Risk factors for infectious complications of ANCA-associated vasculitis: a cohort study

Background Severe infections are common complications of immunosuppressive treatment for antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) with renal involvement. We investigated the clinical characteristics and risk factors of severe infection in Chinese patients with AAV after immunosuppressive therapy. Methods A total of 248 patients with a new diagnosis of ANCA-associated vasculitis were included in this study. The incidence, time, site, and risk factors of severe infection by the induction therapies were analysed. Multivariate Cox proportional hazards models were used to calculate hazard ratios (HRs) with 95% confidence intervals (CI). Results A total of 103 episodes of severe infection were identified in 86 (34.7%, 86/248) patients during a median follow-up of 15 months. The incidence of infection during induction therapy was 38.5% for corticosteroids (CS), 39.0% for CS+ intravenous cyclophosphamide (IV-CYC), 33.8% for CS+ mycophenolate mofetil and 22.5% for CS + tripterygium glycosides, 76 (73.8%) infection episodes occurred within 6 months, while 66 (64.1%) occurred within 3 months. Pneumonia (71.8%, 74/103) was the most frequent type of infection, and the main pathogenic spectrum included bacteria (78.6%), fungi (12.6%), and viruses (8.7%). The risk factors associated with infection were age at the time of diagnosis (HR = 1.003, 95% CI = 1.000–1.006), smoking (HR = 2.338, 95% CI = 1.236–4.424), baseline secrum creatinine (SCr) ≥5.74 mg/dl (HR = 2.153, 95% CI = 1.323–3.502), CD4+ T cell< 281 μl (HR = 1.813, 95% CI = 1.133–2.900), and intravenous cyclophosphamide regimen (HR = 1.951, 95% CI =1.520–2.740). Twelve (13.9%) patients died of severe pneumonia. Conclusion The infection rate during induction therapy was high in patients with AAV. Bacterial pneumonia was the main type of infection encountered. Age at the time of diagnosis, smoking, baseline SCr ≥5.74 mg/dl, CD4+ T cell< 281 μl, and IV-CYC therapy were identified as risk factors for infection.


Patient selection
A total of 248 patients newly diagnosed with AAV and renal involvement who met the criteria of the Chapel Hill Consensus Conference [7] between January 1, 1998 and December 31, 2013 at the National Clinical Research Center of Kidney Diseases Jinling Hospital were included, among whom 194 patients had renal biopsies that showed pauci-immune necrotic and crescentic glomerulonephritis. All patients were ANCA-positive. Patients with secondary vasculitis, including Henoch-Schonlein purpura, allergy, autoimmune disease, tumour, cryoglobulinemia and infection, were not included. Patients with end-stage renal disease (ESRD) or who received only non-immunosuppressive treatment for infection at the time of diagnosis of AAV were excluded from the study. Ethical statement: This study was approved by the Institutional Review Board of our hospital and performed in accordance with the ethical standards laid down in appropriate version of the Declaration of Helsinki. All patients signed informed consent.

Clinical and laboratory data
All clinical and laboratory data were collected retrospectively at diagnosis and during the follow-up period, including the patients' age, gender, medical history, routine blood analysis, 24-h urine protein excretion, urinary sediment red blood cell count, serum albumin and serum creatinine (SCr), liver enzymes, immunoglobulin and T lymphocyte counts, serum ANCAs, lung involvement, Birmingham Vasculitis Activity Score (BVAS) [16], the usage of immunosuppressive agents, methlyprednisone pulse therapy, plasma exchange, and adverse events including major infection. Major infections were diagnosed according to common terminology criteria for adverse events (CTCAE) v4.0 in addition to clinical and radiological manifestations and microorganism cultures.

Immunosuppressive therapies
None of the patients had received any immunosuppressive therapy before diagnosis. Patients without contraindication initially received intravenous methylprednisolone pulse therapy (0.5 g, once daily, for 3 consecutive days) after diagnosis of AAV. Patients with severe manifestations of AAV underwent plasma exchange therapy. All patients received oral prednisone at a dose of 0.6-0.8 mg/ kg/day for 4 weeks, which was then tapered by 5 mg each week to 10 mg/day. Induction immunosuppressive agents included MMF 1-1.5 g/day orally, monthly intravenous cyclophosphamide (IV-CYC) at 0.75-1.0 g/m 2 body surface area in monthly pulses, tripterygium glycosides (TW, extract from the traditional Chinese herb Tripterygium wilfordii, which mainly contains triptolide) and multi-target therapy (prednisone, mycophenolate mofetil and tacrolimus) [8]. Maintenance therapy included prednisone 5 mg/day combined with MMF and azathioprine. Prophylaxis of Pneumocystis Jirovecii pneumonia (PJP) with SMZ-CO (trimethoprim-sulfamethoxazole 400/80 0.48 g per day) was used in patients whose CD4 + T cell counts were less than 200/μl, and the doses were tapered in patients with renal dysfunction [17].

Antimicrobial therapy
All immunosuppressive agents, except prednisone, were discontinued in patients with AAV who suffered from major infection during the follow-up period. Antimicrobial therapy was prescribed according to clinical and radiological manifestations and microbiological characteristics. Patients diagnosed with PJP were treated with SMZ-CO and echinocandin together.

Supportive therapy
Patients with weight loss were prescribed enteral nutrition. The patients with severe acute kidney injury or acute respiratory distress syndrome (ARDS) were treated with continuous blood purification.

Definitions
A recorded severe infectious complication was defined as implying the administration of an antimicrobial medication for an observable clinical, microbiological and radiologic suspected infection requiring hospitalization. Immediate dialysis was defined as the clinical necessity of renal replacement therapy on admission. The first immunosuppressive agent used in addition to corticosteroids was termed induction therapy. The immunosuppressive regimen used during follow-up was termed the maintenance agent. The diagnosis criteria for deep fungal infection included clinical manifestations, such as fever, cough, diarrhoea or lower urinary tract symptoms, and the detection of fungi in sputum, urine, stool or tissue specimens. Cytomegalovirus (CMV) infection was diagnosed by CMV polymerase chain reaction (PCR). The range of quantification of this assay was 600-100,000 copies/ml for CMV. CMV pneumonia was defined as the detection of ground glass opacity by chest X-ray film or computed tomography, the detection of CMV in the bronchoalveolar lavage fluid or lung tissue samples, and clinical signs such as fever, cough, dyspnoea and hypoxemia. The diagnosis of PJP was made clinically or by the identification of Pneumocystis from sputum, bronchoalveolar fluid, tracheal secretions or lung tissue by special stains or a non-nested PCR, specifically designed to diagnose pneumonia rather than colonization [18]. ARDS was defined as the acute onset of hypoxemia (arterial partial pressure of oxygen to fraction of inspired oxygen [P aO2 /F IO2 ] ≤ 200 mmHg) with bilateral infiltrates on chest radiographs, without left atrial hypertension. Multiple organ dysfunction syndrome (MODS) was defined as the simultaneous failure of at least two organs. ESRD was defined as eGFR < 15 ml/min per 1.73m 2 or requiring renal replacement treatment for > 3 months.

Follow-up and endpoints
The follow-up endpoints included the final date of December 31, 2014, dropping out before the final date, reaching ESRD, or death.

Statistical analysis
Statistical analysis was performed with SPSS 20.0 for Windows (SPSS Inc., Chicago, IL, USA). Medians and ranges were reported for non-normally distributed data, and means ± standard deviations were reported for normal-distributed data. The Kruskal-Wallis test was applied for the comparison of non-normal distributed data. Differences between means were tested using the Student's t-test. A Mann-Whitney U test was used for nonparametric distributions. Chi-squared tests were used for the comparison of categorical data. To address the independent predictive value of factors associated with the rate of infections, the variables with P values of less than 0.1 in univariate analysis as well as those reported in the literature were selected for multivariate analysis using the Cox regression model. The group with corticosteroids only was used as a reference group in multivariate analysis. Only the time to first severe infection was evaluated. Laboratory values and BVAS used for modelling were from the time of diagnosis. Receiver operating characteristic (ROC) curve analysis was performed to determine the cut-offs of SCr, haemoglobin, albumin, CD4+ T cells and BVAS. All tests were two-tailed, and P-values of < 0.05 were considered significant. Confidence intervals (CIs) were calculated at the 95% level.

Treatment and outcome of infectious episodes
All 103 episodes were treated with intravenous antibiotics. Twelve (11.7%) of 103 patients died and all due to severe pneumonia. The time to death was from one to sixteen months after the initiation of immunosuppressive therapy. None died due to AAV (Table 4).

Discussion
A link between vasculitis and infection has long been suspected. Bacterial infections can trigger the production of various autoantibodies, including ANCA [19]. Infection is a major concern in the management of AAV and is the most common cause of death, especially in patients with malnutrition or immunosuppressive therapy [1][2][3]. Immunosuppressive therapy is performed with consideration of the disease activity, which is comprehensively evaluated based on the BVAS score [20]. Nonetheless, even in patients with severe ANCA-associated vasculitis, secondary infection, rather than active AAV, is the leading cause of death [21]. There still remains no firm conclusion about the burden and characteristics of major infections in patients with AAV. We retrospectively reviewed the clinical charts of 248 Chinese patients with AAV. Major infections were reported in 34.6% of our single-centre cohort. Approximately 64.1% of these infections developed in the first three months of induction therapy. In the reported studies, corticosteroids contributed to 89% of infections of patients with AAV, and the infection rate decreased when the corticosteroids were tapered [1][2][3][4]. Corticosteroid treatment leads to an immunocompromised status in patients by inhibiting cytokines, neutrophils, and immunologic response and by exerting anti-inflammatory and immunosuppressive effects [22]. Infection is suspected when fever (≥37.3°C) persists for no less than three days and C-reactive protein increases after remission of AAV [22]. The evaluation of infection is based on the presence of organ manifestations. Identificating methods of causative microorganisms, such as common bacteria, viruses, and fungi, include mycological, histological, and genetic tests [20].
The main areas of infection included the lungs and skin. The lung infection rate was as high as 79.6% in this cohort. Most AAV patients had impaired renal function, and lung involvement and diffuse alveolar haemorrhage injure the local protective barrier. Renal injury also increases the risk of severe infection and is closely associated with a poor outcome [6,18]. According to the literature, the most common causative pathogens are bacteria, such as Streptococcus pneumonia and Haemophilus influenza [23,24], followed by fungi and viruses. In our cohort, the main bacteria included Acinetobacter baumannii, Staphylococcus aureus, and Pseudomonas aeruginosa (Table 2), and our rates of infections with fungi and viruses were higher and lower, respectively, than those of previous reports [25]. Characteristics of AAV, such as global inflammation, renal injury, lung involvement, malnutrition, and immunosuppressive therapy, contribute to infections by opportunistic pathogens [26]. CMV, PJP and 13 cases of pneumomycosis developed during induction therapy. It is also possible that a PJP diagnosis may have been   [27][28][29]. Thus, improving the vaccination coverage against streptococcus pneumonia and influenza in high-risk populations could play an important role in pulmonary prevention [30]. The most common computed tomography findings were ground-glass attenuation, reticular pattern, and fibrous bands with infiltration. In cases of bacterial, fungal and viral pneumonia, a consolidation and reticular pattern, patchy consolidation and glass-ground attenuation were most commonly observed, respectively. These characteristics are predominantly seen in pneumonia patients with AAV.
Given the high incidence of infections in patients with AAV, risk factors need to be defined in order to increase surveillance and prescribe prophylactic antibiotic therapy. Many studies have reported that age, female gender, diabetes, impaired renal function, clinical grade category of rapidly progressive glomerulonephritis (RPGN), lymphopenia and immunosuppressive therapy are risk factors for infection in AAV [6, 12-14, 20, 31]. However, there remains no consensus about the infectious risk factors in Chinese patients with AAV. In this cohort, BVAS and the frequency of diabetes in the infectious group were higher than that in the control group, indicating that higher BVAS and diabetes are potential risk factors of infection. Age at the time of diagnosis (HR = 1.003, 95% CI = 1.000-1.006), smoking (HR = 2.338, 95% CI = 1.236-4.424), baseline Scr ≥5.74 mg/dl (HR = 2.153, 95% CI = 1.323-3.502), CD4 + T cell< 281 ul (HR = 1.813, 95% CI = 1.133-2.900), and use of intravenous CYC were independent risk factors of infection. Whether or not the use of CYC was a risk factor for developing infection in AAV patients remains controversial [9,10,14]. Masaharu [20] also reported that the use of CYC was a risk factor for developing infection in AAV patients, but no difference was observed in renal failure between those with or without infection. On the other hand, CYC showed similar adverse events when compared to Rituximab in two randomized controlled trials [9,10].
In our study, the infection-related mortality (11.7%) was less than that reported in most of the literatures [4,13,14,32]. Half of these cases died within the first month after diagnosis. Thus, clinicians should consider adaptive immunosuppressive agents to avoid life-threatening infection.

Limitations
There are some limitations in this retrospective study. First, the treatment protocols were not uniform and lack of data on Rituximab. Only a minority of patients were given SMZ-CO prophylaxis because of the insufficient awareness. None of these patients received prophylaxis for fungal infection. In addition, some cases with pulmonary or central nervous system infection failed to show a definitive pathogen. The frequency and severity of pneumonia should be lowered by prophylactic treatment and early diagnosis.

Conclusion
Infections can develop during every stage of AAV, primarily in the lungs and skin. The pathogens identified in this study mainly consisted of bacteria, candidiasis, CMV and herpes simplex virus, and age at the diagnosis, smoking, baseline SCr higher than 5.74 mg/dl, CD4 + T cell< 281 μl, and CYC therapy were independent risk factors for infection in patients with AAV.