Clinical features , treatments and outcomes of influenza A ( H 1 N 1 ) 2009 among the hospitalized patients in the Clinic for Infectious Diseases in Novi Sad

Background/Aim. Most infections caused by influenza A (H1N1) 2009 virus are presented by mild respiratory symptoms. However, some patients required admission to the intensive care unit (ICU). In this article we aimed to describe the clinical and laboratory characteristics of the patients with influenza A (H1N1) 2009, antiviral therapy use, the disease outcome and risk factors associated with the severe disease. Methods. The patients with the signs and simptoms of novel influenza A (H1N1) 2009, admitted to the Clinic for Infectious Disease in Novi Sad, were evaluated. The study included 293 patients hospitalized between October 2009 and February 2010. Basic demographic data, underlying medical conditions, clinical signs and symptoms, duration of the disease before the admission, laboratory tests, radiographic findings, treatment, and the final outcome (survived, died) were all noted. Factors associated with severe disease requiring ICU admission were determined by comparing the ICU cases with control groups of the patients admitted to the hospital but not to ICU. Results. The average age of the patients was 32.72 years. A total of 114 (38.9%) of the patients had an underlying medical condition. Asthma and chronic obstructive pulmonary disease were present in 44 (15.01%) of the patients, chronic cardiovascular diseases in 28 (9.56%), diabetes mellitus in 16 (5.46%), malignity in 15 (4.44%) of the patients and 11 (3.75%) of the patients were pregnant. Fever was registered in 282 (96.24%), myalgias in 119 (40.61%), headache in 48 (16.38%), cough in 240 (81.91%), sore throat in 25 (8.53%), runny nose and sneezing in 17 (5.8%) and dyspnea in 110 (37.54%) of the patients. A total of 192 (65.53%) had radiological findings that were consistent with pneumonia. A total of 154 (56.61%) of the patients received antiviral therapy within 48 h. A total of 280 (96.24%) patients were discharged and 13 (4.44%) were transferred to ICU. Fatal outcome was noticed in 2/13 (15.3%) ICU treated patients and 11/13 (84.7%) patients survived. The median time from the onset of illness to the initiation of antiviral treatment was 7.1 days for the patients admitted to ICU and 3.2 days for non-ICU patients (p < 0.05). Low blood oxygen saturation (SaO2 92%) was more common in ICU admitted patients, 10/13 (76,92%), compared to 28/280 (10%) non-ICU admitted ones (p < 0.01). Serum C-reactive protein (CRP) levels > 200 mg/L were noticed in 9/13 (69.23%) patients admitted to ICU and 85/280 (30.35%) patients who were not (p < 0.05). Conclusion. Most novel influenza A (H1N1) 2009 infections presented mild respiratory disease. Prompt antiviral therapy in patients with A (H1N1) virus infection seem to be the best approach to avoid serious form of the disease. Special attention should be payed to patients having low level of peripheral oxygen saturation and raised CRP serum level.


Introduction
During the spring of 2009, a novel influenza A (H1N1) virus of swine origin caused human infection and acute respiratory illness in Mexico 1, 2 .After initially spreading among persons in the United States and Canada the virus spread globally, resulting in the first influenza pandemic since 1968 with circulation outside the usual influenza season in the Northern Hemisphere 3,4 .By March 2010, almost all countries had reported cases, and more than 17,700 deaths among laboratory-confirmed cases had been reported to the World Health Organization (WHO) 5 .
Compared with seasonal influenza, the number of hospitalizations, admission to intensive care units (ICU), and invasive life support were disproportionately high among children and young adults, whereas underlying medical conditions, especially pregnancy, immunosuppression, obesity, diabetes, cardiovascular and pulmonary disease were identified as risk factors for hospitalization of patients with pandemic influenza A (H1N1) 2009 [6][7][8][9][10] .Frequently reported complications have included pneumonia, bacterial coinfection and exacerbation of underlying medical conditions [11][12][13] .
The first case of pandemic influenza A (H1N1) in Vojvodina was registered on June 24, 2009.At the end of October 2009, the mandatory outbreak investigation of acute respiratory illness detected new cases of pandemic influenza A (H1N1) among students who had returned from schoolorganised trips to Prague, Bratislava and Vienna.This was considered the beginning of pandemic influenza in the Autonomous Province of Vojvodina 14,15 .
Although most patients presented mild and self-limited symptoms with no sign of pulmonary involvement, some patients required admission to an ICU and received maximal life support measures.Therefore, the information on the clinical spectrum and risk factors for severe form of the disease, treatment and outcome of patients with pandemic influenza A (H1N1) 2009 is still collected 8,[16][17][18] .
The aim of the study was to describe clinical and laboratory characteristics of the patients with pandemic influenza A (H1N1) 2009, antiviral therapy use, disease outcome and risk factors associated with severe disease requiring admission to intensive care unit (ICU).

Methods
We retrospectively studied 293 patients with confirmed or suspected novel influenza A (H1N1) 2009 hospitalized in the Clinic for Infectious Diseases, Clinical Center of Vojvodina, between October 2009 and February 2010.We classified patients according to case definitions (confirmed or suspected) developed by the WHO, Centers for Disease Control and Prevention 19,20 .A suspected case was defined as an influenza-like illness (temperature 37.5°C and at least one of the following symptoms: sore throat, cough, rhinorrhea or nasal congestion) and either a history of travel to a country where infection had been reported in the previous 7 days or an epidemiologic link to a person with confirmed or suspected infection in the previous 7 days.A confirmed case was defined by a positive result of a real-time polymerasechain-reaction (RT-PCR) to identify the virus A (H1N1) from nasopharyngeal swabs of the patients.
Our Indications for hospital admission included radiographic findings that were consistent with pneumonia, exacerbation of underlying medical condition, especially asthma or chronic obstructive pulmonary disease, hypoxemia, hemodynamic instability and dysfunction of other organs.Critically ill patients with low blood oxygen saturation (SaO 2 92%) and with hemodynamic instability with the need for vasopressors were transferred to ICU.
Laboratory analyses were performed in the laboratory of the Clinical Centre of Vojvodina for all the patients including complete blood count, C-reactive protein (CRP), urea, creatinine, alanine aminotransferasae (ALT), gammaglutamyl transpeptidase (GGT), creatine kinase (CK), lactate dehydrogenase (LDH) and gas analysis.
Chest radiography were performed at the Institute of Radiology, Clinical Center of Vojvodina.The diagnosis of influenza A (H1N1) was confirmed by RT-PCR from nasopharyngeal swabs of the hospitalized patients.The test was performed in a reference laboratory in the Institute of Immunology and Virology "Torlak" Belgrade.
Diagnosis of pneumonia was based on clinical data and radiographic infiltrates in the lung parenchyma.The diagnosis of acute respiratory distress syndrome (ARDS) was based on clinical findings consistent with acute respiratory infection, massive bilateral lung infiltrates on chest radiography, the absence of heart failure and low level of oxygen saturation (Sa02 < 92%).
The results were presented as numbers and percentages.The Fisher's exact test was used to compare proportions for categorical variables.For continuous variables, Student's ttest and 2 test to assess the significance in differences between the groups were used.A probability level of p < 0.05 was considered statistically significant.

Results
The age of the hospitalized patients in our study ranged from 2 to 84 years, the average age was 32.72 years.Totally 245 (83.6 %) patients were less than 50 years of age, and 61 (20.7%) were patients under the age of 19 years (Table 1).Both genders were equally represented, 152 (51.88%) of the patients were males and 141 (48.12%) were females.Most patients had clinical symptoms and signs of general infectious syndrome characteristic for influenza (Figure 1).Fever 38°C was registered in 282 (96.24%), myalgias in 119 (40.61%) and headache in 48 (16.38%) of the patients.Most patients manifested more or less pronounced symptoms and signs of the respiratory tract: cough was registered in 240 (81.91%), sore throat in 25 (8.53%), runny nose and sneezing in 17 (5.8%) of the patients, while dyspnea was registered in 110 (37.54%) of the patients.Symptoms of gastrointestinal tract were recorded in less than ¼ of the patients (24.2%).

-The signs and symptoms of a novel influenza A (H1N1) in hospitalized patients
The average time from the onset of illness to hospital admission in our series of patients was 3.7 days (range from 6 hours to 16 days).A total of 116 (39.59%) of them were admitted within 48 hours, 172 (58.7%) within 3 days, 92 (31.4%) were admitted 4-7 days upon occurrence of first symptoms, while 29 (9.9%) patients were hospitalized in the second week (Table 2).
Laboratory data on admission in our series of patients are shown in Table 3 The median age of patients admitted to ICU was 28 years (range 6-52 yars).A total of 6 (46.15%)ICU patients had an underlying medical condition on admission.There was no significant difference in the prevalence of background medical conditions among the patients required ICU hospitalization and those who did not.The median temperature and the duration of fever were similar for both hospitalized groups (  Low blood oxygen saturation (SaO 2 92%) was more common in the patients admitted to an ICU, 10/13 (76.92%), compared to those who were not, 28/280 (10%), and this difference was significant (p < 0.01).
Among the evaluated laboratory parameters, serum CRP levels on admission were the only significantly differentiated factor (p < 0.01) between ICU admitted patients and those who were not (median 93 mg/L vs 198 mg/L).Serum CRP levels > 200 mg/L was noticed in 9/13 (69.23%) patients admitted to an ICU and 85/280 (30.35%) patients who were not; the difference was also significant (p < 0.05).
Chest radiographic findings consistent with pneumonia were more prevalent among patients who required ICU care than among those who did not, but this difference was not statistically significant (100% vs 68.57%, p > 0.05).
A total of 12/13 (92.3%) patients admitted to an ICU received antiviral drugs, and all received antibiotics.The median time from the onset of illness to the initiation of antiviral treatment was 7.1 days (range 3-12) for ICU admitted patients and 3.2 days (range, 1-10) for those not admitted to ICU (the difference was statistically significant, p < 0.05).
Only one (7.69%) of the ICU patients received antiviral therapy within 48 h after the onset of symptoms in contrast to 154/280 (55%) of the non-ICU admitted patients who recevied oseltamivir within 48 h after the onset of symptoms (the difference was significant, p < 0.01).

Discussion
In the present study, we retrospectively analyzed clinical features, treatment and outcome of 293 patients with confirmed or suspected novel influenza A (H1N1) hospitalized between October 2009 and February 2010.The majority of the patients (79.3%) were between the age of 10 and 50 years.This is consistent with other studies on 2009 pandemic influenza which found greater affinity of the novel A (H1N1) virus for the younger population [21][22][23][24][25][26][27] .The median age of the patients in our study was 32.7 years in contrast to 27 years in the study of Louie et al. 28 and 22.6 years in the study of Xiao et al. 29 .Serologic studies suggest that many older people had preexisting antibodies that cross-reacted with the novel pandemic influenza A (H1N1) 2009 virus.This phenomenon may explain why older people were relatively protected against contracting the virus, while younger people, who lacked these antibodies, were more likely infected 30,31 .
Both genders were equally represented in our study.The structure of our patients by gender is consistent with the knowledge of equal sensitivity of both sexes to influenza A viruses, both seasonal and novel A (H1N1) 9 , Miki et al. 32 found twice as much male patients than female, which is consistent with the gender predominance of male patients at the Military Medical Academy in Belgrade.
Infection with pandemic influenza A (H1N1) 2009 virus causes broad spectrum of clinical syndromes, ranging from afebrile upper respiratory illness to fulminant viral pneumonia.Afebrile or atypical presentations of A (H1N1) infection occurred also in pregnant women, patients with immunosuppression and other chronic disorders 33 .Most pa-tients have typical influenza-like illness with fever and cough that are sometimes accompanied by sore throat and rhinorrhea 2,24,[33][34][35][36] .The commonest symptoms in our patients were: fever (96.24%), cough (81.91%), myalgias (40.61%) and dyspnea (37.54%).Our results concur with data from the United States 6,13,14 , Mexico 2 and Shanghai 29 in which more than 80% of cases presented with fever.Mild illness without fever has been reported in 8% to 32% of infected persons 7,34 .According to Burke et al. 37 occurrence of myalgia in addition to dyspnea, raises high suspicion of A (H1N1) infection.The incidence of diarrhea in our study (9.22%) was much lower than previously reported in the United States (25%) and in the United Kingdom (28%) 7,38,39 .In the study of Liang et al. 40 fever was reported in 90%, cough in 70%, myalgia in 30% of patients and none reported diarrhea.
The average time from the onset of illness to hospital admission in our series of patients was 3.7 days (range from 6 h to 16 days).It was not significantly different in comparison to some other studies.In the study of Loui et al. 28 the median time from the onset of symptoms to hospitalization was 2 days (range, 0-31 days), in the study of Kumar et al. 41 4 days (0-18 days) and in the study of Jain et al. 7 it was 3 days (0-18 days).
History of an underlying medical condition was reported in 60%-83% of patients with pandemic influenza A(H1N1) 7 .The most common comorbid illness in our patients were asthma and COPD (15.01%) and chronic cardiovascular diseases (9.56%),In the study of Bewick et al. 42 asthma was also the most common comorbid illness (25.2%).Similarly to our results, chronic cardiovascular disease in the study of Jain et al. 7 was seen in 13% of patients and asthma in 27% of adults patients.Thus, some studies showed that exacerbation of underlying lung disease appears to be a more common indication for hospital admission in patients with seasonal influenza than with pandemic influenza 21 .
A number of studies suggest that certain underlying chronic medical condition represent risk factors for complication and severe form of illness.However, in up to 50% of patients with severe disease, no conventional risk factor could be identified 21 .In our study, 38.9% of patients had an underlying medical condition on admission and we also found no significant difference in the prevalence of background medical conditions among the patients who required hospitalization in the ICU and those who did not.
Laboratory findings at presentation, in patients with influenza include normal or low-normal leukocyte counts with lymphocytopenia and elevations in the levels of serum aminotransferases, lactate dehydrogenase, creatine kinase and creatinine 2,24,25,27 .Studies have shown that in the early and late stages of influenza infection, neutrophils play a vital role in inhibiting viral replication, and an inferior status in neutrophil activity may result in severe form of illness even if the viral strain has only medium virulence 43,44 .In our study, 22.87% of the patients had leucopenia, 36.86% of them had lymphopenia and 19.31% had leucocytosis.The number of patients with leucopenia was similar to that of other authors.For example, in the study of Jain et al. 7  study of Liang et al. 40 leucocytosis was noted in 30%, neutrophilia in 40%, lymphopenia in 50% patients while in the study of Mu et al. 45 almost 52% of patients had a neutrophil level higher than the upper normal limit, only 5.4% had elevated lymphocyte level and 32.6% had lymphopenia.The pandemic influenza A (H1N1) virus also affected hepatic functioning.This collateral damage to the liver may result from the viral activation of the Kupfer cells in the liver 45 .In our study 14% of the patients showed increased levels of serum ALT and 3.41% of the patients had increased GGT.This is consistent with the study of Mu et al. 45 who noticed 7.6% of the patients with increased levels of ALT and 4.9% of the patients with increased levels of GGT.
The diagnostic utility of biomarkers such as CRP and procalcitonin may be useful for differentiation between viral and bacterial mixed infection.Given the tendency to administer both antiviral and antibacterial therapy to patients infected with novel inflenza A (H1N1) virus, low procalcitonin and CRP levels when combined with clinical judgement, may allow earlier cessation of antibiotic therapy 46 .A retrospective observational study performed at an Australian hospital conducted by Ingram et al. 47 suggested a CRP cutoff of > 200 mg/L best identified patients with bacterial mixed infection (sensitivity 100%, specifity 87.5%).In this study the median value for CRP in the bacterial mixed infection was 363 mg/L and 103 mg/L in the A (H1N1) group.In our study 92.49% of the patients had increased CRP levels > 5 mg/L (median 133, range 7-312) while we noticed CRP levels > 200 mg/L in 32.08% of patients.Humoral immunity test in the study of Mu et al. 45 showed that 71.4% patients had a CRP level higher than the upper limit of the normal range (median 10.80 mg/L).In the study of Bewick et al. 42 median CRP level was 85 (range 34-199) and in the study of Liang et al. 40 it was 20 mg/L (range 7-57).
Song et al. 48analysed clinical, laboratory and radiologic characteristics of pandemic influenza A (H1N1) pneumonia and concluded that both procalcitonin and CRP would be helpful to differentiate primary influenza pneumonia from concomitant secondary bacterial pneumonia (CRP cutoff value 86 mg/L, sensitivity 81% and specifity 59% was discriminative between patients with concomitant bacterial pneumonia and patients with primary influenza pneumonia).In the study of Ahn et al. 49 the sensitivity and specifity for detection of mixed bacterial infection pneumonia during the pandemic A (H1N1) influenza were 69% and 63% for CRP levels > 100 mg/L.The results of the study conducted by Zimmerman et al. 50also confirmed that high serum CRP levels were associated with a severe course of influenza A (H1N1) and low initial serum CRP levels were an excellent predictor of good outcome.None of the patients with CRP levels lower than 33 mg/L in this study required ICU care.
Radiographic findings of patients with influenza A (H1N1) virus infection commonly include diffuse mixed interstitial and alveolar infiltrates, particularly in patients with bacterial coinfection 7 .A principal clinical syndrome leading to hospitalization and intensive care is diffuse viral pneumonitis associated with severe hypoxemia, ARDS, and sometimes shock and renal failure 38 .This syndrome accounted for approximately 49% to 72% of ICU admissions 41,51 .In our study, 192/280 (68,57%) of nonICU patients and 13/13 (100%) of patients who admitted to ICU had radiological findings that were consistent with pneumonia.This number is much higher than in findings of Jain et al. 7 who reported 40% of patients with pneumonia.Similarly to our results, among hospitalized patients with influenza A (H1N1) presented by Louie et al. 28 as many as 66% had pneumonia.
The currently circulating influenza A (H1N1) virus is susceptible to the neuraminidase inhibitors oseltamivir and zanamivir.Therapy with a neuraminidase inhibitor is especially important for patients with risk factors, including pregnancy and those with severe or progressive clinical illness.According to the WHO guidelines on the pharmacologic management of influenza virus, patients who are at risk for pneumonia should be treated with oseltamivir or zanamivir as soon as symptoms develop [52][53][54][55] .Available findings suggested the importance of early use of antiviral drugs and antibiotics in the treatment of serious cases 33,38 .
Previous observational studies showed that oseltamivir could reduce the duration of symptoms and shorten the duration of fever in cases of influenza A (H1N1) infection 21,28,29 .Most of our patients (94.88%) were treated with oseltamivir and 56.61% of the patients received antiviral therapy within 48 h.The fever disappeared in our patients for the mean 2.38 days after the initiation of the therapy.This is consistent with the data of Hong et al. 39 who found that the fever disappeared for the mean 2.46 days.Totally 85.3% of patients in this study received oseltamivir within 48 h following the onset of symptoms.
Some studies showed that pulmonary complications are common in patients with influenza A (H1N1) infection admitted to ICU, which required early antibiotic therapy in combination with antiviral treatment 56 .In this regard, in most studies, antibiotic therapy was administered in almost all hospitalized patients 7,32 .In a study of Jain et al. 7 97% of patients with radiographic findings that were consistent with pneumonia were treated with antibiotics.Commonly used antibiotics included ceftriaxone, azithromycin, vancomycin and levofloxacin.In the study of Falagas et al. 57 85% of patients received antibiotics and similarly to this, in our study antibiotics were given in 93.5% of the patients.Commonly used antibiotics included ceftriaxone, azithromycin and ciprofloxacin.We applied antibiotic treatment in all the patients with pneumonia and also in the patients with increased CRP levels because of suspect bacterial mixed infection.According to the literature data, Staphylococcus aureus is an important cause of secondary bacterial pneumonia with a high mortality rate and treatment of pneumonia should include empirical coverage for this pathogen 24 .Influenza A (H1N1) virus infection can induce rapidly progressive lower respiratory tract disease resulting in acute lung injury and acute respiratory distress syndrome wich calls for intensive care.Severe cases can occur at patients with extreme ages or with underlying chronic medical condition but they can also occur in young and previously healthy people [58][59][60][61] .
In pandemic influenza A (HIN1), data suggested that timely antiviral treatment was associated with enhanced viral clearance and improved survival in hospitalized patients.Unfortunately, many patients had a delay before starting antiviral therapy 21 .In the study of Jain et al. 7 25% of the patients were admitted to ICU.These patients had longer time between the onset of illness and the initiations of antiviral therapy (median 6 days), as compared with patients who were not admitted to ICU (median 3 days).Similarly, in our study the median time from the onset of illness to the initiation of antiviral therapy was 7.1 days for patients admitted to ICU and 3.2 days for non-ICU patients.The small number of patients (4.44%) admitted to an ICU in our study might be attributed to the early administration antiviral therapy.Totally 55% of patients not admitted to ICU received oseltamivir within 48 h in contrast to only one of ICU patients who received antiviral therapy within 48 h after the onset of symp-toms.This is consistent with the study of Sertogullarindan et al. 60 who found that none of the patients admitted to ICU had taken oseltamivir within 48 h.

Conclusion
The most novel influenza A (H1N1) 2009 infection presented the mild respiratory disease.Prompt antiviral therapy in patients with A (H1N1) virus infection seem to be the best approach to avoid serious form of disease requiring ICU admission.Special attention should be paid to patients with low level of peripheral oxygen saturation and raised CRP serum level.Early identification of risk patients for developing severe disease is an important aim in preparation for future waves of pandemic A (H1N1) influenza.

Table 1 The age of 293 hospitalized patients with a novel influenza A (H1N1)
. Totally 67 (22.87%) of the patients anak G, et al.Vojnosanit Pregl 2013; 70(2): 155-162.hadleukopeniawhile leukocytosis was noted in 39 (19.31%) of the patients.Thrombocytopenia was registered only in one case, as a part of pancytopenia after chemotherapy in a patient with leukemia.Elevated serum enzymes were found in less than ½ of the patients.ALT was elevated in 41 (14%) of the patients, GGT in 10 (3.41%), LDH in 24 (8.15%) and CK in 65 (22.18%) of the patients.Elevated CRP levels (> 5 mg/L) was noticed in 271 (92.49%) of the patients (median 133, range 7-312 mg/L) and CRP levels > 200 mg/L was noticed in 94 (32.08%) of the patients.admissionto the Clinic.All the patients received oseltamivir.Totally 274 (93.5%) of the patients received antibiotics.Commonly used antibiotics included ceftriaxone, azithromycin and ciprofloxacin.The median time from the onset of illness to the initiation of antiviral therapy was 3.2 days (range, 1-12 days).A total of 154 (56.61%) patients received antiviral therapy within 48 h and 172 (76.1%) of the patients received antiviral therapy within 72 h after the onset of symptoms.The outcome of illness among our patients was favorable in most cases.A total of 280 (96.24%) patients were discharged and 13 (4.44%)were transferred to ICU.Fatal outcome was noticed in 2/13 (15.3%) patients treated in the ICU and 11/13 (84.7%) patients survived.

Table 4 Comparison between variables of ICU and non-ICU patients
ICU -intensive care unit; COPD -chronic obstructive pulmonary disease; WBC -white blood cells; PMN -polymorphonuclears; LYM -lymphocytes; CRP -C reactive protein