Özet
Giriş: İnfluenza virüs tarih boyunca birçok pandemiler yaparak milyonlarca insanın ölümüne yol açan önemli bir hastalık etkenidir.
Akciğerde patolojik değişikliklere neden olarak ağır hastalık ve ölüme neden olabilir. Akciğerlerde oluşan hücresel infiltrat ve doku
hasarında doğal immünitenin bir parçası olan sitokinlerin rolü bilinmektedir. İnfluenza virüsün bazı hastalarda oluşturduğu aşırı
ve kontrolsüz sitokin salınımının (sitokin fırtınası), hastalığın ağır seyrinden sorumlu olabileceği düşünülmektedir.
Materyal ve Metod: Hastaneye pandemik influenza ön tanısıyla yatırılan ve referans laboratuvarında RT-PCR ile teyit edilmiş
influenza A (H1N1) tanısı konan hastalar ve tamamen sağlıklı kontrol grubunun serumlarında, enzim immünoassay (EIA) yöntemiyle
tümör nekroz faktörü-alfa (TNF-α), interlökin 1-beta (IL-1β) ve interlökin 6 (IL-6) çalışıldı.
Bulgular: Çalışmaya 57 konfirme H1N1 hastası, 62 kontrol grubu sağlıklı kişi dahil edildi. Hastaların 6 (%10.5)'sı takipleri sonunda
hayatını kaybederken, 51 (%89.4) hasta şifa ile taburcu edildi. TNF-α ölen hastalarda 43.0 pg/mL, yaşayan hastalarda 20.9
pg/mL, kontrol grubunda 4.1 pg/mL ölçülürken, IL-6 değerleri ölen hastalarda 1074.1 pg/mL, yaşayan hastalarda 191.0 pg/mL,
kontrol grubunda 36.1 pg/mL ölçüldü ve aradaki fark istatistiksel olarak anlamlıydı (sırasıyla; p= 0.003 ve p< 0.001). IL-1β değerleri
ise ölen hastalarda 2.1 pg/mL, yaşayan hastalarda 7.1 pg/mL iken, kontrol grubunda 7.5 pg/mL ölçüldü ve aradaki fark istatistiksel
olarak anlamlı değildi (p= 0.657).
Sonuç: TNF-α ve IL-6 düzeylerinin ölen hastalarda belirgin yüksek saptanması fatalite belirteci olarak kullanılabileceğini düşündürmektedir.
Giriş
In the fight against influenza, vaccines and protective measures are used to prevent transmission of the disease, and antiviral drugs are used for treatment. In addition to antiviral drugs, immunosuppressive drugs have been used to halt excessive and uncontrolled cytokine secretion (cytokine storm) that influenza virus causes in animal models[3].
The aim of this study was to determine the effects of proinflammatory cytokines on mortality in patient with influenza A (H1N1) 2009 infection.
Yöntem
All patients with influenza A virus (H1N1) 2009 infection received oseltamivir on admission. The standard dose (150 mg/day) was administered for patients with mild disease, and a higher dose (300 mg/day) was used for critical patients. Empirical antibiotic therapy was given to all patients with pneumonia, and secondary bacterial pneumonia was not diagnosed in any patient. The study protocol was approved by the Ethics Committee for Clinical Research of Diskapi Yildirim Beyazit Training and Research Hospital, Ankara, Turkey.
Control Group
Sixty-two blood donors with normal physical examination and with no known disease were included in the study.
Cytokine Assay
The blood samples of the patients hospitalized with preliminary diagnosis of pandemic influenza were taken within two hours after admission. After coagulation of the samples, they were centrifuged, and sera were separated immediately and stored at -70°C until use. Cytokine levels were measured with commercially available enzyme immunoassay kits (EIA) (tumor necrosis factor (TNF)-α-EASIA KAP1751, interleukin (IL)-1β-EASIA KAP1211, IL-6-EASIA-CE KAP1261, Diasource, Belgium). With zero attachment, and within 2 standard deviations of average OD values, the lower limits of detection of the tests were as follows: TNF-α: 0.7 pg/mL, IL-1β: 0.35 pg/mL and IL-6: 2 pg/mL.
Statistical Analysis
The Statistical Package for the Social Sciences (SPSS) 15.0 package program was used for statistical analysis. ANOVA, chi-square and Mann-Whitney U tests were used for comparisons. A value of p< 0.05 was accepted as statistically significant.
Bulgular
Table 2: Clinical characteristics of the patients
Measured TNF-α, IL-1β and IL-6 levels of the patients and control group are given in Table 3. Levels of TNF-α and IL-6 were higher in H1N1 patients than in the control group and were higher in patients who died during follow-up than in surviving patients, and the difference between them was statistically significant (Table 3, Figures 1,2). While IL-1β levels were higher in the control group than in H1N1 patients, they were at the lowest level in exitus patients, but the difference was not statistically significant (Table 3, Figure 3).
Tablo 3: TNF-α, IL-1β and IL-6 levels in patients and control group
Tartışma
The role of inflammatory cytokines and chemokines in the pathogenesis of influenza was shown in human and animal experiments[2, 5]. The role of cytokines in human H5N1 influenza disease accompanied by high mortality is well known, and it was also suggested that an increase in cytokine levels might have been responsible for the high mortality rate in the 1918 H1N1 pandemic[6, 3].
During experimental influenza infection in animals models, it was shown that while IFN-α, IL-6, and TNF-α levels increased significantly correlated with disease severity, increases in IL-8 and IL-1 levels were less obvious[ 7]. In other studies with mice, although the protective role of TNF-α was shown, a significant increase in TNF-α was determined as a sign of fatal disease and poor prognosis[8, 9]. Moreover, it was shown in patients with influenza that especially TNF-α and IL-6 levels were related to the clinical findings of the disease[10, 11].
Similarly, in our study, TNF-α and IL-6 levels were found to be higher in patients with influenza than in the control group and were even higher in fatal patients. IL-1β levels were found to be lower in fatal patients. In the study carried out by Giamarellos-Bourboulis et al., it was suggested that a decrease in IL-1 level was related to the anti-inflammatory effect of IL-6[12].
Hagau et al. performed a study during the 2009- 2010 influenza season with 21 patients with influenza A (H1N1)-related acute respiratory distress syndrome (ARDS), 11 patients with influenza A (H1N1) mild disease, and 15 healthy volunteers as a control group[13]. They found that the levels of IL-6, IL-8, IL-9, IL-12, IL-15, IL-10 and TNF-α were significantly increased in critically ill patients versus the control group. When mild and critical cases were compared, IL-6, IL-8, IL-15, and TNF-α were significantly higher in critical ARDS patients as hallmarks of disease severity. The higher levels of IL-6 and TNF-α in critically ill patients were similar to our results in fatal patients. Another study conducted by To et al. demonstrated that IL-6 levels were higher in those patients with more severe disease throughout the disease period, whereas IL-1α and TNF-α levels were higher only in the later phase of the disease[14]. In contrast to that study, we found higher TNF-α levels in the early phase of the disease, especially in the fatal patients.
The influenza A (H1N1) 2009 pandemic caused serious morbidity and mortality. Unlike the seasonal flu, the pandemic influenza A (H1N1) 2009 caused mortality in patients without underlying risk factors. Therefore, it is important to predict in which patients the disease may be fatal. The significant increase in TNF-α and IL-6 levels in patients who did not survive demonstrated these markers to be useful as indicators of fatality. This study conducted among patients with H1N1 influenza is important in this respect, and we suggest that it could be important for the prediction of fatality.
Limitations of This Study
Due to limited resources during the pandemic, only hospitalized patients were sampled for RT-PCR test for the definitive diagnosis of influenza. Since there was no possibility for definitive diagnosis of the influenza, outpatients with mild disease were not included in this study.
ACKNOWLEDGEMENT
This study was supported by grants from the Scientific Studies Grants Evaluation Commission of Diskapi Yildirim Beyazit Training and Research Hospital. It was presented as a poster in the 15th Turkish Clinical Microbiology and Infectious Disease Congress, 23-27 March 2011, Antalya.