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Clinical Characteristics of 26 Human Cases of Highly Pathogenic Avian Influenza A (H5N1) Virus Infection in China
Hongjie Yu1#, Zhancheng Gao2#*, Zijian Feng1#, Yuelong Shu3#, Nijuan Xiang1, Lei Zhou1, Yang Huai1, Luzhao Feng1, Zhibin Peng1, Zhongjie Li1, Cuiling Xu3, Junhua Li4, Chengping Hu5, Qun Li6, Xiaoling Xu7, Xuecheng Liu8, Zigui Liu9, Longshan Xu10, Yusheng Chen11, Huiming Luo12, Liping Wei13, Xianfeng Zhang14, Jianbao Xin15, Junqiao Guo16, Qiuyue Wang17, Zhengan Yuan18, Longnv Zhou19, Kunzhao Zhang20, Wei Zhang21, Jinye Yang22, Xiaoning Zhong23, Shichang Xia24, Lanjuan Li25, Jinquan Cheng26, Erdang Ma27, Pingping He28, Shui Shan Lee29, Yu Wang1, Timothy M. Uyeki30, Weizhong Yang1*
1 Office for Disease Control and Emergency Response, Chinese Center for Disease Control and Prevention (China CDC), Beijing, China, 2 Department of Respiratory Medicine, Peking University People's Hospital, Beijing, China, 3 State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China, 4 Hunan Provincial Center for Disease Control and Prevention, Changsha, China, 5 Xiang Ya Hospital of Central South University, Changsha, China, 6 Anhui Provincial Center for Disease Control and Prevention, Hefei, China, 7 Anhui Provincial Hospital, Hefei, China, 8 Sichuan Provincial Center for Disease Control and Prevention, Chengdu, China, 9 Huaxi Hospital, Sichuan University, Chengdu, China, 10 Fujian Provincial Center for Disease Control and Prevention, Fuzhou, China, 11 Fujian Provincial Hospital, Fuzhou, China, 12 Guangdong Provincial Center for Disease Control and Prevention, Guangzhou, China, 13 Third Affiliated Hospital, Guangzhou Medical College, Guangzhou, China, 14 Hubei Provincial Center for Disease Control and Prevention, Wuhan, China, 15 Hankou Union Hospital, Hubei Province, Wuhan, China, 16 Liaoning Provincial Center for Disease Control and Prevention, Shenyang, China, 17 First Affiliated Hospital, China Medical University, Shenyang, China, 18 Shanghai Center for Disease Control and Prevention, Shanghai, China, 19 Ninth Affiliated Hospital, Shanghai Transportation University, Shanghai, China, 20 Jiangxi Provincial Center for Disease Control and Prevention, Nanchang, China, 21 First Affiliated Hospital, Nanchang University, Nanchang, China, 22 Guangxi Provincial Center for Disease Control and Prevention, Nanning, China, 23 First Affiliated Hospital, Guangxi Medical University, Nanning, China, 24 Zhejiang Provincial Center for Disease Control and Prevention, Hangzhou, China, 25 First Affiliated Hospital, Zhejiang University, Hangzhou, China, 26 Shenzhen Center for Disease Control and Prevention, Shenzhen, China, 27 Xinjiang Uygur Autonomous Region Center for Disease Control and Prevention, Urumqi, China, 28 Department of Epidemiology and Biostatistics School of Public Health, Health Science Center, Peking University, Beijing, China, 29 Centre for Emerging Infectious Diseases, Chinese University of Hong Kong, Hong Kong Special Administrative Region, China, 30 Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia, United States of America
Abstract
Background
While human cases of highly pathogenic avian influenza A (H5N1) virus infection continue to increase globally, available clinical data on H5N1 cases are limited. We conducted a retrospective study of 26 confirmed human H5N1 cases identified through surveillance in China from October 2005 through April 2008.
Methodology/Principal Findings
Data were collected from hospital medical records of H5N1 cases and analyzed. The median age was 29 years (range 6?62) and 58% were female. Many H5N1 cases reported fever (92%) and cough (58%) at illness onset, and had lower respiratory findings of tachypnea and dyspnea at admission. All cases progressed rapidly to bilateral pneumonia. Clinical complications included acute respiratory distress syndrome (ARDS, 81%), cardiac failure (50%), elevated aminotransaminases (43%), and renal dysfunction (17%). Fatal cases had a lower median nadir platelet count (64.5×109 cells/L vs 93.0×109 cells/L, p = 0.02), higher median peak lactic dehydrogenase (LDH) level (1982.5 U/L vs 1230.0 U/L, p = 0.001), higher percentage of ARDS (94% [n = 16] vs 56% [n = 5], p = 0.034) and more frequent cardiac failure (71% [n = 12] vs 11% [n = 1], p = 0.011) than nonfatal cases. A higher proportion of patients who received antiviral drugs survived compared to untreated (67% [8/12] vs 7% [1/14], p = 0.003).
Conclusions/Significance
The clinical course of Chinese H5N1 cases is characterized by fever and cough initially, with rapid progression to lower respiratory disease. Decreased platelet count, elevated LDH level, ARDS and cardiac failure were associated with fatal outcomes. Clinical management of H5N1 cases should be standardized in China to include early antiviral treatment for suspected H5N1 cases.
Citation: Yu H, Gao Z, Feng Z, Shu Y, Xiang N, et al. (2008) Clinical Characteristics of 26 Human Cases of Highly Pathogenic Avian Influenza A (H5N1) Virus Infection in China. PLoS ONE 3(8): e2985. doi:10.1371/journal.pone.0002985
Editor: Joel Mark Montgomery, U.S. Naval Medical Research Center Detachment/Centers for Disease Control, United States of America
Received: January 24, 2008; Accepted: July 19, 2008; Published: August 21, 2008
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: This study was supported by grants from the Ministry of Science and Technology of China (2004BA519A17, 2004BA519A71 and 2006BAD06A02), and the China-U.S. Collaborative Program on Emerging and Re-emerging Infectious Diseases. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
* E-mail: gaozhancheng5446@163.com (ZG); yangwz@chinacdc.cn (WY)
# These authors contributed equally to this work.
Introduction
As of July 13, 2008, 385 confirmed human cases of infection with highly pathogenic avian influenza A (H5N1) virus with 243 deaths had been reported from 15 countries since November, 2003 [1]. Although largely a panzoonotic among poultry and wild birds, avian-to-human transmission of H5N1 virus has resulted in most human cases [2], with rare instances of limited, non-sustained human-to-human H5N1 virus transmission [3]?[5]. The continuing propagation of highly pathogenic H5N1 viruses among poultry [6] and migratory birds [7], [8] poses a continuing and potentially escalating threat to human populations. Preparedness for a possible H5N1 pandemic requires not only enhanced prevention efforts but also a heightened awareness of the clinical characteristics of H5N1 cases among physicians.
To date, limited H5N1 clinical data are available in case reports and limited case series from Hong Kong Special Administrative Region (SAR), China in 1997 [9] and 2003 [10], and Vietnam [11], [12], Thailand [13]?[15], Indonesia [4], [16], Cambodia [17], Azerbaijan [18], [19], and Turkey [20] during 2004?2006. These observational studies described symptoms, signs, and laboratory findings at hospital admission. Few data are available on the clinical characteristics of cases throughout the course of H5N1 disease. Data on the natural history of H5N1 disease may allow risk stratification and identification of prognostic factors for outcomes of H5N1 virus infection. We describe the natural history and report the clinical characteristics at illness onset, hospital admission, and throughout hospitalization for 26 H5N1 cases identified by surveillance between October 2005 and April 2008.
Methods
National surveillance system and case definitions
In China, all suspected H5N1 cases are reported to the Chinese Center for Disease Control and Prevention (China CDC, Beijing, China) through a national surveillance system, which is based upon reporting of hospitalized cases of pneumonia of unknown origin, and by enhanced 1-month surveillance for cases of influenza-like illness at all health-care facilities within a 3-km radius after the occurrence of a suspected or confirmed H5N1 poultry outbreak with high bird mortality.
A case of pneumonia of unknown origin was defined as a patient with all of the following criteria without specific laboratory diagnosis: fever (temperature ?38°C); radiological evidence of pneumonia or acute respiratory distress syndrome (ARDS); normal white blood cell count (WBC; range 4?10×109 cells per L), leukopenia (WBC <4×109 cells per L), or lymphopenia (lymphocyte count <0.8×109 cells per L) at clinical presentation; and absence of clinical improvement after treatment with broad-spectrum antibiotics. A case of influenza-like illness was defined as a patient with fever (temperature ?38°C) and cough or sore throat, in the absence of any other confirmed diagnosis.
A confirmed case of H5N1 was defined as a patient with pneumonia or influenza-like illness and laboratory evidence of H5N1 virus infection diagnosed by viral isolation or reverse transcriptase (RT) PCR by testing respiratory specimens, or a four-fold or greater increase in H5N1 antibody titre in paired acute and convalescent sera.
Case-patients
All suspected H5N1 case-patients were interviewed by staff of the local CDC, and respiratory specimens, and acute- and convalescent-phase sera were obtained if available for laboratory investigations following the WHO protocol [21]. Respiratory specimens were tested by conventional [22] and real-time RT-PCR [23] to detect H5-specific viral RNA in biosafety level (BSL) 2 facilities at the National Influenza Center (NIC) of China CDC, and were inoculated into amniotic and/or allantoic cavities of specific pathogen free (SPF) embryonated chicken eggs for viral isolation [24] in enhanced BSL 3 facilities at the NIC. H5N1 antibody testing was performed on sera at the NIC by microneutralization (MN) assay [25] in a BSL-3 laboratory, and modified hemagglutination-inhibition (HI) assay using horse red blood cells [26] in BSL-2 conditions.
In mainland China, 30 confirmed human H5N1 cases have been identified to date. We included 26 laboratory-confirmed H5N1 cases identified by surveillance in 12 provinces in China between October 2005 and April 2008. Our analyses included limited data from 2 case reports [27], [28] and 6 urban cases reported previously in a brief epidemiological dispatch [29]. Of the 26 cases, H5N1 virus infection was confirmed by both virus isolation and RT-PCR in 20 (77%) cases, one (4%) case by virus isolation only, three (11%) by RT-PCR and serology, and two (8%) by serology only. Twenty-four cases in southern China were infected with clade 2.3.4 H5N1 viruses, and two cases from northern China had clade 2.2 H5N1 virus infections [2]. We excluded four H5N1 cases, including 2 military cases with unavailable clinical data and 2 cases in a cluster with limited person-to-person transmission reported elsewhere [5].
Clinical investigations
A trained team from the China CDC interviewed all confirmed H5N1 cases or their proxies, and collected clinical data through review of hospital medical records. A standardized form was used to collect information on demographic characteristics and clinical data, including clinical findings, blood chemistry testing and chest radiograph results performed during clinical management, complications, treatments, and outcomes. Data were collected during field investigations by China CDC staff, and was part of a continuing public-health outbreak investigation and determined by the Ministry of Health to be exempt from institutional review board assessment in China.
We used the following definitions: cardiac failure was defined as requiring use of inotropic agents; respiratory failure was defined as the need for assisted ventilatiory support; ARDS was defined as clinical deterioration with severe arterial hypoxaemia and diffuse bilateral infiltrates on chest radiograph; disseminated intravenous coagulation (DIC) was defined as elevated prothrombin time (PT) with elevated activated partial thromboplastin time (APTT), and decreased fibrinogen (FIB) level with thrombocytopenia; liver function impairment was defined as aminotransferase (ALT or AST) levels ? 2× upper range of normal values; renal dysfunction was defined as creatinine level >178mmol/L for adults or ? 2× upper limit of normal for age. High-dose corticosteroid use was defined as ?250 mg hydrocortisone or equivalent intravenous (IV) administration daily. For children <13 years old, high-dose corticosteroid use was defined as ?5 mg hydrocortisone or equivalent IV/kg/day.
Statistical analysis
Medians and interquartile ranges (IQRs) were calculated for continuous variables, and compared between fatal and nonfatal cases using Wilcoxon rank sum test. For categorical variables, percentages of case-patients in each category were compared using Fisher's exact test. Fatal cases were compared to nonfatal cases by demographic characteristics, H5N1 virus clade, underlying medical conditions, medical care practices, haematological and biochemical markers at admission or during hospitalization, clinical complications, and treatments, in the bivariate analyses using logistic regression. All statistical tests were two-sided with a significance level set at ? = 0.05. Data were analyzed with SPSS (version 13.0, SPSS Inc, Chicago, IL, USA).
Results
Twenty-six confirmed H5N1 cases had illness onset beginning in October, 2005 through February 2008. The median age of the 26 cases was 29 years (range 6?62) and 58% were female. Five (19%) were children aged <10 years old, one (4%) was 16 years old, and 20 (77%) were adults aged >18 years.
Clinical presentation
The earliest reported symptoms and signs of 26 patients at illness onset and noted at hospital admission are shown in Table 1. Many patients reported fever (92%) or cough (58%) initially, but very few reported upper respiratory symptoms such as rhinorrhea or sore throat. All patients developed cough a median of 1 day (IQR 1?3) from illness onset, and 85% had sputum production a median of 3 days (IQR 1?5.3) after illness onset. Lower respiratory tract signs and symptoms such as tachypnea and dyspnea increased substantially from illness onset to hospital admission. Most patients (88%) had tachypnea a median of 5 days (IQR 4?7) from illness onset and 46% reported dyspnea a median of 6.5 days (IQR 4.5?8.5) from illness onset. Diarrhea was reported in only one adult case at illness onset, and in two cases at hospital admission, but developed in six patients (one child and five adults) after hospitalization. The duration of diarrhea in these nine (35%) cases was a median of 1 day (IQR 1?4).
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Table 1. Signs and symptoms of 26 H5N1 cases at illness onset and at hospital admission, China.
doi:10.1371/journal.pone.0002985.t001
All case-patients had abnormal chest radiographs at admission; unilateral or bilateral infiltrates were observed in 10 (38%) and 16 (62%) case-patients, at a median of 6.5 days (IQR 4?7.3) and 7.5 days (IQR 6.3?9) from illness onset, respectively. The 10 case-patients with unilateral infiltrates at admission all developed bilateral pneumonia (Table 2). Chest radiographs showing rapid progression from unilateral to bilateral pulmonary infiltrates and ARDS in adult and paediatric cases are shown in Figure 1. Radiographic findings included patchy or diffuse infiltrates or consolidation with air bronchograms in multi-segmental or lobular distribution.
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Figure 1. Progression of pulmonary disease in chest radiographs from adult (35-year-old male, Panels A day 6 and B day 23 of illness) and pediatric (6-year-old male, Panels C day 6 and D day 14 of illness) H5N1 cases.
doi:10.1371/journal.pone.0002985.g001
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Table 2. Initial chest radiographic findings and progression during hospitalization of 26 H5N1 cases, China.
doi:10.1371/journal.pone.0002985.t002
Laboratory findings
Laboratory findings on initial testing, at admission and during hospitalization are shown in Table 3. The prevalence of patients with abnormal haematological findings at admission [leukopenia (46%), lymphopenia (62%), and moderate thrombocytopenia (50%)] increased to 92%, 89% and 73%, respectively, during hospitalization. At admission, the median leukocyte count was 3.5×109 cells/L (IQR 2.3?4.5) and median lymphocyte count was 0.6×109 cells/L (IQR 0.4?1.0). These declined during hospitalization to a median leukocyte count of 2.3×109 cells/L (IQR 1.5?2.8) and median lymphocyte count of 0.3×109 cells/L (IQR 0.3?0.5), after a median of 8.0 days.
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Table 3. Laboratory findings of 26 H5N1 cases* on initial testing, at hospital admission, and during hospitalization, China.
doi:10.1371/journal.pone.0002985.t003
Abnormal percentage, peak measurement and median days from illness onset of biochemical markers on initial testing, at hospital admission, and during hospitalization are shown in Table 3. Elevated ALT, AST, creatine kinase (CK), creatine phosphokinase isoenzymes (CPK), lactic dehydrogenase (LDH), and plasma glucose concentration, and decreased albumin levels were observed in more than half of cases at admission, and developed in nearly all cases during hospitalization. Elevated creatine level was observed in 25% of cases during hospitalization. Seventeen (77%) cases developed proteinuria at a median of 9.0 days (IQR 7.0?11) after illness onset.
Treatment
All cases received empiric treatment with broad-spectrum antibiotics during hospitalization, including ceftriaxone (n = 6), moxifloxacin (n = 8) and azithromycin (n = 15). Corticosteroids (median methylprednisolone dosage 1.6 [1]?[5] mg/kg per day IV) were initiated at a median of 6.5 days (IQR 6.0?8.0) from illness onset and administered to 24 (92%) cases for a median of 6 days (IQR 3?13). Of these, 21 (88%) received high-dose corticosteroids.
Four children received late antiviral treatment. One was treated with amantadine (100 mg per os (po) twice daily (BID) on illness days 10?15) and ribavirin (200 mg IV/d on illness days 9?16), and one received rimantadine (100 mg po each day (qD) on illness days 9?11); both cases survived. One child received oseltamivir (37.5 mg po BID) on illness days 12?14, and one child was treated with oseltamivir (40 mg po qD) on illness day 10; both died. Eight adults received late oseltamivir treatment, including two fatal cases ? one received 75 mg po BID on illness days 8?11, and one received both oseltamivir (75mg po BID on illness days 11?20) and rimantadine (200mg po qD on illness day 11). Six adults treated with oseltamivir survived: one was treated with 75 mg/day on illness days 8?12, four received 75 mg BID on illness days 4?11, illness days 8?14, illness days 10?14, and illness days 8?12, respectively, and one was treated with 75mg BID and amantadine (100 mg po BID) on illness days 8?12.
Two critically ill adult H5N1 cases (31-year-old male, 44-year-old female) with ARDS were treated with convalescent plasma obtained from one of two fully recovered H5N1 adult donor cases. Plasma was obtained 129 days after illness onset from an adult female case and 81 days after illness onset from an adult male case. Both donors' convalescent plasma tested negative for hepatitis B, hepatitis C, and HIV, and were separated and heat-inactivated at 56°C for 10 h before transfusion. The male ARDS case received three units (200 mL/unit) of transfused convalescent plasma from the female donor for 2 days, beginning on illness day 13. His H5N1 viral titre in bronchial-alveolar lavage fluid declined substantially and was undetectable for the next 3 consecutive days after receipt of the third convalescent plasma dose. The female ARDS case, who had a history of bronchiectasis, received one unit (200 mL) of transfused convalescent plasma from the male donor once daily for 3 days, starting on illness day 13. Further virological testing has not been done for this case. Both cases also received oseltamivir (75 mg po BID) on illness days 10?14 and days 8?12, respectively. Both cases recovered fully and were discharged home.
Complications and outcomes
Twenty-three (88%) cases required ventilatory support for respiratory failure. ARDS developed in 21 (81%) cases at a median of 8 days (IQR 7?9) after illness onset. Liver function impairment, renal dysfunction and cardiac failure occurred in 9 (43%), 4 (17%) and 13 (50%) patients.
Seventeen (65%) cases died (2 children, 1 adolescent and 14 adults), including one pregnant woman at 4 months' gestation [28] after a median of 10 days (IQR 8?20.5). Nine (35%) nonfatal cases were discharged at a median of 41 days (IQR 31.5?64.0) after illness onset. Five (24%) of the 21 cases with ARDS survived, including one pregnant woman, two adults who received convalescent H5N1 plasma, and two other previously healthy adults. The pregnant woman survived after developing ARDS and experiencing a spontaneous abortion during mechanical ventilation. Her pulmonary status subsequently improved and her temperature normalised quickly; the patient was extubated and recovered completely. All 17 fatal cases had multi-organ failure, including respiratory failure (94%), cardiac failure (71%), renal failure (27%) and 24% had disseminated intravenous coagulation (Table 4).
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Table 4. Comparison of demographic and clinical features of 17 fatal and 9 nonfatal H5N1 cases, China.
doi:10.1371/journal.pone.0002985.t004
In the bivariate analyses, demographic characteristics, year of illness onset, clade of H5N1 virus infection, and underlying medical conditions were similar between fatal and nonfatal cases (Table 4). Fatal cases had significantly lower median nadir platelet count during hospitalization (64.5×109 cells/L vs 93.0×109 cells/L, p = 0.02), higher median peak LDH level during hospitalization (1982.5 U/L vs 1230.0 U/L, p = 0.001), higher frequency of ARDS (94% [n = 16] vs 56% [5], p = 0.034), more frequent cardiac failure (71% [n = 12] vs 11% [1], p = 0.011), and shorter median duration of corticosteroid therapy (4.0 days vs 12.0 days, p = 0.025) compared to cases that survived. A higher proportion of cases survived that received any antiviral treatment compared to those that did not receive antivirals (67% [8/12 patients] vs 7% [1/14 patients], p = 0.003), with a positive correlation between antiviral therapy and disease outcome (Gamma coefficient = 0.664, p = 0.005).
Discussion
Our findings suggest that H5N1 disease in Chinese patients generally begins with fever, cough, and sputum production, and progresses rapidly to lower respiratory disease. Upper respiratory symptoms of rhinorrhea and sore throat were less common in China than observed in Hong Kong SAR, China [9], Thailand [13], Turkey [21], Azerbaijan [18], and Egypt [2]. Studies suggest that the lower respiratory tract is the major site for H5N1 viral replication, although initial infection may occur in either the upper or lower respiratory tract [30]?[33].
Diarrhea was present in only two H5N1 cases at admission, but developed in a quarter of cases during hospitalization. Diarrhea was a common presenting symptom among H5N1 cases in Vietnam [11], [12] and Thailand [13], but was reported infrequently among cases in Hong Kong SAR, China [9], [10], and Indonesia [4], [16]. H5N1 virus and viral RNA have been detected in feces and intestines of human H5N1 cases [12], [17], [30], [33]. Whether the gastrointestinal tract is a primary site for H5N1 virus infection is currently unknown.
Disease course in Chinese H5N1 cases was rapidly progressive; the median time from illness onset to death in our case series is consistent with WHO findings [2]. All H5N1 cases presented with pulmonary infiltrates, and all cases progressed rapidly to bilateral disease. Many cases experienced respiratory failure, ARDS, and multi-organ failure, with hepatic dysfunction and cardiac failure. Leukopenia and lymphopenia were also common. A recent molecular pathology study on two cases documented that in addition to the lungs, H5N1 virus infects the trachea and disseminates to other organs including the brain [30]. Our findings are consistent with other reports [11]?[20]. The pathogenesis of some clinical complications could be immunologically mediated, as suggested by high levels of proinflammatory cytokines and chemokines in vitro and cytokine dysregulation in fatal cases in observational studies [10], [33], [34].
Five H5N1 cases were younger than 10 years old and one was aged 16-years, in contrast to other case series [16], [19]?[20] and the WHO finding that the highest frequency of cases was aged 10?19 years old [35]. The age profile of Chinese H5N1 cases may reflect exposure differences due to traditional social and cultural behaviours. Visiting wet poultry markets in urban areas and exposure to sick or dead backyard poultry in rural areas before illness onset are H5N1 risk factors in China (unpublished data, China CDC). Paediatric cases lived in rural areas of China, and likely had more exposures to sick/dead backyard poultry than children in urban areas. In rural areas, young Chinese children are much more likely to play with backyard poultry than older children. Adults are much more likely to visit poultry markets in urban areas of China than children and all urban adult H5N1 cases had visited a wet poultry market prior to illness onset (unpublished data, China CDC).
In contrast to the WHO finding that cases aged 10?19 years old had the highest case-fatality [2], mortality of H5N1 cases in China was not associated with median age, sex or underlying medical conditions in the bivariate analysis. Isolates from 24 cases in southern China were characterized as H5N1 clade 2.3.4 viruses with consistent genetic and antigenic properties from 2005 through 2008 (unpublished data, China CDC). There were no significant differences in case-fatality ratios between years during 2005?2008 or between cases with clade 2.2 and clade 2.3.4 H5N1 virus infection. However, fatal outcomes were associated with decreased platelet counts, increased LDH, ARDS, cardiac failure, and lack of antiviral treatment in the bivariate analyses. In Thailand [13] and Hong Kong SAR [9], mortality was associated with late presentation, lower admission leukocyte, platelet, and lymphocyte counts, bilateral pulmonary findings on chest X-ray, and development of ARDS. Decreased leukocyte and lymphocyte counts, and increased d-dimer levels were associated with fatal outcomes in other studies [4], [17], [20], [33].
Survival was significantly higher in cases that received any antiviral treatment than in untreated cases, and 5 of 8 adult cases that received standard oseltamivir treatment survived even though all were treated late in their illnesses. However, it should be noted that treatment was uncontrolled and our findings lack sequential virological data on antiviral susceptibilities or quantitative H5N1 viral shedding, and favorable outcomes and clinical courses of some H5N1 cases cannot be attributed definitively to antiviral treatment. In contrast to clade 1 H5N1 viruses isolated in Vietnam and clade 2.1 viruses in Indonesia [2], the clade 2.3.4 and clade 2.2 H5N1 viruses isolated from cases in China were susceptible to both M2 inhibitors and neuraminidase inhibitors (unpublished data, China CDC). These findings suggest roles for either class of antiviral drugs as well as combination antiviral therapy for H5N1 cases in China [36], [37].
Very few Chinese H5N1 cases received early antiviral treatment because only one patient was admitted within two days of illness onset, and no patients received outpatient antiviral treatment. Antivirals were not administrated to most Chinese H5N1 cases until they were hospitalized with pneumonia. Oseltamivir was not available in some hospitals for treatment of some cases that died. Therefore, education of health-care providers about the epidemiological risk factors and clinical characteristics of H5N1 patients, and wider availability of antiviral drugs could help facilitate earlier detection and treatment of H5N1 cases in China. Although little data on early versus late oseltamivir treatment for H5N1 patients are available, current WHO guidance recommends initiating oseltamivir treatment as early as possible, including consideration of higher dosing for severe disease and longer treatment duration because of prolonged viral replication [37].
Although antiviral therapy is the primary treatment, most clinical management of H5N1 disease is supportive. For severely ill Chinese H5N1 patients with ARDS or multiorgan failure, management has focused on appropriate mechanical ventilation, correction of hypoxemia, fluid management, and treatment of other complications such as DIC. Corticosteroids were administered empirically to most H5N1 cases in China. A reduction in the proportion of cases reporting with fever from illness onset (92%) to hospital admission (69%) may reflect an early use of corticosteroids or non steroidal anti-inflammatory drugs. Compared to fatal cases, nonfatal cases in China had a longer duration of corticosteroid treatment. However, we cannot conclude that corticosteroid therapy resulted in survival and such treatment has not been shown to be effective in H5N1 patients [2]. Furthermore, prolonged or high-dose corticosteroid therapy may result in serious adverse events, including infection with opportunistic pathogens. Recent WHO H5N1 treatment guidance recommends against routine use of corticosteroid treatment [37].
Two cases with ARDS survived after receiving passive immunotherapy with transfused convalescent plasma from surviving H5N1 cases. This is compelling, but since passive immunotherapy and other treatments were administered in an uncontrolled manner, no definitive conclusions can be made about the benefit of such treatment [38]. A third Chinese H5N1 case survived after receiving post-vaccination plasma from an H5N1 vaccine clinical trial participant and combination antiviral treatment [5]. A meta-analysis of studies of convalescent plasma treatment during the 1918 influenza pandemic [39], evidence from animal experiments [40]?[42], and the limited experience in three Chinese H5N1 cases suggest that passive immunotherapy may be a viable option for the treatment of H5N1. Further research is needed to investigate the efficacy and effectiveness of passive immunotherapy with H5N1 convalescent plasma treatment for H5N1 patients, including cases with severe complications such as ARDS.
Our study was limited to available data for H5N1 cases identified through surveillance during the study period. Due to the small number of H5N1 cases, the study was too underpowered to compare differences between fatal and nonfatal cases. National surveillance and laboratory testing might not have identified all H5N1 cases that occurred, especially if the cases were clinically mild. Clinical management was uncontrolled, H5N1 viral shedding data, immunological and pathological data were not available, and any differences in outcomes cannot be interpreted to be due to the use of antiviral drugs, corticosteroids, or other uncontrolled treatments.
To improve clinical management of H5N1 patients in China, physicians should be educated about the natural history of H5N1 disease and epidemiological risk factors, and therapy should be standardized based upon current knowledge [37]. Early antiviral treatment and expanded testing should be considered for suspected H5N1 patients, with wider availability of antiviral medications at all health care facilities. In the absence of any definitive treatment for H5N1, preventive education to reduce risk behaviours for H5N1 exposures (e.g. avoiding direct contact with sick or dead poultry) must be emphasized more strongly.
Acknowledgments
We thank the provincial health bureaus of Hunan, Anhui, Sichuan, Fujian, Guangdong, Hubei, Liaoning, Shanghai, Jiangxi, Guangxi, Zhejiang and Xinjiang for assistance in coordinating field investigations and provision logistics support, and the Ministry of Health in China for generously facilitating this study. We thank Chin-Kei Lee from the WHO Beijing Office for helping us prepare the article. The views expressed in this article are those of the authors and do not represent the official policy of the China CDC or US CDC.
Author Contributions
Wrote the paper: HY TU. Designed the protocal of investigation, set up the field clinical investigation, contacted all investigators: HY ZG ZF YS WY. Responsible for the virus isolation, microneutralisation, hemagglutination inhibition assay, RT PCR, and real-time RT PCR testing, including the experimental design and analysis of data: YS CX. Participated in collection and management of data: NX LZ YH LF ZP ZL JL CH QL XX XL ZL LX YC HL LW XZ JX JG QW ZY LZ KZ WZ JY XZ SX LL JC EM PH SSL YW. Provided technical assistance for the clinical investigations and helped to review the data: TU.
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Vaccines
Univ. of Pittsburgh scientists receive $3.6M to test vaccine against deadliest strain of avian flu
8/25/08 Univ. of Pittsburgh--Scientists at the University of Pittsburgh Center for Vaccine Research have been awarded $3.6 million from the National Institute of Allergy and Infectious Diseases to conduct animal studies of vaccines designed to protect against the most common and deadliest strain of avian flu, H5N1. Recent outbreaks of H5N1 have prompted health officials to warn of its continued threat to global health and potential to trigger an avian flu pandemic.
"Worldwide avian flu control efforts have been mostly successful, but like seasonal influenza, avian flu changes year to year, creating new subtypes and strains that could easily and quickly spread among humans," said Ted M. Ross, Ph.D., principal investigator of the grant and assistant professor, Center for Vaccine Research, University of Pittsburgh.
Unlike other avian flu vaccines, which are partially developed from live viruses, the vaccines Dr. Ross and colleagues will test in non-human primates are based on a virus-like particle, or VLP, that is recognized by the immune system as a real virus but lacks genetic information to reproduce, making it a potentially safer alternative for a human vaccine. Given the evolving nature of H5N1, the vaccines have been engineered to encode genes for many influenza viral proteins to offer enhanced protection against possible new strains of the virus.
"VLPs may be advantageous over other vaccine strategies because they are easy to develop, produce and manufacture," said Dr. Ross. "Using recombinant technologies, within ten weeks, we could generate a vaccine most effective towards the current circulating strain of virus, making it a cost-effective counter-measure to the threat of an avian influenza pandemic."
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Co-investigators at the University of Pittsburgh include Simon M. Barratt-Boyes, Ph.D., Department of Infectious Diseases and Microbiology; Gerard J. Nau, M.D., Ph.D. and Jodi K. Craigo, Ph.D., Department of Microbiology and Molecular Genetics; Elodie Ghedin, Ph.D., Department of Medicine; and Clayton A. Wiley, M.D., Department of Pathology.
The Center for Vaccine Research (CVR) at the University of Pittsburgh houses both the Regional Biocontainment Laboratory and the Vaccine Research Laboratory. Researchers at the CVR, directed by Donald S. Burke, M.D., dean of the University of Pittsburgh Graduate School of Public Health and UPMC Jonas Salk Professor of Global Health, develop new methods and strategies to prevent and treat infectious diseases, potentially improving and protecting global health.
Regional Reporting and Surveillance
Sierra Leone: Health Ministry on Bird Flu
8/25/08 Sierra Leone News--The Ministry of Health and Sanitation and security personnel along the border chiefdoms in the Kenema District held a one-day Health Education Division Sensitization meeting for personnel in cross border districts on Avian Flu (Bird Flu).
The consultative meeting took place at the Kenema district council hall along Maxwell Khobe Street in Kenema city. Speaking at the opening ceremony, Manager of Health Education Division Sahr Hemon said the Education Division of the Ministry is meant to educate people about epidemic in various areas.
?The Ministry,? he disclosed, ?is the forerunner in making sure that the epidemic is not spread in the locality.?
Hemon stated that they are calling on people to empower them to sensitize their people about disease. He appealed to participants to spread the messages to friends, neighbors and other people of what they are gaining from the meeting.
?Bird Flu has destroyed many farm animals, such as fowls in other parts of Africa,? said Deputy Health Coordinator Lansana Conteh, ?and as such, the meeting was to discuss the ways of preventing the disease in the country.?
Conteh therefore called on police and military personnel at the borders to report any strange disease affecting the community, so that the ministry can intervene quickly.
The Kenema District Medical Officer (DMO), Dr. Yankuba Madina Bah, in his statement said the disease ?is not one man?s business but the business of everybody,? noting that the disease can easily be transferred to human beings. He appealed to participants to disseminate the messages of what they were gaining from the meeting to the public.
Dr. Bah added that they were treating Lassa fever, which comes from rats, in the hospital and most of the patients were from Lower Bambara chiefdom in the Kenema district. He said they are now working with the bodies in minimizing rabies. The sensitization campaign, he said, is all over the World and as such, he pleaded with the coordinator to expand the meeting to somewhere else in the border region.
Antivirals
H1N1 flu viruses growing more resistant to Tamiflu
8/25/08 CIDRAP--With influenza season well under way in the southern hemisphere, one of the three kinds of seasonal influenza virus is becoming increasingly resistant to the antiviral drug oseltamivir (Tamiflu), the World Health Organization (WHO) reported last week.
Thirty-one percent (242 of 788) of influenza A/H1N1 isolates from 16 countries that were tested in recent months carried a mutation associated with oseltamivir resistance, the WHO said. In South Africa, all of the 107 isolates tested had this mutation, known as H274Y, the agency reported.
Other countries and areas that tested 10 or more isolates and found resistance included Australia, 100% (10 of 10 isolates); Ghana, 20% (2 of 10) Hong Kong, 17% (97 of 583); and Chile, 13% (4 of 32 isolates).
The findings strengthen a trend that that was first observed last January in Norway and subsequently in many other countries. Overall for the last quarter of 2007 and the first quarter of this year, 16% (1,182 of 7,528) of tested H1N1 isolates carried the resistance mutation, according to WHO figures. Resistance was found in 35 countries, mostly in the northern hemisphere, including in 12% of tested US isolates and 26% of tested Canadian isolates.
"What we're seeing is the evolution of the resistance gene and the distribution of it throughout the world," said Lance Jennings, a clinical virologist with the Canterbury District Health Board in Christchurch, New Zealand, and chair of the Asia-Pacific Advisory Committee on Influenza, as quoted in an Aug 22 Bloomberg News report.
In South Africa, Terry Besselaar, director of the National Influenza Centre in Johannesburg, said, "The patients are from across the country, so the resistant strain is widespread," according to the Bloomberg report.
The WHO said only 1 of the 107 patients in South Africa was taking oseltamivir, and no unusual clinical features or underlying conditions were found.
No increase in oseltamivir resistance has been reported in the other two types of seasonal flu viruses, A/H3N2 and B. Recent WHO updates have not indicated which types are most common overall in the southern hemisphere this season, but the Aug 20 statement said flu was widespread in New Zealand, with H3 and B viruses predominant. The statement also cited sporadic flu activity in Argentina, with H1 viruses most common.
Many countries have stockpiled oseltamivir, which is used to treat people infected with the H5N1 avian flu virus and is generally considered the most promising antiviral to use in case H5N1 evolves into a human pandemic strain. The WHO statement did not mention any reports of resistance to zanamivir (Relenza), the other drug in the neuraminidase inhibitor class.
A spokeswoman for Roche, the maker of Tamiflu, said H5N1 viruses remain sensitive to the drug, according to the Bloomberg report. The spokeswoman, Claudia Schmitt, said the company plans to conduct surveillance on resistant and susceptible flu viruses during the 2008-09 flu season.
In a summary of H1N1 resistance to oseltamivir in the the 2007-08 flu season, the WHO said in June that no link between "oseltamivir exposure and resistance at the individual patient level was noted."
The increasing oseltamivir resistance in H1N1 viruses has puzzled experts. In an editorial published by Eurosurveillance in January, authorities said resistant viruses with the H274Y mutation had been seen in previous flu seasons but were rare and did not spread easily. But the more recent H1N1 isolates with the mutation were "fitter" and were spreading in the community, they wrote.
A recent update by the European Centre for Disease Prevention and Control (ECDC) observed, "At this stage the significance of these [resistance] findings remains uncertain. The emergence of drug resistance in the context of limited drug use is unexpected, and the extent of future circulation is difficult to predict."
AI Research
Protein Structure Discovery Opens Door For Drugs To Fight Bird Flu, Other Influenza Epidemics
8/25/08 ScienceDaily--Researchers at Rutgers University and The University of Texas at Austin have reported a discovery that could help scientists develop drugs to fight the much-feared bird flu and other virulent strains of influenza.
The researchers have determined the three-dimensional structure of a site on an influenza A virus protein that binds to one of its human protein targets, thereby suppressing a person's natural defenses to the infection and paving the way for the virus to replicate efficiently. This so-called NS1 virus protein is shared by all influenza A viruses isolated from humans ? including avian influenza, or bird flu, and the 1918 pandemic influenza virus.
A paper detailing this breakthrough discovery appears in the PNAS (Proceedings of the National Academy of Sciences) Early Edition and will be published in an upcoming issue of the PNAS print edition.
About 10 years ago, Professor Robert M. Krug at The University of Texas at Austin discovered that the NS1 protein binds a human protein known as CPSF30, which is important for protecting human cells from flu infection. Once bound to NS1, the human protein can no longer generate molecules needed to suppress flu virus replication. Now, researchers led by Rutgers Professor Gaetano T. Montelione and Krug identified the novel NS1 binding pocket that grasps the human CPSF30 protein.
"Our work uncovers an Achilles heel of influenza A viruses that cause human epidemics and high mortality pandemics," said Montelione, professor of molecular biology and biochemistry. "We have identified the structure of a key target site for drugs that could be developed to effectively combat this disease."
X-ray crystallography, which was carried out by Kalyan Das, Eddy Arnold, LiChung Ma and Montelione, identified the three-dimensional structure of the NS1 binding pocket. "The X-ray crystal structure gives us unique insights into how the NS1 and human protein bind at the atomic level, and how that suppresses a crucial antiviral response," said Das, research professor at Rutgers.
Rei-Lin Kuo, Jesper Marklund, Karen Twu and Krug at The University of Texas at Austin verified the key role of this binding pocket in flu replication by genetically engineering a change to a single amino acid in the NS1 protein's binding pocket, which in turn eliminated the protein's ability to grasp the human protein that is needed to generate antiviral molecules. These investigators then produced a flu virus with an NS1 pocket mutation and showed that this mutated virus does not block host defenses, and as a consequence has a greatly reduced ability to infect human cells.
"These experiments validate the NS1 pocket as a target for antiviral drug discovery," said Krug, professor and chair of molecular genetics and microbiology. "Because this NS1 pocket is highly conserved in all influenza A viruses isolated from humans, a drug targeted to the pocket would be effective against all human influenza A strains, including the bird flu."
This project was supported by two different institutes at the National Institutes of Health (NIH), demonstrating how several NIH initiatives can complement each other. Support for the Rutgers research was provided in part by the Protein Structure Initiative (PSI) of the NIH Institute of General Medical Sciences, a follow-on to the human genome project, which is providing large numbers of protein samples and three-dimensional structures of biologically important proteins to the broad scientific community.
"This work underscores the value of scientific collaborations between large-scale structural centers and individual biomedical research labs," said John Norvell, director of the PSI.
The University of Texas at Austin research was supported by a long-standing grant from the NIH Institute of Allergy and Infectious Disease (NIAID). In addition, NIAID has recently awarded a grant to investigators at The University of Texas at Austin and Rutgers to develop antiviral drugs directed against this NS1 binding pocket.
Other Rutgers faculty members on the research team were James Aramini, Rong Xiao, Li Zhao and Brian Radvansky. Most of the Rutgers investigators are also researchers at the Center for Advanced Biology and Medicine, a joint research institute of Rutgers and the University of Medicine and Dentistry of New Jersey?Robert Wood Johnson Medical School. Krug is also a member of the Institute for Cellular and Molecular Biology at The University of Texas at Austin.
Regional Reporting and Surveillance
Bird flu strain in India, Bangladesh similar
8/25/08 Times of India--The bird flu virus, that caused India's worst Avian Influenza (AI) outbreak this year, has been found to be "a lot similar" to the one that created havoc in Bangladesh.
This has been confirmed by Indian scientists after it studied the genetic make-up of Bangladesh's H5N1 virus strain. India's eastern neighbour, after much persuasion, finally shared the genetic sequencing data of its virus with India earlier this month.
The sequencing was completed in the OIE Reference Laboratory for Avian Influenza in Weybridge, UK. The H5N1 outbreak, that broke out in West Bengal in January this year, spread to nearly 13 of the state's 19 districts.
A animal husbandry department source told TOI: "Both India and Bangladesh finally exchanged genetic information of its H5N1 strains. We have found that both the strains are related. However, we can't say that Bangladesh was the cause of the outbreak in West Bengal because we don't have documented proof to show that infected poultry was smuggled into India."
He added: "We now know that both the viruses was of clade 2.2 variety which is a sub-lineage of the highly pathogenic Qinghai strain. The strain, however, is different to the one that caused the Manipur and Maharashtra outbreaks."
India was almost certain that the virus came from Bangladesh through illegal poultry trade. Even agriculture minister Sharad Pawar had openly said so. The animal husbandry department then made a formal request to the Bangladesh government through the ministry of external affairs to share the genetic history of its virus.
The H5N1 virus was first detected in Bangladesh in March 2007. Since then, over 47 of the country's 64 districts had been affected by bird flu.
Even though Bangladesh reported a human infection, India was lucky on that count and did not see any humans getting infected. The outbreak in Bengal saw over 42 lakh birds being culled.
Sources said that India had also made a formal complaint to FAO and OIE (World Organisation of Animal Health) about Bangladesh's slack handling to contain the virus, seriously putting at risk India's internal security. A team of the Border Security Force, manning the West Bengal-Bangladesh border, had once reported to the Centre how Bangladeshi citizens were seen dumping dead birds in no-man's land.
Vaccines
Novavax Says Vaccine Protects Humans Against Deadly Bird Flu
8/26/08 Bloomberg--Novavax Inc. said its experimental vaccine spurred an immune response in humans that can protect against a deadly strain of bird flu linked to more than 100 deaths.
In the study, 160 patients each received two injections, in doses ranging from 15 to 90 micrograms. At the highest dose, the vaccine produced a response against one version of the lethal H5N1 bird flu in 94 percent of patients, Novavax said in a statement today.
Novavax, based in Rockville, Maryland, has been working with General Electric Co. to develop a vaccine that can be mass- produced quickly. Outbreaks of lethal avian flu have spread from birds to humans in 15 countries, mostly in Asia, and are ``not expected to diminish significantly in the short term,'' according to the U.S. Centers for Disease Control and Prevention's Web site.
``What we've shown in this study is that the vaccine is immunogenic in humans,'' said Rahul Singhvi, Novavax's president and chief executive officer, in an interview. ``It will allow countries around the world to produce a custom vaccine on demand within their own borders.''
Novavax fell 1 cent to $2.97 yesterday in Nasdaq Stock Market composite trading. General Electric fell 80 cents, or 2.8 percent, to $28.32.
There have been 385 confirmed cases of bird flu in humans, resulting in 243 deaths, from late 2003 through June 19 -- the latest data available -- according to the World Health Organization's Web site. The study involved what is known as the Indonesian strain of H5N1. That version has accounted for 135 cases, mostly fatal, of avian flu in humans, according to Novavax.
Insect Cell Cultures
Novavax's process, which uses insect-cell cultures, avoids the need to grow viruses in eggs by making vaccines from particles that mimic viruses. With its process, Novavax can produce seven to 10 times as much vaccine in the same time as techniques that rely on eggs or cells from mammals, the company said. Novavax said it can make vaccine within 10 to 12 weeks of identifying a strain.
``This data milestone marks good progress in the viability of Novavax's vaccine,'' said Peter Ehrenheim, president and chief executive of life sciences for GE Healthcare, based in Chalfont St. Giles, England, near London, in a statement.
Other drug companies, including GlaxoSmithKline Plc and Sanofi-Aventis SA, are developing vaccines that could be produced rapidly during a flu pandemic. Traditional flu shots are made in chicken eggs, a process that can take up to six months after a strain of the virus is identified. Scientists have predicted that an avian flu strain could spread across the globe within days.
Safe to Continue
``The data are encouraging that this new vaccine approach can help prevent pandemic influenza,'' said Robert B. Belshe, an immunologist and infectious disease specialist at the Saint Louis University School of Medicine, who served on an independent safety monitoring board for the study, in a statement.
No ``serious'' side effects have been reported for the Novavax vaccine study and an independent monitoring board has supported continuing the study, Novavax said in its statement. Complete safety data for the study aren't yet available, the company said.
Novavax also is conducting two preliminary studies of a vaccine for seasonal influenza, which causes more than 500,000 deaths worldwide annually. One of the studies will test the vaccine on healthy young adults and another on people age 65 and over. Results of those studies are expected in late 2008 or early 2009.
