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Clinical features, treatments and prognosis of the initial cases of pandemic influenza H1N1 2009 virus infection in Shanghai China

Y.P. Mu, Z.Y. Zhang, X.R. Chen, X.H. Xi, Y.F. Lu, Y.W. Tang, H.Z. Lu
DOI: http://dx.doi.org/10.1093/qjmed/hcq012 311-317 First published online: 25 February 2010

Abstract

Background and Objective: As of 13 December 2009, more than 208 countries and overseas territories or communities have reported laboratory-confirmed cases of pandemic influenza H1N1 2009, which have resulted in at least 10 582 deaths. As of 7 December 2009, 4328 severe cases were reported in Mainland China, resulting in 326 deaths. This study’s objective was to determine the clinical features, treatments and prognosis of the initial cases of Pandemic influenza H1N1 2009 virus infection in Shanghai, China, and how its clinical features related to patient gender.

Methods: A total of 224 confirmed 2009 influenza A/H1N1-infected patients treated and discharged by Shanghai Public Health Clinical center between 24 May and 20 July 2009 were included in the study. Patients’ personal information, signs and symptoms, laboratory and imagery data, disease course, hospitalization period and seroconversion duration for viral nucleic acid after antiviral treatment were analyzed.

Results: Among the 224 patients, 118 were male and 106 were female, yielding a sex ratio of 1.1:1. Approximately 52% of the patients came from Australia, and 63.8% were between 18 and 40 years old. Clinical manifestations included fever, cough and congestion of the throat, and lab findings were characterized by elevated C-reaction protein (CRP) and neutrophils. Female patients had significantly lower serum Prealbumin (PA) levels than male patients (P < 0.05). The patients’ serum CRF levels significantly decreased after treatment (P < 0.05), while the levels of CD3, CD4 and CD8 significantly increased after treatment (P < 0.01). Approximately 29.9% of the patients had abnormal signs on chest computer tomography scan, and 21.9% had obvious signs indicating pneumonia. However, blood cultures were negative in these patients. The average disease course was 3.9 ± 1.4 days, the average hospitalization period was 5.0 ± 1.7 days, and the seroconversion duration for viral nucleic acid after antiviral treatment was 3.8 ± 1.3 days.

Conclusion: Initial cases of pandemic influenza H1N1 2009 were characterized by fever, cough and throat congestion, with elevated CRP and neutrophils being the most significant lab findings. The pandemic influenza H1N1 2009 strain was able to affect multiple organs, including the hepatic synthesis of PA and immune functioning. The novel 2009 Influenza A/H1N1 virus was mild clinically, with a short disease course and good prognosis.

Introduction

The outbreak of pandemic influenza H1N1 2009 in Mexico and the United States from late March to mid-April 2009 caused the World Health Organization on 11 June 2009 to raise the warning level for the virus to the highest available—Phase Six—indicating that this episode of influenza had entered a pandemic stage.1 Different from strains in the past and containing a unique combination of gene segments from swine, avian and human lineages,2 this new virus is able to spread among human beings, leading to influenza-like symptoms and progressing in a few cases to viral pneumonia, respiratory failure, multiple organ failure and death. As of 13 December 2009, more than 208 countries and overseas territories or communities have reported laboratory-confirmed cases of pandemic influenza H1N1 2009, resulting in at least 10 582 deaths.3 As of 7 December 2009, 4328 severe cases of pandemic influenza H1N1 2009 were reported in Mainland China, including at least 326 deaths. This study retrospectively analyzed the clinical features, disease course, hospitalization period, negative seroconversion duration of viral nucleic acid following antiviral treatment, and the association between the clinical features and patient gender of 224 confirmed pandemic influenza H1N1 2009 patients, who were treated and discharged by Shanghai Public Health Clinical center between 24 May and 20 July 2009.

Methods

Diagnosis

All 224 patients were diagnosed by influenza A (H1N1) virus mRNA detection at their first visit to the Center for Disease Control and Prevention (CDC) of Shanghai. The mRNA was assessed using quantitative reverse transcription polymerase chain reaction (RT–PCR). Primers and oligonucleotide probes: Forward, 5′-GTA CTA TAA ACA CCA GCC TYC CA-3′; Reverse, 5′-CGG GAT TTA CCT TAA TCC TGT RGC-3′; Probe, 5′-CA GAA TAT ACA TCC RGT CAC AAT TGG ARA A-3′; Y=C or T; R=A or G.

Laboratory detection

Blood routine, liver function, humoral and cellular immunity indicators were measured at the Clinical Diagnostic Center of Shanghai Public Health Clinical center affiliated with the Fudan University (Shanghai, China).

Chest computer tomography scan

All 224 patients underwent a chest computer tomography (CT) scan immediately after hospital admission.

Blood cultures

Patients who had abnormal chest images by CT scan were evaluated using blood cultures.

Therapy

Because cases of death caused by pandemic influenza H1N1 2009 were reported in Europe, causing great alarm in China, the patients who had no pneumonia according to their CT scan were given a single treatment of Oesltamivir (75 mg b.d.) or Oesltamivir plus traditional Chinese medicine (TCM). TCM used included Shuanghuanglian oral Concoction, composed of honeysuckle, Scutellaria baicalensis Georgi and Fructus Forsythiae; or Antiviral Oral Concoction, composed of isatis root, reed rhizome, radices rehmanniae, radix curcumae, rhizoma anemarrhenae, Acorus gramineus Soland., Agastache and Forsythia suspensa Vahl; or Qutanling Oral Concoction, composed of bamboo juice and cordate houttuynia; or Xiaochaihu Granule, composed of Radix Bupeuri, Rhizoma Pineliae, Rasix Ginseng, Radix Scutelariae, Rhizoma Zingiberis Recens, Fructus Ziziphi Jujubae and Radix Glycirrhizae). Totally 63 out of 67 pneumonic patients diagnosed by chest CT scan were treated with Oseltamivir (75 mg b.d. p.o.) plus Azithromycin (0.5 g q.d. p.o.) and TCM. Four patients with mild increased lung markings were not treated with Azithromycin.

Statistical analysis

Data were expressed as means ± standard errors (SE). Data were analyzed using one-way analysis of variance (ANOVA). Q-test and T-test were used for parameter comparisons and correlation analyses, respectively.

Results

Personal data

Among the 224 cases, 218 were from sources outside of China and six were second-generation cases originating in Mainland China. Among 224 cases, 118 (52.7%) were male and 106 (47.3%) were female, yielding a sex ratio of 1.1:1 (M:F). Mean patient age was 24.0 ± 11.7 years (range 2–75), with 63.8% of cases between 18 and 40 years old, 25.9% between 2 and 17 years old and 10.3% greater than 40 years old. Seven patients had a history of chronic hepatic diseases, asthma, or gout, and one case was pregnant. Over half of the patients (116 cases, 51.8%) came from Australia, with the remainder from Europe, America and Asia (Table 1).

View this table:
Table 1

Patients characteristics

Male (%)Female (%)
Gender118 (52.7)106 (47.3)
Age (years)
    2–1728 (12.5)30 (13.4)
    18–4076 (33.9)68 (30.4)
    >4014 (6.3)8 (3.6)
Origin of the patients
    Australia60 (26.8)56 (25.0)
    Other countries or regions59 (26.3)49 (21.9)
Signs
    Temperature (°C, mouth)
        Normal (<37.3)4 (1.8)0
        Mild fever (37.3–38.0)19 (8.5)27 (12.1)
        Medium fever (38.1–39.0)69 (30.8)56 (25)
        High fever (39.1–41.0)26 (11.6)23 (10.3)
        Super high fever (>41.0)00
    Congestion of the throat113 (50.4)102 (45.5)
    Tonsil swelling26 (11.6)32 (14.3)
Clinical manifestations
    Fever111 (49.6)101 (45.1)
    Cough96 (42.9)82 (36.6)
    Sore throat53 (23.7)44 (19.6)
    Expectoration15 (6.7)29 (12.9)
    Nasal congestion23 (10.3)24 (10.7)
    Running nose24 (10.7)17 (7.6)
    Myalgia11 (4.9)19 (8.5)
    Chills8 (3.6)16 (7.1)
    Headache9 (4.0)11 (4.9)
    Poor appetite1 (0.4)10 (4.5)
    Dry mouth4 (1.8)4 (1.8)
    Bitter taste2 (0.9)2 (0.9)
    Sweating04 (1.8)
    Diarrhea01 (0.4)
Basic diseases
    Chronic Hepatitis B02 (0.9)
    Steatohepatitis01 (0.4)
    Asthma1 (0.4)1 (0.4)
    Hypertension02 (0.9)
    Hyperthyroidism01 (0.4)
    Pregnant/1 (0.4)
    Obesity1 (0.4)0
    Heart diseases00
    Lung diseases00
Complications00
hospitalization period (days)5.0 ± 1.65.0 ± 1.8

Clinical manifestations

As Table 1 shows, the clinical manifestations observed included fever (212, 94.6%) and cough (178, 79.5%), which could be accompanied by sore throat (97, 43.3%), expectoration (44, 19.6%), nasal congestion (47, 21%), running nose (41, 18.3%), myalgia (30, 13.4%), chills (24, 10.7%), headache (20, 8.9%), poor appetite (11, 4.9%), dry mouth (8, 3.6%), bitter taste (4, 1.8%), sweating (4, 1.8%) and diarrhea (1, 0.4%).

Signs

A total of 215 (96.0%) patients manifested congestion of the throat and 58 (25.9%) presented tonsil swelling. The average patient temperature was 38.6 ± 0.69 (range 37.2–39.7). Four (1.8%) patients had normal temperature (<37.3°C), 46 (20.5%) had mild fever (37.3–38°C), 126 (56.3%) had medium fever (38.1–39°C) and 49 (21.9%) had high fever (39.1–41°C). There were no super high fever (>41°C) cases (Table 1).

Blood routine test (BRT)

A total of 26 (11.6%) patients had decreased leukocyte levels (<4.0 × 109/l), and 13 (5.6%) had increased levels (>10 × 109/l). A total of 116 (51.8%) patients had an increased neutrophil percentage (>70%), and 19 (8.5%) had a decreased percentage (<50%). A total of 12 (5.4%) patients had their lymphocyte percentage (>40%) increased, and 73 (32.6%) had their percentage (<20%) decreased.

Further BRT analysis indicated that the average leukocyte count, average lymphocyte percentage and average neutrophil percentage were 6.25 ± 2.33 × 109/l, 22.87 ± 11.18%, and 68.72 ± 11.58%, respectively. Furthermore, male patients had significantly higher leukocyte counts than female patients (6.71 ± 2.45 vs. 5.74 ± 2.10, P < 0.01; Table 2).

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Table 2

Routine blood testing and hepatic functions (mean ± SD)

MaleFemale
Routine blood testing
    Leukocyte (×10 9/l)6.71 ± 2.45**5.74 ± 2.10
    Neutrophil (%)69.14 ± 11.3668.26 ± 12.47
    Lymphocyte (%)21.91 ± 11.4023.73 ± 10.99
Hepatic functions
    ALT (U/l)27.88 ± 23.2123.38 ± 22.97
    AST (U/l)29.09 ± 25.3328.97 ± 28.24
    pre-A (mg/l)272.06 ± 93.51*244.80 ± 76.05
  • *P < 0.05; **P < 0.01, compared with female.

Imaging findings

Totally 67 (29.9%) patients were found to be abnormal by chest CT, including indications of pneumonia, pleural thickening and increased lung markings. Forty-nine (21.9%) patients manifested pulmonary inflammation; 22.9% (n = 27) of males and 20.8% (n = 22) of females.

Blood culture

To prevent pandemic influenza H1N1 2009 virus complicated with bacterial infection, 63 patients diagnosed with pneumonia by chest CT scan were treated with Oesltamivir plus Azithromycin and TCM. At the same time, the blood bacteriological culture was administered; results were all negative.

Effect of pandemic influenza H1N1 2009 on hepatic functions

Serum alanine amiotransferase (ALT), aspartate aminotransferase (AST) and PA levels were determined in an effort to learn the affect of the pandemic influenza H1N1 2009 virus on hepatic functions. The average levels of these indicators were 25.75 ± 23.10 U/l, 29.03 ± 26.65 U/l and 258.99 ± 86.44 mg/l, respectively (all in the normal range). Some patients manifested abnormal hepatic functions; increased levels of ALT, AST, total bilirubin (TBil), lactate dehydrogenase (LDH), γ-glutamyl transpeptadase (GGT), and alkaline phosphatase (AKP) were seen in 17 (7.6%), 9 (4.0%), 2 (0.9%), 11 (4.9%), 11 (4.9%) and 5 (2.0%) patients, respectively. Totally 66 patients (29.5%) had decreased PA, and the PA was significantly lower in female patients compared with male patients (272.06 ± 93.51 vs. 244.80 ± 76.05, P < 0.05; Table 2).

Effect of the pandemic influenza H1N1 2009 virus on immunity

Humoral immunity tests showed that 160 (71.4%) patients had a CRP level higher than the upper limit of the normal range, and 15 (22.3%) patients had a rheumatoid factor (RF) higher than the upper limit of the normal range. In addition, the male patients had a CRP level significantly higher than that of the females (16.32 ± 18.27 vs. 12.05 ± 11.44, P < 0.05; Table 3).

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Table 3

Humoral and cellular immunity indicators (mean ± SD)

GroupnCRP (mg/l)RF (IU/ml)CD3 (cell/μl)CD4 (cell/μl)CD8 (cell/μl)
Gender
    Male11816.32 ± 18.27#20.67 ± 2.98829.73 ± 410.46445.85 ± 245.80341.39 ± 193.44
    Female10612.05 ± 11.4421.25 ± 3.37825.63 ± 363.36442.64 ± 196.42341.63 ± 176.08
Treatment
    Before22410.80 ± 12.9220.57 ± 1.45700.50 ± 307.46370.08 ± 157.62288.88 ± 164.98
    After2243.81 ± 4.93*20.50 ± 1.151449.46 ± 446.71**762.69 ± 255.66**592.38 ± 238.24**
  • CRP, C-Reactive Protein; RF, rheumatoid factor.

  • #P < 0.05, compared with group female.

  • *P < 0.05; **P < 0.01, compared with before treatment.

Average CD3, CD4 and CD8 of T-lymphocyte subsets in peripheral blood were normal. Levels lower than the lower limit of the normal range of CD3, CD4 and CD8 were seen in 105 (46.9%), 116 (51.8%) and 50 (22.3%) patients, respectively. There were no significant gender differences (P > 0.05) in the three subsets (Table 3). Following treatment, patients showed a significant decrease in their CRP level to 35% of the value prior to treatment (P < 0.05) and a doubling of their CD3, CD4 and CD8 levels (P < 0.01; Table 3). These findings suggest that influenza A (H1N1) virus impair immune functioning of the human body during early stages of infection.

Disease course, hospitalization length and seroconversion duration for viral nucleic acid

On average, the disease course lasted 3.9 ± 1.4 (range 1–8) days, the hospitalization period was 5.0 ± 1.7 (range 1–9) days, and the time for seroconversion for the inability to detect viral nucleic acid was 3.8 ± 1.3 (range 2–8) days.

Forty patients were treated with Oesltamivir, 110 with Oesltamivir plus a mixture of TCM, including Shuanghuanglian Oral Concoction, or antiviral Oral Concoction, Qutanling Oral Concoction or Xiaochaihu Granule), 63 with Oesltamivir plus Azithromycin plus a mixture of TCM (the same as those listed above), eight with Oesltamivir plus Azithromycin, and three with Shuanghuanglian Oral Concoction alone. Retrospective, observational cohort analysis of the patients receiving the first three treatments indicated that there was no significant difference between the treatments with regards to disease course and seroconversion duration for the 2009 novel Influenza A/H1N1 (P > 0.05; data on the patients undergoing the last two treatments were not included in the statistical analysis because of too few cases). However, patients receiving the third treatment had a significantly longer hospitalization period (P < 0.01; Table 4).

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Table 4

Clinical outcome of different treatment strategies (mean ± SD)

GroupnDisease course (days)Negative duration of the nucleic acid (days)Hospitalization period (days)
Oesltamivir403.8 ± 1.63.6 ± 1.14.6 ± 2.0
Oesltamivir+TCM1103.8 ± 1.43.7 ± 1.34.7 ± 1.6
Oesltamivir+Azithromycin + TCM634.0 ± 1.33.9 ± 1.25.6 ± 1.3**
  • TCM, traditional Chinese medicine, including Shuanghuanglian oral Concoction, composed of honeysuckle, Scutellaria baicalensis Georgi, and Fructus Forsythiae; or Antiviral Oral Concoction, composed of isatis root, reed rhizome, radices rehmanniae, radix curcumae, rhizoma anemarrhenae, Acorus gramineus Soland., Agastache, and Forsythia suspensa Vahl; or Qutanling Oral Concoction, composed of bamboo juice and cordate houttuynia; or Xiaochaihu Granule, composed of Radix Bupeuri, Rhizoma Pineliae, Rasix Ginseng, Radix Scutelariae, Rhizoma Zingiberis Recens, Fructus Ziziphi Jujubae and Radix Glycirrhizae.

  • **P < 0.01, compared with Oesltamivir and Oesltamivir + TCM.

Discussion

The influenza A virus changes genetically by antigen drift or antigen shift, and the latter can lead to the development of a novel strain of influenza capable of causing a global epidemic. The three episodes of global pandemic in 1918, 1957 and 1968 have been described previously as the Spanish flu (H1N), Asian flu (H2N2) and the Hong Kong flu (H3N2), respectively. The latter two primarily showed higher morbidity and mortality among infants, the elderly and people suffering from chronic diseases, and the former predominantly showed high morbidity and mortality among youth and middle-aged aged people, 20–40 years of age and was characterized by leukopenia and hemorrhage. Acute pneumochysis and hemorrhagic pneumonia are two elements that quickly have claimed the lives of the youth or middle-aged people.4

Different from other strains, the pandemic influenza H1N1 2009 strain contains a combination of gene segments from swine, avian and human lineages.2 Based on our current understanding of the novel 2009 H1N1 strain there is serious concern in the public that an epidemic similar to those of 1918, 1957 and 1968 may occur. The 224 patients infected with this new type of virus who were treated and discharged by our hospital were mostly from sources outside of China (e.g. overseas students or employees), the majority of whom (over 70%) were between the ages of 18 and 40 years old. Their clinical manifestations were characterized by fever, cough, congestion of the throat, and elevated neutrophil and CRP, and sometimes accompanied by running nose, nasal congestion, sore throat, headache, myalgia, malaise, poor appetite, diarrhea, antiadoncus and low lymphocyte level. In general, the clinical manifestations were mild and lasted a short period without severe complications or mortality, and the prognosis was good.

Neutrophils have long been regarded as a major effector cell fighting against bacterial and fungal infections, as the cell plays an important role in the inflammatory reaction to viral infection. Its elevation is an indication of early influenza infection among humans, ferrets and mice.5 Studies have shown that neutrophils are capable of fighting the influenza strain A/PR/8/34(A/H1N1),6,7 showing a protective effect in the early stage of infection.8,9 Furthermore, studies have demonstrated that in the early and late stages of influenza infection, the neutrophil plays a vital role in inhibiting viral replication, and an inferior status in neutrophil activity may result in severe aftermath even if the viral strain has only medium virulence.10

Almost 52% of the 224 patients described in this paper had a neutrophil level higher than the upper normal limit, while only 5.4% had elevated lymphocyte level and 32.6% had decreased lymphocyte levels, which may be one of the clinical features of the new virus type. In addition, retrospective observational cohort analysis revealed that although some patients received treatment combined with Azithromycin due to the presence of pneumonia, the combined use did not significantly shorten the conversion duration for the detection of viral genomic RNA or decrease the duration of the disease. On the contrary, the disease course increased significantly, suggesting that the patients with pneumonia in the early stage of infection may have had a more severe disease, which increased the duration of the disease, while not necessarily requiring antibiotics administration.

Research has shown that three kinds of viral RNA (vRNA) can be detected in the lung, heart, thymus, liver and spleen of mice after they are infected with the viral strains of A/Dunedin/4/73(H3N2), A/Mississippi/ 1/85(H3N2) or A/PR/8/34(H1N1), with the latter resulting in significant damage of the lung and thymus.11 Based on CT imaging, 30% of the patients showed involvement of the upper respiratory tract and abnormalities of the pleura due to infection with the pandemic influenza H1N1 2009. This novel virus also affected hepatic functioning. Nearly 30% of the patients showed decreases in their PA level and increases of their levels of ALT, AST, LDH, AKP, GGT and Tbil. In addition, male patients showed significantly higher PA levels than female patients. This collateral damage to the liver may result from the viral activation of the Kupffer cells in the liver.12 The novel virus also affected the immune system, as patients showed elevated CRP and RF (male patients had significantly higher CRF levels than the female patients) and an overall decrease in CD3 and CD4 levels (although the averages still fell within the normal range). These findings indicate that the pandemic influenza H1N1 2009 strain can spread to multiple organs. However, the mechanism as to why male patients had higher leukocyte counts, PA and CRF levels than female patients is still unknown.

Although the patients treated by our hospital presented mild manifestations and short disease course without severe complications or mortality, historical records have shown variations and worsening situations in the months following initial identification and isolation of novel strains,13,14 indicating that some uncertainties—including viral evolution—may occur in the next 6 months.15 In fact, a second outbreak of the epidemic has emerged in China, where severe cases are rapidly increasing, and the scope of the epidemic is still expanding and remains grim for the next 1 to 2 months.

Experiences from our American peers have shown that the extensive use of Oesltamivir may increase the prevalence of drug resistance.16 Measures should be taken to deal with possible drug resistance, which may include producing a mass supply of pandemic influenza H1N1 2009 vaccine and reducing routine drug use.

In summary, evaluations of 224 patients treated at our hospital demonstrated that early pandemic influenza H1N1 2009 infection is mild clinically but able to affect hepatic and immune functions. While the neuraminidase inhibitor Oesltamivir can help shorten the fever period16 and quickly improve symptoms and signs, it is necessary to consider drug resistance issues and resolutions brought about by its use. Meanwhile, the international health care community should remain vigilant to the possibility of epidemics and heavy death tolls caused by genetic drift of the pandemic influenza H1N1 2009 strain.

Funding

Grant for Research on Mutation and Epidemic laws of the Pandemic Influenza H1N1 2009 Virus from the Science and Technology Commission of Shanghai through grant number 09DZ1906600 (2009).

References

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