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Efficiency of interleukin 6 and interferon gamma in the differentiation of invasive pulmonary aspergillosis and pneumocystis pneumonia in pediatric oncology patients

Open AccessPublished:May 18, 2016DOI:https://doi.org/10.1016/j.ijid.2016.05.016

      Summary

      Objective

      Invasive pulmonary aspergillosis (IPA) and Pneumocystis pneumonia (PCP) are two types of pulmonary fungal infection that are not easy to differentiate. The purpose of this study was to investigate the role of inflammatory cytokines in the differential diagnosis of IPA and PCP.

      Methods

      A total of 227 pediatric oncology patients diagnosed with acute pneumonia were enrolled. They were divided into three groups: IPA, PCP, and ‘others’. The cytokine levels in these groups were compared, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-10, IL-6, IL-4, and IL-2.

      Results

      Of the six cytokines, only IL-6 and IFN-γ levels were elevated in patients with acute pneumonia. IL-6 was comparable between patients with IPA and PCP (52.0 pg/ml vs. 25.8 pg/ml, p = 0.092), while IFN-γ was much higher in patients with PCP (19.9 pg/ml vs. 8.9 pg/ml, p = 0.001). The accuracy of IL-6 and the ratio of IL-6/IFN-γ in predicting IPA were 69.0% and 72.0%, respectively, while the accuracy of IFN-γ to predict PCP was 67.2%. IL-6 >140 pg/ml and IL-6/IFN-γ >9.0 presented specificities of 90% in predicting IPA, while IFN-γ >40 pg/ml presented specificity of 90% in predicting PCP.

      Conclusions

      IL-6 is predominantly elevated in IPA, while IFN-γ is significantly increased in PCP. These are helpful tools for the differential diagnosis of IPA and PCP.

      Keywords

      1. Introduction

      Pulmonary infection is a common cause of mortality among pediatric oncology patients, especially those with neutropenia. Fungi are common pathogens, with an increasing morbidity and high mortality.
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      The epidemiology of fungal infections in patients with hematologic malignancies: the SEIFEM-2004 study.
      For example, patients with hematological malignancies are the population most frequently affected by invasive aspergillosis,
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      and aspergillosis is the most common fungal infection in children, accounting for about 75% of invasive fungal disease.
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      Pneumocystis jirovecii is a ubiquitous fungus that can cause significant respiratory disease in immunocompromised hosts, with a mortality rate of 35–55% in HIV-negative patients.
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      Analysis of underlying diseases and prognosis factors associated with Pneumocystis carinii pneumonia in immunocompromised HIV-negative patients.
      Both of these fungi are common in pediatric oncology patients after intensive chemotherapy. However, due to the non-specific clinical manifestations and the poor sensitivity or specificity of most conventional laboratory tests and radiological imaging, the diagnosis is often difficult and delayed.
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      Laboratory diagnosis of invasive aspergillosis.
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      Although these organisms can be identified using light microscopy, immunofluorescence, or molecular methods,
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      PCR diagnosis of Pneumocystis pneumonia: a bivariate meta-analysis.
      it is often difficult to obtain the necessary samples from these patients, such as bronchoalveolar lavage fluid, induced sputum, and lung biopsy specimens.
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      Pneumocystis pneumonia in children—the relevance of chemoprophylaxis in different groups of immunocompromised and immunocompetent paediatric patients.
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      Pneumocystis infection in humans: diagnosis and treatment.
      Recently, Samuel et al. reported that the PCR results for Pneumocystis pneumonia (PCP) using upper respiratory samples presented high consistency with those obtained using lower respiratory samples in children, which may make the diagnosis of PCP easier.
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      • et al.
      Improved detection of Pneumocystis jirovecii in upper and lower respiratory tract specimens from children with suspected Pneumocystis pneumonia using real-time PCR: a prospective study.
      However, wider implementation of PCR on upper respiratory samples for the diagnosis of PCP is warranted to confirm its accuracy.
      Inflammatory cytokines including interleukin (IL)-6, IL-10, tumor necrosis factor (TNF), and interferon (IFN)-γ are important biomarkers for distinguishing infections caused by various pathogens. A prospective study was previously conducted on the role of rapid cytokine profile determination by flow cytometry in pediatric hematology–oncology patients who had experienced a febrile illness since 2005. Previous studies have shown that these cytokines are very helpful in discriminating Gram-positive and Gram-negative bacteremia, viral and bacterial infections.
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      • Song H.
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      • et al.
      Inflammatory cytokine measurement quickly discriminates Gram-negative from Gram-positive bacteremia in pediatric hematology/oncology patients with septic shock.
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      • et al.
      Evaluation of Th1/Th2 cytokines as a rapid diagnostic tool for severe infection in paediatric haematology/oncology patients by the use of cytometric bead array technology.
      In order to investigate the role of inflammatory cytokines in differentiating pulmonary infections caused by Aspergillus and P. jirovecii, the clinical data of patients diagnosed with invasive pulmonary aspergillosis (IPA) and PCP were collected retrospectively and the cytokine profiles of the two entities were compared.

      2. Patients and methods

      2.1 Data collection

      From January 2011 through June 2015, patients in the Department of Hematology–Oncology of the Children's Hospital of Zhejiang University School of Medicine diagnosed with acute pneumonia and with positive microbiological results were enrolled. The patients were divided into three groups according to the pathogen causing the pneumonia: IPA group, PCP group, and ‘others’ group. The ‘others’ group included patients with pneumonia caused by pathogens other than Aspergillus and P. jirovecii. Fifty pediatric oncology patients who underwent Th1/Th2 cytokine measurement at admission and who were without symptoms or signs of infection served as a control group. Information including patient demographic characteristics and clinical and laboratory findings at diagnosis was collected for analysis in this study.

      2.2 Definition of IPA and PCP

      The diagnosis of IPA was obtained when the following criteria were fulfilled: (1) host factors such as neutropenia, corticosteroid or immunosuppressant use, or chemotherapy; (2) clinical symptoms including cough, respiratory distress, dyspnea, and fever resistant to antibiotics for more than 72 h; (3) pulmonary computed tomography (CT) scan showing typical signs, such as nodular lesions with or without halo, cavities, and air crescent sign; (4) positive result for serum galactomannan (GM) antigen, direct stain, or culture as mycological evidence of IPA; (5) empirical therapy and definitive therapy with one or a combination of antifungal drugs was effective.
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      • Walsh T.J.
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      • Stevens D.A.
      • Edwards J.E.
      • Calandra T.
      • et al.
      Revised definitions of invasive fungal disease from the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG) Consensus Group.
      The clinical diagnosis of PCP was retained when (1) at least two of the three following signs were present: non-productive cough or cough producing clear sputum, fever, and dyspnea increasing over time; (2) chest X-ray revealed the characteristic diffuse infiltrations in both lung fields, or a chest high-resolution CT scan showed diffuse frosted glass opacities and thickened alveolar sputum in both lungs; (3) a favorable outcome was obtained under co-trimoxazole therapy, provided that no other infectious agent was found.
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      • Decaux O.
      • et al.
      Diagnosis of Pneumocystis jirovecii pneumonia in immunocompromised patients by real-time PCR: a 4-year prospective study.

      2.3 Th1/Th2 cytokine measurement

      The Th1/Th2 cytokines were measured at diagnosis. Concentrations of IFN-γ, TNF-α, IL-10, IL-6, IL-4, and IL-2 were determined quantitatively using a cytometric bead array (CBA) kit (CBA Human Th1/Th2 Cytokine Kit II; BD Biosciences, San Jose, CA, USA), as described previously.
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      Diagnostic accuracy of a specific cytokine pattern in hemophagocytic lymphohistiocytosis in children.

      2.4 Statistical analysis

      Comparisons were performed using the Chi-square test or Fisher's exact test for categorical variables, and using the Mann–Whitney U-test (for two groups) or Kruskal–Wallis H-test (for three groups) for continuous variables. Receiver operating characteristic (ROC) curves were used to evaluate the discriminatory power (represented by the area under the curve) of IL-6, IFN-γ, and their ratios to differentiate IPA and PCP and to determine the cut-off values with optimal sensitivity and specificity. All statistical analyses were performed using IBM SPSS Statistics version 20.0 software (IBM Corp., Armonk, NY, USA). A p-value of <0.05 was considered to be statistically significant.

      3. Results

      3.1 Patient characteristics

      A total of 277 pediatric oncology patients (227 patients with acute pneumonia and 50 control cases) were included in this retrospective analysis: 115 patients had IPA, 37 had PCP, 75 had pneumonia caused by other pathogens (50 by Mycoplasma pneumoniae, 15 by bacteria, and 10 by viruses and others), and 50 were control cases without symptoms or signs of infection. The demographic characteristics of the IPA, PCP, and other pneumonia groups are shown in Table 1. The age and sex distribution were comparable between the IPA and PCP groups. Acute lymphoblastic leukemia and acute myeloid leukemia were the major underlying diseases in this cohort. More than 70% of patients with IPA were in a neutropenic state when the diagnosis was made, while only about one-third of patients with PCP were neutropenic. On the other hand, more patients with PCP needed oxygen support (14 with a nasal cannula, five with a mask, and three with mechanical ventilation) than patients with IPA (14 with nasal cannula and four with a mask). Four patients died of pulmonary infection. The death rate was comparable between the two groups.
      Table 1Comparisons of demographic characteristics and laboratory findings between patients with IPA and PCP
      CharacteristicsIPAPCPOther pneumoniap-Value
      Comparison between IPA and PCP groups.
      Sex, male/female66/4924/1345/300.421
      Age, years, median (range)7.3 (0.9–15.4)5.3 (1.3–15.9)4.0 (1.0–12.3)0.072
      Underlying disease0.763
       ALL99 (86.1%)32 (86.5%)53 (70.7%)
       AML14 (12.2%)3 (8.1%)11 (14.7%)
      Neutropenia81 (70.4%)13 (38.2%)7 (9.3%)<0.001
      Oxygen support18 (15.6%)22 (59.5%)5 (6.7%)<0.001
      Death2 (1.7%)2 (5.4%)0 (0%)0.084
      IL-2 (pg/ml)2.9 (1.0–5.7)2.7 (1.0–6.0)3.2 (1.0–7.1)0.954
      IL-4 (pg/ml)3.0 (1.0–91.0)3.0 (1.0–80.0)3.3 (1.2–30.0)0.507
      IL-6 (pg/ml)52.0 (2.5–2484.0)25.8 (4.6–3738.1)13.7 (1.3–552.9)0.092
      IL-10 (pg/ml)6.7 (1.0–147.0)7.7 (2.2–117.4)4.9 (1.7–61.2)0.701
      TNF-α (pg/ml)2.1 (1.0–63.0)2.2 (1.0–52.3)2.2 (1.0–48.0)0.870
      IFN-γ (pg/ml)8.9 (1.0–1100.6)19.9 (2.0–186.0)10.2 (1.2–164.7)0.001
      CRP (mg/l)52 (1–200)34 (1–165)7.0 (1–161)0.827
      PCT (μg/l)0.22 (0.02–48.7)0.19 (0.06–8.7)0.11 (0.02–2.59)0.903
      IPA, invasive pulmonary aspergillosis; PCP, Pneumocystis pneumonia; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; CRP, C-reactive protein; PCT, procalcitonin.
      a Comparison between IPA and PCP groups.

      3.2 Th1/Th2 cytokines in patients with IPA and PCP

      The distribution of Th1/Th2 cytokines at the time of diagnosis is shown in Figure 1 and Table 1. The normal median and range values were as follows: IL-2, 2.9 (1.0–4.8) pg/ml; IL-4, 3.0 (1.2–5.1) pg/ml, IL-6, 5.6 (2.3–8.6) pg/ml, IL-10, 7.5 (2.9–13.7) pg/ml, TNF-α, 3.8 (1.7–9.7) pg/ml, and IFN-γ, 5.5 (2.0–9.2) pg/ml. Of the six cytokines, IL-2, IL-4, IL-10, and TNF-α values were comparable among patients with IPA, PCP, other pneumonia, and control cases, while IL-6 and IFN-γ levels were elevated in patients with IPA, PCP, and other pneumonia when compared with control cases (Figure 1). The IL-6 concentration in patients with IPA was significantly higher than that in patients with other pneumonia (p < 0.001), and appeared to be higher than that in patients with PCP, but without statistical significance (p = 0.092). The IFN-γ concentration was much higher in patients with PCP compared with patients with IPA (p = 0.001) or other pneumonia (p = 0.011). C-reactive protein (CRP) was moderately elevated in most patients with IPA and PCP, and was both higher than that in patients with other pneumonia (both p < 0.001) . A similar trend was found for procalcitonin (PCT), although the PCT level was within the normal range in most patients. Both CRP and PCT were comparable between patients with IPA and PCP.
      Figure thumbnail gr1
      Figure 1Distribution of IL-6, IFN-γ and IL-6 to IFN-γ ratio in control and patients with acute pneumonias. IPA, invasive pulmonary aspergillosis; PCP, Pneumocystis pneumonia.

      3.3 Differentiating IPA from PCP by IL-6 and IFN-γ

      As IL-6 and IFN-γ were elevated in patients with acute pneumonia, their role in predicting IPA and PCP was then investigated. As shown in Figure 2, the diagnostic accuracy of IL-6 and IFN-γ in predicting IPA and PCP was 69.0% (95% confidence interval (CI) 62.1–76.0%) and 67.2% (95% CI 56.9–77.5%), respectively. Next, whether the ratio of IL-6 to IFN-γ performed better than either one parameter was investigated. As shown in Figure 1, Figure 2, the median and range IL-6/IFN-γ ratio was 4.9 (0.02–776.3) in patients with IPA, 1.6 (0.09–189.7) in patients with PCP, and 1.33 (0.09–32.5) in patients with other pneumonia. The diagnostic accuracy of the IL-6/IFN-γ ratio in predicting IPA was 72.0% (95% CI 65.4–78.7%). Based on the ROC analysis, as shown in Table 2, an IL-6 concentration higher than 52 pg/ml and an IFN-γ concentration higher than 24 pg/ml yielded a sensitivity of about 50% and a specificity of about 80% when predicting IPA and PCP, respectively. When the cut-off value of IL-6 was set at 140 pg/ml and the cut-off value of IFN-γ was set at 40 pg/ml, the specificities could reach as high as nearly 90%. The IL-6/IFN-γ ratio presented higher accuracy to predict IPA when compared with IL-6, yielding a sensitivity of 59.1% and a specificity of 78.6% at the cut-off value of 3.4. If the ratio was higher than 9.0, the specificity could be higher than 90%. In contrast, among patients with IPA and PCP, if the IFN-γ/IL-6 ratio was higher than 1.6 (IL-6/IFN-γ ratio less than 0.63), the specificity to diagnose PCP could be nearly 90%.
      Figure thumbnail gr2
      Figure 2Accuracy of IL-6 (A) and IL-6 to IFN-γ ratio (B) to predict IPA and IFN-γ to predict PCP (C). IPA, invasive pulmonary aspergillosis; PCP, Pneumocystis pneumonia.
      Table 2Predictive accuracy of IL-6 and IFN-γ for PCP and IPA
      Cut-offTargetSensitivitySpecificity
      IL-617.5 pg/mlIPA80.0% (71.8–86.3)48.2% (39.2–57.4)
      52.0 pg/mlIPA50.4% (41.4–59.4)77.7% (69.1–84.4)
      140 pg/mlIPA21.7% (15.2–30.1)88.4% (81.1–93.1)
      IFN-γ6.5 pg/mlPCP81.1% (65.8–90.5)30.0% (23.9–36.9)
      24.0 pg/mlPCP51.4% (35.9–66.6)82.6% (76.6–87.6)
      40.0 pg/mlPCP35.1% (21.8–51.2)89.5% (84.3–93.1)
      IL-6/IFN-γ1.0IPA80.9% (72.7–87.0)40.2% (31.6–49.4)
      3.4IPA59.1% (50.0–67.7)78.6% (70.1–85.2)
      9.0IPA36.5% (28.3–45.6)91.1% (84.3–95.1)
      IFN-γ/IL-6
      Includes only IPA and PCP groups.
      0.25PCP83.8% (68.9–92.4)59.1% (50.0–67.7)
      0.43PCP43.2% (28.7–59.1)80.0% (71.7–86.3)
      1.6PCP35.1% (21.8–51.2)89.6% (82.6–93.9)
      IL, interleukin; IFN, interferon; PCP, Pneumocystis pneumonia; IPA, invasive pulmonary aspergillosis.
      a Includes only IPA and PCP groups.

      4. Discussion

      IPA and PCP are two types of potentially life-threatening fungal infection seen predominantly in immunocompromised individuals. Despite progress in understanding these diseases and the availability of new fungicidal agents, the mortality rates in pediatric hematology–oncology patients remain high. This is in part related to the difficulty in making a sensitive and specific diagnosis. Delayed diagnosis has an obvious impact on the initiation of early treatment and consequently on the patient's prognosis.
      • Guo F.
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      • Yang S.L.
      • Xia H.
      • Li X.W.
      • Tong Z.H.
      Pneumocystis pneumonia in HIV-infected and immunocompromised non-HIV infected patients: a retrospective study of two centers in China.
      For example, although a definite PCP diagnosis can be made when the P. jirovecii organism is found in respiratory samples using light microscopy, immunofluorescence, or molecular methods, obtaining the samples necessary for these techniques from pediatric hematology–oncology patients, including bronchoalveolar lavage fluid, induced sputum, and biopsies, is really difficult. The serum β-1,3-glucan level is a marker of Pneumocystis infection that can contribute to the diagnosis of PCP;
      • Li W.J.
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      however, it takes a long time for results to become available and it is not helpful to differentiate various fungal infections. The serum galactomannan assay is reported to be sensitive and specific for the diagnosis of invasive aspergillosis.
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      However, it appears to be insufficiently sensitive when performed in many Chinese institutions due to reagents or problems with the technique.
      During Aspergillus infection, monocytes, macrophages, and neutrophils mostly contribute to the antifungal innate immune response through phagocytosis and direct pathogen killing. Then, the development of specific T-helper cell (Th) responses is protective against the development of IPA.
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      IFN-γ is a highly potent activator of phagocytes against opportunistic fungal pathogens in humans. IL-6 appears to have a protective proinflammatory and immunomodulatory role in pulmonary host defense.
      For Pneumocystis infection, alveolar macrophages are the primary resident phagocytes that mediate clearance of Pneumocystis from the lung, which release TNF, IL-1, and IL-6.
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      These cytokines initiate the CD4+ T-cell response during PCP. CD4+ T-cells proliferate in response to Pneumocystis antigens and generate IFN-γ, which augments the Pneumocystis-associated host response. However, different from IPA, PCP is an immunorestitution disease that symptomatically or paradoxically deteriorates during recovery of the immune system .
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      This indicates that immunopathological damage contributes to the development of this disease, especially during the reversal of the immunosuppressive processes. The overwhelming T-cell activation contributes to the predominant level of IFN-γ. The finding in the present study that about two-thirds of patients with PCP were not in a neutropenic state is consistent with this theory.
      IPA and PCP share many similar features, which leads to difficulty in differentiating the two entities; these include similar clinical manifestations and radiological imaging. Furthermore, as many laboratory tests are not specific or not easily available, it is a great challenge for clinicians to make a quick diagnosis based on the limited evidence. The role of serum inflammatory markers such as CRP, PCT, and IL-6 in the diagnosis of fungal infection has been evaluated in a few retrospective studies; however, these studies have generated heterogeneous findings.
      • Robinson J.O.
      • Lamoth F.
      • Bally F.
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      • Calandra T.
      • Marchetti O.
      Monitoring procalcitonin in febrile neutropenia: what is its utility for initial diagnosis of infection and reassessment in persistent fever?.
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      • Lafon I.
      • Ferrant E.
      • Legouge C.
      • et al.
      Evolution of procalcitonin, C-reactive protein and fibrinogen levels in neutropenic leukaemia patients with invasive pulmonary aspergillosis or mucormycosis.
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      In this study, the role of inflammatory cytokines in the diagnosis of pulmonary fungal infection was investigated and it was found that IL-6 and IFN-γ are frequently elevated in patients with IPA and PCP. However, the two entities present different cytokine profiles. IL-6 is predominantly increased in patients with IPA, while IFN-γ is more significantly increased in patients with PCP. As the two markers do not increase in parallel in these two diseases, the ratio of IL-6/IFN-γ is an even more accurate marker to differentiate IPA and PCP. When the ratio is higher than 9.0 or less than 0.63, the specificities to diagnose IPA and PCP can reach as high as 90%. This appears to be the first report on the role of IL-6 and IFN-γ in diagnosing fungal infection. The results of Th1/Th2 cytokine measurements can be obtained within 5 h of sample collection when using CBA technology, which makes this a helpful approach for the rapid diagnosis of critical diseases like IPA and PCP.
      There are some limitations to this study. Histopathological results were lacking for most of the patients in this cohort diagnosed with IPA and PCP. Therefore, the possibility that some of the patients had another type of fungal infection, such as candidiasis, cannot be excluded. Also, this was a retrospective study and the cytokine results that were used were those measured at the time the fungal infection was suspected; the cytokines were not dynamically monitored thereafter, thus the concentrations of cytokines measured may not be the peak levels and they may not have been obtained at the same stage of disease. This may lead to heterogeneity or bias.
      In conclusion, IL-6 and IFN-γ are increased in the serum of patients with IPA and PCP. IL-6 is predominantly elevated in IPA, while IFN-γ is significantly increased in PCP. Thus, the concentrations of IL-6 and IFN-γ and the ratio of IL-6/IFN-γ are helpful for the differential diagnosis of IPA and PCP.

      Acknowledgements

      This study was supported in part by grants from the National Natural Science Foundation of China (No. 81470304) and the Zhejiang Provincial Natural Science Foundation of China (No. LZ12H08001).
      Conflict of interest: The authors declare no conflict of interest.

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