antigen [ENA], antiphospholipid antibodies, anti- glomerular basement membrane [anti-GBM] antibody, perinuclear anti-neutrophil cytoplasmic antibody [P-ANCA], and cytoplasmic anti-neutrophil cytoplasmic antibody [C-ANCA]). The workup for infectious causes was also unrevealing (throat culture, antistreptolysin O titer, hepatitis B and C viruses). Further laboratory workups showed normocytic normochromic anemia (Hemoglobin 7 g/dL) (Table 1).

Consequently, she was diagnosed with lupus-like nephritis and continued management with high-dose prednisolone, ten sessions of plasmapheresis, one course of rituximab, and six doses of cyclophosphamide.

After 2 weeks of diagnosis, she presented with a high BP of 170/120 mmHg and one episode of focal convulsion. A CT scan of her brain showed findings suggestive of posterior reversible encephalopathy syndrome (PRES), most likely due to hypertension rather than lupus cerebritis. The BP was effectively controlled with an intravenous infusion of labetalol, leading to no further seizures and a return to a normal level of consciousness.

Initially, during the first four weeks, she showed mild improvement with a reduction in serum creatinine to 230 µmol/L. However, despite intensive immunosuppressive therapy, she progressed to chronic kidney disease stage V within 4 months of diagnosis. During follow-up, the autoantibodies and other lupus workup remained persistently negative up to 12 months follow-up. Moreover, a WES had been done and showed negative results.

The study was approved by the King Abdulaziz University Biomedical Ethics Committee (approval number 454-22). Moreover, an informed consent was obtained from both patients and there legal parents.

Juvenile SLE is a common autoimmune disorder that can affect children of any age, although it is rarely present before the age of 5 and is more common in females. The prognosis for jSLE is less favorable compared to adult-onset SLE due to the longer duration of the disease in children, the early involvement of multiple systems, and the adverse effects of systemic therapy [2].

Most children with SLE test positive for ANA; however, there are reported cases of ANA-negative SLE in children, a concept that remains controversial and warrants further evaluation. The presence of additional autoantibodies such as anti-dsDNA, anti-Smith (anti-Sm), Ro, La, and ribonucleoprotein (RNP) can help confirm the diagnosis of SLE, but they are not always present. It is important to note that these antibodies may also be found in children who do not meet the classification criteria for SLE, and some of these children may go on to develop SLE or other rheumatic diseases, while others may remain asymptomatic.

In the two cases presented, kidney biopsy confirmed the diagnosis of LN, and negative WES ruled out known monogenic SLE. In case 1 the presence of CNS symptoms and vasculitis changes in the MRI brain favor the diagnosis of SLE. However, according to the latest ACR criteria, these patients could not be classified as having SLE, leading to a diagnosis of lupus-like nephritis. In Case 1, the profound complement activation (low C3, C4) despite negative ANA, underscores its significance in jSLE severity, independent of traditional serological markers [1].

Several cases of juvenile lupus-like with negative serology have been reported (35 cases) (Table 2) [5-14], with initial presentations varying from renal symptoms, like sub-nephrotic or nephrotic proteinuria, to systemic manifestations such as vasculitis. All these cases have been diagnosed using renal biopsy, showing evidence of LN. Treatment responses have varied, with many patients responding to prednisone or more intensive treatments like cyclophosphamide and azathioprine. Follow-up data showed that 20% of these cases developed positive ANA over time that range from 4 weeks to 10 years, while 63% remained negative, and the status of the remaining 17% was not reported. Regarding our patients, both cases were followed up for almost 12 months duration after the diagnosis and both remained negative for ANA. Our cases add to the previously reported cases and emphasize the variability of this entity and the heterogeneity of disease prognosis that warrants early treatment and closemonitoringas a proportion of these cases could evolve into SLE with time.

Table 2 . A literature review of pediatric ANA-negative lupus-like cases.

Citation	Year	Presentation	Treatment	Outcome	Follow-up ANA result
Wen and Chen [6]	2010	A 14-year-old female presented with nephrotic proteinuria.	PRED	Not reported	Negative (20 months at follow-up)
		A 14-year-old female presented with non-nephrotic proteinuria.	PRED	Developed malar rash and anti-DNA Ab at follow-up after 8 months.	Not reported
Sharman et al. [7]	2004	A 16-year-old female presented with sub-nephrotic proteinuria with a very high risk of progressive renal damage due to her histological appearances.	PRED, Cyc, AZA	Within 4 years, she had reached ESRD and subsequently was transplanted without recurrence of glomerular disease.	Not reported
		A 15-year-old female presented with sub-nephrotic proteinuria and preserved renal function.	PRED, AZA	Six years with no change in renal function.	Not reported
Enriquez et al. [8]	1989	Three children presented with nephrotic proteinuria.	Not reported	Short-term follow-up showed no other systemic manifestation.	Negative
Cairns et al. [9]	1979	A 16-year-old female presented with nephrotic syndrome.	PRED	At 5 months the ANA became positive and then 6 years later the anti-DNA Ab was elevated.	Positive (5 months at follow-up)
Gianviti et al. [10]	1999	Seventeen cases (11 females and six males) eight cases presented as AKI.	Variable therapy	Three cases became positive in ANA and anti-dsDNA after years of follow-up. However, two patients had persistent proteinuria, one had CKD, and one was transplanted after progressing to ESRD.	- Three cases positive (3, 5, and 10 years at follow-up) - Fourteen cases negative
Hunley et al. [11]	1998	A. 14-year-old female presented with nephrotic syndrome.	Not reported	Not reported	Positive (7 months at follow-up)
Gomathy et al. [12]	2017	A 10-year-old female presented with hypertension and steroid-resistant nephrotic proteinuria.	PRED, MMF, Tac	After 2 years she became positive for ANA and Anti-dsDNA.	Positive (2 years at follow-up)
Maziad et al. [13]	2017	A 5-year-old male presented with hypertension, nephrotic proteinuria, and AKI.	PRED, MMF, Cyc	Not reported	Negative
Ahmed et al. [14]	2022	Three cases (two females and one male), all cases presented with sub-nephrotic proteinuria, and one of them also had AKI.	Not reported	Not reported	Not reported
Khan et al. [5]	2013	A 13-year-old male presented as vasculitis with rash, gastrointestinal bleeding, and acute renal failure.	IV-MP, dipyridamole, Cyc, AZA and PRED	The patient improved and his ANA and anti-dsDNA titers remained negative.	Negative
	2007	A 10-year-old female presented with recurrent abdominal pain on further workup, she was diagnosed as a case of SLE nephritis.	IV-MP, Cyc	ANA remained negative. However, the patient progressed into stage 4 CKD after 17 months.	Negative
	2021	A 15-year-old male presented with fever, progressive swelling of the body for weeks, and reduced urine output for 1 week.	PRED	Not reported	Positive (4 weeks at follow-up)
	2009	A 6-year-old female presented with interstitial vasculitis and proteinuria.	IV-MP, Cyc	ANA remained negative and she had persistent low-grade proteinuria.	Negative

AKI: acute kidney injury, ESRD: end-stage renal disease, CKD: chronic kidney disease, PRED: prednisolone, IV-MP: intravenous methylprednisolone, Cyc: cyclophosphamide, AZA: azathioprine, MMF: mycophenolate mofetil, Tac: tacrolimus, ANA: antinuclear antibody, anti-dsDNA: anti-double-stranded DNA, SLE: systemic lupus erythematosus..

The variability in ANA results over time and the presence of other diagnostic criteria in these lupus-like cases raise questions about the ongoing relevance of immunological serology as a central diagnostic criterion in SLE.

In conclusion, our cases present a rare entity in childhood, named lupus-like with negative serology. They show variability in clinical presentation, treatment responses, and the risk of poor prognosis, even with aggressive immunosuppressive treatment, as seen in the second case. This paper underscores the limitations of ANA serology as a mandatory criteria and advocates for reassessing the weight given to serological tests in the diagnostic criteria for SLE. Such an approach can enhance diagnostic accuracy and patient outcomes. This study emphasizes the importance of continuous research and reassessment of diagnostic criteria in the dynamic field of autoimmune diseases.

We would like to acknowledge the two presented patients and their parents.

No potential conflict of interest relevant to this article was reported.

Conceptualization: M.A.N., A.M.A.; Data curation: A.A.A., E.B.B, M.A.S, A.A.Ash.; Supervision: J.A.K. M.A.N., A.M.A.; Writing: A.A.A.. A.M.A. All authors have read and approved the final version.