Introduction

Treatment of refractory childhood nephrotic syndromes, such as frequently relapsing nephrotic syndrome/steroid-dependent (FRNS/SDNS) and steroid-resistant nephrotic syndrome (SRNS), remains a challenge. Although various immunosuppressive agents are effective, a substantial number of children are intractable. Recently, rituximab (RTX), a monoclonal antibody that targets the B cell specific antigen CD20, has been demonstrated to be effective for FRNS/SDNS and SRNS in children [1–3]. RTX is relatively well tolerated; however, occasionally severe or life-threatening adverse events occur, including progressive multifocal leukoencephalopathy [4], interstitial pneumonia [5], and ulcerative colitis [6]. Moreover, a decreased number of B cells potentially induces opportunistic infection and deterioration of infection. Pneumocystis jiroveci, formerly known as Pneumocystis carinii, pneumonia (PCP) is a rare but serious cause of mortality in patients with acquired immunodeficiency syndrome (AIDS), as well as in immunocompromised hosts. RTX increases susceptibility to PCP. Previous cases of PCP following rituximab treatment have been reported for various indications, such as B cell lymphoma [7–9], rheumatoid arthritis [10], Wegener’s granulomatosis [11], autoimmune hemolytic anemia [12, 13], pure red cell aplasia [14], pemphigus [15], and acute rejection of kidney transplants [16, 17]. PCP usually presents as a bilaterally diffuse and fairly symmetric interstitial pattern on chest X-ray and as patchy ground-glass opacities on high-resolution computed tomography (hrCT). However, immunocompromised hosts who exhibit hematological malignancy with or without bone marrow transplants, chemotherapy including RTX, and AIDS infrequently present with atypical PCP with multiple nodular granulomatous lesions [18–20]. We report the first pediatric case of nodular granulomatous PCP in which the patient was treated with a single dose of 375 mg/m2 of RTX against SDNS. RTX potentially increases the risk of granulomatous PCP. Written informed consent for publication of this information was obtained from the child’s family.

Case report

The patient is a 3-year-old boy with SDNS. He had been healthy before he presented with NS at 2 years of age. At primary onset, he was initially treated with 25 mg/day (60 mg/m2/day) of daily prednisolone (PSL) for 4 weeks. NS was in remission 10 days after initiation of PSL. PSL was then reduced to 16 mg/day (40 mg/m2) on alternate days, but NS had relapsed soon after PSL reduction. He was treated with 25 mg of daily PSL again. Proteinuria resolved in 7 days. However, as PSL was reduced to 20 mg/day on alternate days, he experienced a second relapse. Thereafter, we stopped reducing PSL by administration on alternate days. Nevertheless, his NS repeatedly relapsed whenever daily PLS was reduced to less than 15 mg. Cyclosporine, losartan and mizoribine were added after 5, 8, and 9 months after diagnosis, respectively. Renal biopsy was performed 5 months after the onset of NS.

One year after onset, at his fifth relapse, the patient was treated with daily PSL 18 mg/day (32 mg/kg/m2), cyclosporine 50 mg/day (3.3 mg/kg/day), and losartan 15 mg/day (1 mg/kg/day). He was then treated with daily PSL 25 mg/day (1.8 mg/kg/day) for 4 weeks. The dose of PSL was slightly less than that used previously, because he was intolerant to the previous full dose of PSL owing to a mood disorder. After 4 weeks, his urinary protein/creatinine ratio was still 6, and serum albumin level was 2.5 g/dl. Additionally, he suffered from serious steroid-related toxicities, including growth retardation, obesity, and bilateral glaucoma. He was transferred to our institute because of SRNS.

After admission, he was treated with one course of methylprednisolone pulse therapy (30 mg/kg/day, three consecutive days). Four days after this therapy, his NS completely remitted. Thereafter, we administered 375 mg/m2 of RTX 2 days after the remission, and we added two more courses of methylprednisolone pulse therapy for reduction of steroids and prevention of further relapses. The patient did not have a reaction to RTX infusion, and was finally discharged. After RTX infusion, daily PSL 1 mg/kg/day was continued for 1 month, and it was reduced to 0.25 and 0.5 mg/kg/day every other day. Cyclosporine was continued, and its 2-h blood concentration was maintained between 400 and 600 ng/dl. However, his NS relapsed 50 days after RTX infusion, and he was admitted to our clinic again. Upon admission, he experienced no symptoms. His temperature was 36.3 °C, heart rate was 110/min and blood pressure was 100/50 mmHg. However, his respiratory rate was slightly fast (30/min). Oxygen saturation remained above 95 % while the patient was awake, but dropped to 92 % during sleep. Other physical examinations did not exhibit any remarkable findings. However, a chest radiograph exhibited patchy and solid infiltrates spread over both lungs (Fig. 1a). Chest helical computed tomography (CT) revealed multiple solid opacities in bilateral lobes.

Fig. 1
figure 1

Chest X-ray and high-resolution computed tomography (CT) scan of the lung. a Chest X-ray shows patchy and solid infiltrates spread over both lungs on admission. b A high-resolution CT scan of the chest 3 weeks after admission shows exacerbation of solid opacities in both lobes

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A peripheral blood examination showed the following: hemoglobin, 13.0 g/dl; platelets, 34.6 × 104/μl; and white blood cells, 6.1 × 104/μl with 72.9 % neutrophils, 18.2 % lymphocytes, 5.2 % monocytes, and 1.5 % eosinophils. The biochemistry profile indicated normal liver and renal function. Lactate dehydrogenase was elevated to 500 IU/l and the isoenzyme patterns suggested that the origin was the lung. The CRP level was 1.0 mg/dl, and erythrocyte sedimentation rate was 44.0 mm/1 h. Serum creatinine and blood urea nitrogen were 0.15 and 11.6 mg/dl, respectively, and serum total protein and albumin were 5.1 and 3.5 g/dl, respectively. Urinary protein was 208.5 mg/dl, and the urinary protein/creatinine ratio was 2.1. The amount of CD19- and CD20-positive B cells in peripheral blood was zero (before RTX infusion, they were 552 and 604/μl, respectively). β-d-Glucan was elevated to 322 pg/ml (normal: <20 pg/ml). Cytomegalovirus antigenemia, Epstein–Barr virus genome, and QuantiFERON (test for tuberculosis) results were all negative. Urinalysis exhibited mild albuminuria (urinary protein: 208 mg/dl, urinary creatinine: 99 mg/dl). Because of marked elevation of β-d-glucan, infection with fungus or P. jiroveci was suspected. We performed bronchoalveolar lavage (BAL) under general anesthesia on day 3. After bronchoscopy, the patient was treated with oral TMP-SMX for Pneumocystis at a dose of TMP 5 mg/kg every 6 h and voriconazole for fungus, including Aspergillus 15 mg/kg/day. PSL was maintained at 10 mg/day to prevent a withdrawal reaction, and cyclosporine was halted. Microscopy was negative but polymerase chain reaction analysis revealed Pneumocystis DNA. No other pathogens were detected in his blood culture and BAL. We stopped voriconazole and continued a full dose of TMP-SMX for 3 weeks followed by a prophylactic dose of TMP (5 mg/kg/day). Although CD19- and CD20-positive cells in peripheral blood recovered 8 months after RTX, he was treated with prophylactic TMP for 10 months because he was still being treated with MMF.

Although the patient’s oxygen saturation improved, and β-d-glucan returned to a normal range, lactate dehydrogenase levels remained elevated. Another chest hrCT scan 3 weeks from the initiation of TMP-SMX demonstrated a further increase in the number and size of the pulmonary nodules (Fig. 1b). To rule out any other causes that were overlooked, the patient underwent open thoracic wedge resection of the nodule lesion. A biopsy sample showed necrotic granulomas containing a few cysts of P. jiroveci and Ziehl-Neelsen staining was negative (Fig. 2a/e). Immunohistochemistry revealed that the granulomas primarily consisted of CD3-positive T cells and CD68-positive histiocytes; however, there was an absence of B cells (Fig. 2b, c, d). His respiratory status remained stable, and lactate dehydrogenase soon returned to the normal range. A chest hrCT on the 52nd day exhibited an apparent decrease in nodular lesions. The patient’s NS also spontaneously remitted along with the recovery of pulmonary findings.

Fig. 2
figure 2

Pathological findings of lung biopsy. The biopsy sample shows necrotic granulomas (a, hematoxylin-eosin stain, magnification ×100), consistent with CD3-positive T cells (b) and CD68-positive histiocytes (c) (positive cells were stained brown), but without CD20-positive B cells (d). Several cysts of P. jiroveci were identified inside of the granulomas by Grocott’s stain (e, arrows)

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Discussion

This is the first childhood case of granulomatous PCP. Additionally, this is the first case of PCP following treatment with rituximab against SDNS. PCP is a serious cause of mortality in immunocompromised patients, affecting up to 50 % of patients. Our patient was treated with a high dose of daily PSL, cyclosporine, and mizoribine for an extended period. Additional rituximab potentially strengthens immunosuppression and increases the susceptibility to PCP. However, our patient’s clinical and radiological findings substantially differed from typical PCP, and we had to rule out viral or fungal infection, tuberculosis, and rituximab-induced interstitial pneumonia.

Accumulating evidence suggests that, under some circumstances, RTX may significantly increase the risk of PCP. Recently, cyclophosphamide, doxorubicin, vincristine and prednisone with rituximab (R-CHOP) against B-cell lymphoma was shown to increase the prevalence of PCP compared with classical CHOP (identical treatment without RTX). Additionally, PCP after rituximab has been previously reported in patients with rheumatoid arthritis, Wegener’s granulomatosis, autoimmune hemolytic anemia, pure red cell aplasia, pemphigus, and acute rejection of kidney transplants. Therefore, B cell depletion by RTX may increase the risk of PCP. They suggested the necessity of PCP prophylaxis after RTX therapy.

The host’s defense against Pneumocystis is thought to be critically dependent on CD4+ helper T cells. The incidence of PCP in human immunodeficiency virus (HIV) infected patients is elevated when the level of circulating CD4 cells falls below 200/μl. However, B cells potentially play a significant role in protection against Pneumocystis. Transgenic B-cell functionally deficient mice, in which B cells do not express major histocompatibility complex class 2 antigens (and thus were unable to act as antigen-presenting cells), are susceptible to Pneumocystis carinii (formerly the species Murina). These mice fail to clear the Pneumocystis infection, most likely because of the inefficient generation of protective CD4+ memory and effector T cells in the lungs [21]. The deficiency of B cells potentially results in attenuated immunoprotection against P. jiroveci.

Our patient was likely infected with P. jiroveci. However, he developed granulomatous PCP after RTX treatment; therefore, RTX potentially contributed to the development of granulomatous PCP. A defect in B cells induces high susceptibility to P. jiroveci, as well as development of granulomatous PCP. Granulomatous PCP is an emerging concept. Granulomatous PCP has been previously reported in up to 4–5 % of PCP patients. A total of 35 adult patients have previously exhibited granulomatous PCP. They suffered from AIDS, hematological neoplasms, and solid malignancy, but none of these patients had NS. Interestingly, three of them were treated with RTX prior to the development of PCP [19, 20]. B cell function appears to be impaired in most of these patients, as in our patient. In our patient, immunohistochemical staining revealed that granulomas primarily consisted of T cells and histiocytes without B cells. B cells may be essential for the clearance of P. jiroveci, and macrophages may substitute for the clearance of P. jiroveci by forming granulomas. In fact, we found several cysts of P. jiroveci inside the granulomas. B cell deficiency following RTX potentially modifies the immune response to P. jiroveci and induces a granulomatous reaction. Furthermore, Totet et al. reported that granuloma formation is not related to any specific genotype of P. jiroveci [22, 23].

Although our patient exhibited no symptoms and his general condition was well on admission, the mortality rate can be 35–50 % in patients without HIV compared with 10–20 % in those with HIV [24, 25]. The diagnosis of conventional PCP is traditionally performed by BAL. However, in the case of granulomatous PCP, BAL frequently fails to detect P. jiroveci [18]. An open lung biopsy is required to generate the correct diagnosis if BAL fails to detect P. jiroveci.

Our case study suggests that RTX may modulate T cell immunity and increase susceptibility to PCP. Furthermore, RTX treatment may cause a granulomatous pattern and make the correct diagnosis of PCP difficult. Indication of rituximab for refractory NS has been recently expanding. Therefore, physicians should be aware of the risk of PCP and its atypical manifestations under combined immunosuppressive therapy, including RTX. Prophylaxis for PCP should be considered after RTX against refractory NS.