Introduction

Avian influenza is an infectious poultry disease caused by avian influenza A viruses, which are classified as low pathogenic avian influenza A (LPAI) and high pathogenic avian influenza A (HPAI) viruses. LPAI viruses rarely cause severe avian diseases, whereas HPAI viruses can cause deadly infectious diseases in birds, spreads rapidly, and can infect humans. Almost all HPAI viruses belong to the H5, H7, and H9 subtypes. The main subtypes of avian influenza viruses known to infect humans are H7, H5, H9, and H10 and N1, N2, N3, and N7. There have been over 100 documented cases of humans infected with H7 viruses from poultry or wildfowl mainly in the United States, United Kingdom, Italy, the Netherlands, Canada, and other European countries [1-6], while N9-subtype viruses had never been reported in humans until recently. Most human infections with avian influenza A (H7) viruses exhibit mild symptoms or present as latent infections. Most cases result in conjunctivitis and symptoms of respiratory tract infection that are mild to moderate in severity, with only one fatal case reported in a patient with H7N7 virus infection [7]. From 1999 to 2003, Puzelli found that 7 of 185 poultry workers (3.8%) were positive for specific antibodies to avian influenza A H7 viruses, of which only 1 case resulted in conjunctivitis, whereas the remaining 6 cases exhibited no clinical symptoms [4]. By May 28, 2013, 123 patients were diagnosed with laboratory-confirmed H7N9 virus infections and admitted to the hospital, and 37 (30%) patients died of the infection; this outcome is inconsistent with those of previous reports on avian influenza A H7 virus infections [8].

In February 2013, the first ever case of human infection with H7N9 was encountered at the respiratory department of the Fifth People’s Hospital of Shanghai [9]. During the following 2-week period, the hospital received another 5 patients with H7N9 infection. All cases were localized to the Minhang District of Shanghai, and the disease onset in all cases occurred within a 2-week period. Of the 6 patients, 2 emerged from a single family, suggesting that aggregation exists to some extent. In addition, H7N9 avian influenza and H5N1, both of which are fatal, show similar clinical symptoms and disease progression [10,11]. Therefore, early diagnosis and treatment, with an improved cure rate are important to control these infections [11]. Here, we summarize the findings of 6 cases of pneumonia caused by H7N9 infection.

Methods

Results

History of exposure to birds and residence state

Among the 6 patients, 5 were residents of the Minhang District of Shanghai and had not left Shanghai prior to the onset of illness. One patient, who was a pork seller at a market in the Minhang District, was originally from the Jingsu province and had been a resident of Minhang District for 9 months at the time of disease onset. All 6 patients lived in the proximity of 2 food markets where poultry were traded and H7N9 virus carrier birds had been discovered. Trading of poultry was banned in the markets on April 4. Two patients had a history of exposure to live birds and 1 had a history of suspected exposure. (Figure 1)

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 1. A map of patient residences and the 2 neighboring markets where H7N9 was detected in poultry.

https://doi.org/10.1371/journal.pone.0077651.g001

Imaging features

Chest radiography and lung computed tomography suggested that consolidation and multiple ground-glass opacities emerged in the early stage of disease and rapidly extended bilaterally. In 2 of 6 cases, pulmonary symptoms such as acute respiratory distress syndrome (ARDS) were detected. Furthermore, minor pleural effusion and subcutaneous and mediastinal emphysema were observed (Figure 2).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Chest radiographic findings and progression of pneumonia in 6 patients with H7N9 infection.

Case 1: Panels A, B, and C; Case 4: Panels D, E, and F; Case 2: Panels G, H, and I. Early stage (panels A, D, and G): Chest computed tomography and radiograph show progressive consolidation in the lungs; Progressing stage (panels B, E, and H): Chest computed tomography and radiograph show bilateral ground-glass opacity and consolidation; Final stage (panels C, F, and I): Chest computed tomography and radiograph show diffuse lesions and bilateral involvement of the lungs (white lungs) in panels C and F, subcutaneous emphysema with pneumomediastinum in panel F, and diffuse lesions are obviously seen in panel I.

https://doi.org/10.1371/journal.pone.0077651.g002

Discussion

H7N9 is a subtype of the avian influenza A virus. Until 2013, 25 types of H7N9 viruses had been detected in birds worldwide. Previously detected H7N9 viruses only infected birds, and these infections were generally mild. However, animal experiments showed that both HPAI H7N3 and LPAI H7N9 could effectively replicate in mice without adaptation, as well as in the upper and lower respiratory tract of ferrets. HPAI H7N3 was also found to be lethal in mice and showed transmission through contact in the ferret model, whereas LPAI H7N9 could not be transmitted in the ferret model [12-15]. In March 2013, 3 cases of human H7N9 avian influenza infection were reported in Shanghai and Anhui, China. Research showed that the H7N9 virus reported at this time was a novel, reassortant influenza A virus [9]. The virus contained 8 gene segments, with only the NA gene being closely related to that of another H7N9 virus (KO14). The HA gene was similar to that of an H7N3 virus (ZJ12) from a nearby region (Zhejiang province) in China. All the internal gene segments were closely related to those from other avian H9N2 viruses.

The sources of avian influenza infection are mainly poultry or wild birds, which are carriers or sufferers of the disease. However, humans can be infected with avian influenza viruses via several transmission routes such as through direct avian-to-human contact, which is the most common mode of transmission; environment-to-human transmission; and mother-to-child vertical transmission [16-19]. Our study showed that 6 cases of pneumonia caused by H7N9 were detected in the area with close proximity to the Fifth People’s Hospital of Shanghai within a 2 week-period from 19 February and 5 March 2013. Among the 6 patients, 2 were related (father and son); in addition, several days prior to disease onset, the younger son (age, 55 years) developed high fever, cough, dyspnea, and rapidly developed ARDS, which was similar to the disease course of the other 2 family members who had confirmed cases of H7N9 infection. However, the H7N9 virus was not detected in his blood and respiratory specimens.

The 6 patients lived in the proximity of the 2 markets where live birds were traded, and 2 patients had a history of exposure to birds; 1 had a history of suspected exposure to birds, and 1 patient worked in one of the markets. After the outbreak, the health department identified H7N9 viruses in carrier poultry in the 2 markets. Live bird trading was then forbidden. Thereafter, no additional cases of H7N9 infection were reported in the Minhang District. The above evidence suggests that the outbreak of infection caused by H7N9 viruses in the district was likely because of live bird carriers of H7N9 viruses in the 2 markets. The patients of this study had close contact with 122 people, who were mainly family members, relatives, and medical staff. These individuals were kept under medical observation for 1 week, and similar signs and symptoms to those of the patients were not observed. The lack of clinical infection signs in the contact individuals suggests that the efficiency of human-to-human transmission is low. Even without appropriate quarantine measures, the possibility of hospital infection is small. Therefore, a similar exposure history among direct relatives is believed to be the cause of clustering cases in a family.

A general lack of immunity to avian influenza viruses leads to human infection, which can result in severe outcomes. Previously detected LPAI H7 avian influenza viruses were reported to cause mild symptoms, mainly conjunctivitis or flu-like symptoms, in humans [1,6,20,21], although H7N7 avian influenza viruses have been reported to also cause more serious complications such as pneumonia and ARDS [21,22]. For example, in the 2003 outbreak of the H7N7 virus in the Netherlands, 82 of the 89 cases (92.1%) manifested as conjunctivitis, whereas the remaining patients presented with an influenza-like illness [5]. HPAI H5N1 avian influenza viruses have caused more severe illnesses, from asymptomatic infection to fatal pneumonia and multiple organ failure than the H7 subtype. The characteristics of H5N1 avian influenza virus infection include a high case rate, high fatality rate, and development of shock in the early stages of disease [23,24]. The 6 patients infected with H7N9 viruses in this report showed signs and symptoms similar to those of patients infected with H5N1 viruses; all infected patients had pneumonia, with most rapidly progressing to ARDS. In the initial stage of disease, patients presented with fever and bloody sputum accompanied by chills, chest pain, nausea, and vomiting in some of the patients. Dyspnea developed 3–7 days after disease onset. Upper respiratory tract infection symptoms such as nasal congestion, runny nose, and sore throat, were not observed in any case, which was probably because of the lack of early diagnosis in patients with mild symptoms. Laboratory testing in the early stage showed that the WBC count was normal or lower than normal; the lymphocyte and platelet counts were low; and serum CRP, procalcitonin, CK, and LDH levels were elevated. Blood gas analysis also suggested hypoxemia and different degrees of excessive ventilation. Chest imaging suggested that consolidation and multiple ground-glass opacities of the lung developed in the early stage of the disease, and rapidly extended bilaterally. Patients with severe disease also showed ARDS-like symptoms. Furthermore, minor pleural effusion as well as subcutaneous and mediastinal emphysema were observed in some patients with severe disease. The above evidence suggests that fever, cough, hemoptysis, normal or low WBC levels, low lymphocyte counts, elevated CK and LDH levels, and pulmonary exudative lesions are significant characteristics of H7N9 infection.

Within 7 days of disease onset, H7N9 infection was confirmed in 4 of the 6 cases by RT-PCR assay and viral isolation using respiratory samples. The remaining 2 cases were confirmed using a specific antibody to the H7N9 virus in double serum samples from the acute and recovery stages. The results suggest that human infection with H7N9 viruses can be diagnosed early by RT-PCR of respiratory samples such as throat-swabs and sputum specimens, and by viral isolation from these samples. After admission, all patients were treated with broad-spectrum antibiotics, which were not effective in resolving symptoms. On days 3–8, all patients were treated with oral oseltamivir, and severely ill patients were treated with intravenous glucocorticoid, immunoglobulin, and thymosin. Several complications were observed, including ARDS, secondary bacterial infection, pleural effusion, left heart functional failure, neuropsychiatric symptoms, and rhabdomyolysis. ARDS developed on days 3–9 (mean, 6 days), the time course of which is consistent with that of reported cases of H5N1 infection in Thailand [25].

Factors affecting the prognosis of H7N9 viruses are unknown. Case 2 was a 69-year-old man with a history of hypertension and no history of drinking, and he recovered from the infection. Case 5 was a 41-year-old man with a history of smoking (smoking index, 4 pack-years) and no history of drinking or underlying disease, who also recovered. The ages of the other 4 patients were 87, 74, 27, and 63 years, and each had at least 1 underlying disease and a history of smoking and drinking; these patients ultimately died of the infection. These findings suggest that age may not be related to prognosis; however, underlying disease does appear to impact prognosis. The patients who recovered had no dyspnea, whereas those who died developed different degrees of dyspnea. In terms of laboratory testing, compared to the patients who died, the patients who recovered had only slightly varying platelet counts and CK and PO₂ levels. In terms of complications, 1 patient who recovered had no complications, whereas the other developed secondary bacterial infections. In contrast, patients who died had 2–4 complications each. The time to treatment with oseltamivir therapy in the recovered cases was days 7 and 8, whereas it was days 3–8 in the patients who died. The above evidence implies that smoking, drinking, and underlying diseases may be related to the prognosis of H7N9 infection; dyspnea, decreased platelet counts, elevated CK levels, and hypoxemia may be associated with poor prognosis. The time to initiation of antiviral therapy seems to have no impact on prognosis, whereas the presence of complications may be associated with poor prognosis [10].

In conclusion, the first ever patient infected with H7N9 was treated at the Fifth People’s Hospital of Shanghai in February 2013. Within a 2-week period, several other cases of H7N9 infection emerged around 2 markets near the hospital. Fever, cough, sputum with blood, low lymphocyte counts, elevated CK and LDH levels, and pulmonary exudative lesions are significant characteristics of H7N9 infection, which easily progresses to ARDS. Among the cases, there was family clustering, which led to a high suspicion of contagious respiratory virus infection. In the early stage, human infection with H7N9 can be diagnosed by RT-PCR and viral isolation from respiratory specimens. Smoking, drinking, underlying diseases, dyspnea, low platelet counts, elevated CK levels, hypoxemia, and complications may be related to poor prognosis. However, diagnosis and treatment may be delayed because of the limited experience with this infection and small number of cases, which were among the first cases of H7N9 infection to be identified. Future studies are needed to elucidate the pathogenicity, transmissibility, and clinical features of H7N9 infection. In addition, techniques for early diagnosis to enable early administration of antiviral therapy and determination of the the factors affecting prognosis require further investigation.

Acknowledgments

The authors would like to thank the Fifth People’s Hospital of Shanghai and Shanghai Public Health Clinical Center, Fudan University for providing medical resources.