Articles Probable limited person-to-person transmission of highly pathogenic avian influenza A (H5N1) virus in China Hua Wang*, Zijian Feng*, Yuelong Shu*, Hongjie Yu*, Lei Zhou, Rongqiang Zu, Yang Huai, Jie Dong, Changjun Bao, Leying Wen, Hong Wang, Peng Yang, Wei Zhao, Libo Dong, Minghao Zhou, Qiaohong Liao, Haitao Yang, Min Wang, Xiaojun Lu, Zhiyang Shi, Wei Wang, Ling Gu, Fengcai Zhu, Qun Li, Weidong Yin, Weizhong Yang, Dexin Li, Timothy M Uyeki, Yu Wang Summary Background In December, 2007, a family cluster of two individuals infected with highly pathogenic avian influenza A (H5N1) virus was identified in Jiangsu Province, China. Field and laboratory investigations were implemented immediately by public-health authorities. Methods Epidemiological, clinical, and virological data were collected and analysed. Respiratory specimens from the patients were tested by reverse transcriptase (RT) PCR and by viral culture for the presence of H5N1 virus. Contacts of cases were monitored for symptoms of illness for 10 days. Any contacts who became ill had respiratory specimens collected for H5N1 testing by RT PCR. Sera were obtained from contacts for H5N1 serological testing by microneutralisation and horse red-blood-cell haemagglutinin inhibition assays. Findings The 24-year-old index case died, and the second case, his 52-year-old father, survived after receiving early antiviral treatment and post-vaccination plasma from a participant in an H5N1 vaccine trial. The index case’s only plausible exposure to H5N1 virus was a poultry market visit 6 days before the onset of illness. The second case had substantial unprotected close exposure to his ill son. 91 contacts with close exposure to one or both cases without adequate protective equipment provided consent for serological investigation. Of these individuals, 78 (86%) received oseltamivir chemoprophylaxis and two had mild illness. Both ill contacts tested negative for H5N1 by RT PCR. All 91 close contacts tested negative for H5N1 antibodies. H5N1 viruses isolated from the two cases were genetically identical except for one non-synonymous nucleotide substitution. Interpretation Limited, non-sustained person-to-person transmission of H5N1 virus probably occurred in this family cluster. Funding Chinese Ministry of Science and Technology; US National Institute of Allergy and Infectious Diseases, National Institutes of Health; China-US Collaborative Program on Emerging and Re-emerging Infectious Diseases. Introduction As of April 2, 2008, 376 cases of infection with highly pathogenic avian influenza A (H5N1) virus, with 238 deaths, had been reported from 14 countries since November, 2003.1 Although most cases have been sporadic, about 25% have occurred in clusters of two or more epidemiologically linked people.2,3 Clusters occurred in 19974 and 20035 in Hong Kong (special administrative region [SAR] of China), and during 2004–07 in Indonesia,6–8 Turkey,9 Azerbaijan,10 Vietnam,11 and Thailand.11,12 Limited person-to-person transmission of the virus has been strongly suggested in the largest cluster in Indonesia6 and in Thailand.12 Previous cluster investigations did not adequately assess whether person-to-person transmission had occurred among exposed contacts. Illness surveillance combined with seroepidemiological investigations in exposed contacts allows a comprehensive assessment of H5N1 virus transmission. In December, 2007, two cases of infection with H5N1 virus in one family were identified within a week in Nanjing, Jiangsu Province, China. Field and laboratory investigations were implemented immediately by public-health authorities. We report the epidemiological, clinical, and virological findings of this family cluster of confirmed H5N1 cases, including assessment of potential spread to exposed contacts. Methods Patients and procedures Epidemiological and clinical data were collected through interviews and review of medical records. Investigation staff interviewed case two and relatives of both cases to verify reported exposure histories during the 2 weeks before the onset of symptoms, to validate timelines of events, and to identify close contacts. We were unable to interview the index case (case one) because he was severely ill at the time of diagnosis and died on the next day. Households and places known to have been visited by the cases in the 2 weeks before the onset of illness were investigated to assess poultry and environmental exposures. Respiratory and stool specimens were collected from the two patients during hospitalisation and placed in sterile viral transport medium for H5N1 testing.13 RNA was extracted from specimens with the RNeasy mini kit (Qiagen, Valencia, CA, USA) as per the www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6 Published Online April 8, 2008. DOI:10.1016/S01406736(08)60493-6 See Online/Comment DOI:10.1016/S01406736(08)60494-8 *Contributed equally Jiangsu Provincial Centre for Disease Control and Prevention, Nanjing, China (H Wang MD, R Zu MD, C Bao MD, M Zhou MD, H Yang MD, Z Shi MD, L Gu MD, F Zhu MD); Office for Disease Control and Emergency Response, Chinese Centre for Disease Control and Prevention (China CDC), Beijing, China (Z Feng MD, H Yu MD, L Zhou MD, Y Huai MD, P Yang PhD, Q Liao MD, Q Li MD, W Yang MD, Prof Y Wang PhD); State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, China CDC, Beijing, China (Y Shu PhD, J Dong MD, L Wen MD, L Dong PhD, M Wang MD, W Wang MD, Prof D Li MD); Jiangsu Provincial People’s Hospital, Nanjing, China (W Hong MD); Nanjing Secondary People’s Hospital, Nanjing, China (W Zhao MD); Najing Centre for Disease Control and Prevention, Nanjing, China (X Lu MD); Sinovac Biotech Co, Beijing, China (W Yin MBA); and Influenza Division, National Center for Immunization and Respiratory Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA (T M Uyeki MD) Correspondence to: Prof Yu Wang, Chinese Center for Disease Control and Prevention, 27 Nanwei Road, Beijing, 100050, China wangyu@chinacdc.cn 1 Articles manufacturer’s protocol and tested by conventional reverse transcriptase (RT) PCR as recommended14 and by real-time RT PCR with H5N1-specific primers and probes.15 These assays were done in biosafety level (BSL) 2 Index case Case two General Age (years) and sex 24, male 52, male Temperature (ºC)* 40·1 (40·4) 38·9 (40·0) White blood cells (×10⁹ per L) 7·1 (0·7) 6·7 (4·0) Lymphocytes (×10⁹ per L) 0·49 (0·12) Blood counts* Platelets (×10⁹ per L) 1·06 (0·5) 88 (37) 99 (54) Alanine aminotransferase (U/L) 17·3 (107·0) 30·7 (110·4) Aspartate aminotransferase (U/L) 43·8 (374·0) 61·5 (84·7) Albumin (U/L) 36·8 (34·7) 37·7 (37·7) Creatinine (U/L) 96·8 (367·1) 98 (98) Serum biochemistry* Creatine kinase (U/L) Lactate dehydrogenase (U/L) 85 (341) 138 (138) 328 (3036) 548 (548) Coagulation* Prothrombin time (seconds) 16·6 (16·6) 11·7 (10·06) Activated partial thromboplastin time (seconds) 87·2 (180·1) 39 (39·0) Fibrinogen (g/L) 2·01 (3·28) 4·4 (4·02) Arterial blood* PaCO2 (mm Hg) 23·3 (36) 27 (28) PaO2 (mm Hg) 42 (44) 59 (66) Bicarbonate (mEq/L) 14·6 (26·4) 20·1 (19·5) Alveolar-arterial oxygen gradient (mm Hg) 79 (83) 92 (94) Other Chest radiography Left lower-lobe infiltrate Right upper, bilateral lower-lobe infiltrates Gastrointestinal symptoms More than five episodes of watery diarrhoea without blood or mucus on the night of day 4 and ≥15 episodes on day 5 Two episodes of watery diarrhoea without blood or mucus on day 3 Complications Acute respiratory distress syndrome, respiratory failure, cardiac failure, disseminated intravenous coagulation, liver function impairment, renal dysfunction Respiratory failure, liver dysfunction Treatment Mechanical ventilation Intubation Mask positive pressure ventilation Corticosteroids Methylprednisolone 40 mg intravenously twice daily on days 6–9 Methylprednisolone 40 mg intravenously twice daily on days 3–8 Rimantadine No 100 mg orally twice daily on days 3–7 Oseltamivir No 75 mg orally daily on day 1 of illness, 150 mg orally twice daily on days 2–6 Passive immunotherapy No 2×200 mL transfusion of post-vaccination plasma on day 5 Days from onset to death or discharge 8, died 23, discharge *Data are measurement at admission (peak or nadir measurement during hospitalisation). Table 1: Clinical features of the two cases 2 facilities at Jiangsu Centre for Disease Control and Prevention (CDC), Nanjing, China, and the National Influenza Centre of the Chinese CDC (China CDC) in Beijing. Respiratory specimens were inoculated in amniotic cavities of pathogen-free embryonated chicken eggs for viral isolation16 in enhanced BSL 3 facilities at the National Influenza Centre. Full genomic sequencing was done on extracted viral RNA. cDNA synthesis and PCR amplification of the coding region of the eight gene segments were done with a one-step RT PCR kit (Qiagen) with gene specific primers (available on request from the authors). The PCR products were purified with the Qiagen QIAquick gel extraction kit and used as templates for nucleotide sequencing. Sequencing reactions were done with the ABI BigDye terminator sequencing kit with reaction products resolved on an ABI 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences were analysed with the DNASTAR package (Lasergene, Madison, WI, USA). Phylogenetic analysis was done by neighbour-joining method with MEGA version 4. The nucleotide sequences obtained from this study are available from GenBank (accession number EU434686-EU434701). Close contacts were placed under daily surveillance— by telephone or in person—for fever and respiratory symptoms for 10 days after their last exposure to a patient infected with H5N1 virus. Close contacts were defined as individuals known to have been within 1 m, or had contact with respiratory secretions or faecal material, of a patient with H5N1 any time from the day before the onset of illness to when the index case died or during the period that case two was hospitalised. Chemoprophylaxis with oseltamivir (75 mg orally once a day for 7 days) was recommended and distributed to contacts. Following written informed consent, a structured questionnaire was used to gather demographic information and data on use of personal protective equipment, oseltamivir chemoprophylaxis, illness symptoms, and potential H5N1 risk factors (eg, poultry contact, visiting poultry markets, contact with individuals with febrile respiratory symptoms) during the 2 weeks before the last exposure to patients with H5N1. Respiratory specimens were gathered from close contacts with febrile respiratory illness during the 10-day observation period for H5N1 testing. Contacts were asked to have acute and convalescent sera (≤1 week, and ≥3–4 weeks after the last exposure to a patient with H5N1, respectively) collected for H5N1 serological testing. H5N1 serological testing was done by microneutralisation assay in a BSL 3 enhanced laboratory at the National Influenza Centre,17 and modified horse redblood-cell hamagglutinin inhibition assay in BSL 2 conditions at the National Influenza Centre.18 Antigens for the assays were produced from the index case’s virus isolate. Sera were tested in duplicate by two separate www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6 Articles microneutralisation assays done on different days. An individual was deemed to be seropositive for H5N1 antibody if H5N1 neutralising antibody titres of 1:80 or greater were detected for single serum, or four-fold or greater rises for paired sera, and confirmed by horse redblood-cell haemagglutinin inhibition assay.14,19 Data collection for H5N1 cases was determined by the Chinese Ministry of Health to be part of a continuing public-health outbreak investigation and exempt from institutional review board assessment. Case two granted permission for data collection on him and his son (case one) for research purposes. The protocol for collection of epidemiological data and serological testing of close contacts was approved by the China CDC institutional review board. Written, signed, informed consent was obtained from 91 adult contacts to participate in the study. Role of the funding source The study sponsors had no role in the design or conduct of the study, or in the collection, analysis, or interpretation of the data. Hongjie Yu had full access to all data included in the study and is responsible for the integrity of the data and accuracy of data analyses. Yu Wang made the final decision to submit the manuscript for publication. Results The index case, a 24-year-old male salesman, was well until August, 2007, when he experienced transient chills and sweats once or twice a month. On Nov 24, he developed fever (38·8°C), malaise, and chills, and was treated with oral antibiotics as an outpatient the next day. On Nov 27, he was hospitalised with persistent fever, chills, headache, myalgia, sore throat, cough, and sputum production. On admission, the patient had lymphopenia, moderate thrombocytopenia, and left-lower-lobe pneumonia (table 1). A blood culture taken on Nov 28 yielded Salmonella choleraesuis; he was thus treated for bacterial infection. The patient developed progressive dyspnoea, copious frothy sputum production, watery diarrhoea, and pneumonia. Despite administration of broad-spectrum antibiotics, corticosteroids, and mechanical ventilation, the patient died of acute respiratory distress syndrome, disseminated intravascular coagulation, and multiorgan failure on the fifth day in hospital. An endotracheal aspirate obtained on the fifth day of hospitalisation, just before death, was positive for H5N1 by RT PCR and H5N1 virus was isolated. Case two (the index patient’s father), a 52-year-old retired engineer with hypertension, developed fever (38·1°C), chills, and cough on Dec 3. That night he took one dose of oseltamivir (75 mg orally) that had been distributed for chemoprophylaxis to contacts of the index case. The next morning, he was hospitalised with fever, mild thrombocytopenia, and bilateral pneumonia, and treated with levofloxacin, corticosteroids, and oseltamivir (150 mg orally twice daily for 5 days; table 1). Rimantadine A B Figure 1: Improvement of pulmonary lesions in chest radiographs from case two (A) Bilateral lower-lobe infiltrates on day 4 of illness. (B) Reduction of pulmonary lesions in the right upper and bilateral lower lobes on day 10 of illness. (100 mg orally twice daily for 5 days) was started on day 3 of his illness. His respiratory status worsened, requiring positive pressure ventilation. On Dec 7, he received two 200 mL transfusions of plasma at 0100 h and 0500 h from a 30-year-old woman who had received two doses of inactivated whole-virion H5N1 vaccine (days 0 and 28) in a phase I clinical trial.20 Plasma obtained 280 days after the second vaccine dose was negative for hepatitis B virus, hepatitis C virus, and HIV, and was heat-inactivated at 56°C for 10 h (neutralising antibody titres 1:40 against the clade 1 vaccine strain A/Vietnam/1194/2004-RG and 1:20 against case two’s virus strain A/Jiangsu/2/2007). The patient’s fever resolved that night. A chest radiograph on Dec 12 (day 10) showed improvement in the right upper and bilateral lower lobes (figure 1). H5N1 virus was isolated from a throat swab collected on day 4 of the patient’s illness, and H5N1 viral RNA was detected in throat and stool specimens up to 10 days after the onset of illness. The patient recovered fully and was discharged 22 days after admission. Complete genomic sequencing showed that the H5N1 viruses isolated from the index case (A/Jiangsu/1/2007) and case two (A/Jiangsu/2/2007) were identical, except for one non-synonymous nucleotide substitution in the NS gene (glutumate to glycine at aminoacid position 82) coding for the NS2 protein. All genes were entirely of avian origin and both isolates were characterised as H5N1 clade 2.3.4 viruses.2 Sequence analyses indicated that these two isolates were highly homologous (sharing 97·2–98·9% homology in aminoacid sequences of the haemagglutinin gene) with viruses isolated from H5N1 cases in southern China. The haemagglutinin receptor-binding site was similar to that of other H5N1 viruses, and a polybasic aminoacid cleavage site (LRERRRKRG) was present. Sequencing of the M2 and www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6 3 Articles neuraminidase genes of both viruses suggested susceptibility to adamantane and neuraminidase inhibitor antiviral drugs. The index case lived with his mother in an apartment located in an urban area 10 km from his father’s home, and rarely shared meals with him. No poultry were raised in the home or neighbourhood, and no live poultry were ever brought home. He had been bitten by a healthy pet dog 25 days before the onset of H5N1 illness and received four doses of rabies vaccine. He had consumed cooked poultry four times at restaurants during the 2 weeks before the onset of illness. No live poultry were present or slaughtered at these restaurants. He did not have any known direct contact with live poultry or ill individuals in the 2 weeks before the onset of illness. 6 days before the onset of illness, he visited a market to purchase vegetables and freshly killed pork with his girlfriend. She reported that the index case had not gone near the area where live poultry were sold and slaughtered in the market, 10 m from the section they had visited. The index case’s father did not raise poultry and had not brought live poultry into the home. He visited a market to purchase vegetables and bean curd 15 days and 8 days before the onset of illness. Live poultry were sold and slaughtered at this market, but the patient denied going near this area, which was 20 m from the vegetable stalls. The patient did not have any known contact with ill individuals except for his son during the 2 weeks before the onset of illness. After the index case became ill, the patient had close contact with him five times, including eating dinner together, providing care, and attending his funeral (table 2). He provided unprotected bedside hospital care for the index case between Nov 27 and 29. The longest continuous time he spent caring for his son was 20 h. During this period, the index case had high fever (40·0ºC), frequent coughing, extensive sputum production, and Activities Exposure details frequent episodes of watery diarrhoea. Case two had helped to change his son’s soiled clothes and bedsheets, and had cleaned the toilet that had been used to dispose of diarrhoeal stool and a spittoon that contained copious sputum. The patient did not use personal protective equipment until after H5N1 had been confirmed in the index case late on Dec 1. A summary of the patient’s exposure to one another is shown in figure 2. 100 close contacts of the H5N1 cases were identified and followed up daily for 10 days. Of these, 91 (91%) gave consent for collection of data, completed a questionnaire, and provided serum specimens, including nine (10%) household contacts, five (5%) social contacts, and 77 (85%) health-care workers who cared for the two cases (table 3). Eight contacts (the index patient’s mother and girlfriend, four family members, one colleague of the mother, and one health-care worker) were exposed to both cases. The median duration of exposure to at least one of the cases was 7 h (IQR 2–36). 78 (86%) of the close contacts reported taking oseltamivir chemoprophylaxis beginning on Dec 3, and some reported always wearing protective equipment, including surgical masks, N95 respirators, gloves, face shields, glasses, or gowns while caring for the two cases (table 3). No adverse effects attributed to oseltamivir chemoprophylaxis were reported. Only two of the 100 close contacts who were followed up—the index patient’s girlfriend and one doctor— developed acute respiratory symptoms during the 10-day surveillance period. The girlfriend had a temperature of 37·3°C and onset of cough 5 days after her last known exposure to the index case and 1 day after her last known exposure to the index case’s father. The doctor had a temperature of 37·3°C 3 days after the last known exposure to the index case. Both ill contacts had normal chest radiographs, and throat swabs collected from them on the first day of illness were negative for H5N1 by RT PCR. Duration (h) Index case 2 Case two Days after onset of illness Signs and symptoms Days before onset of illness Personal protective equipment used Same day Fever (38·8ºC), chills, malaise 8 None Nov 24 Dinner together in restaurant Close contact, talking Nov 27 Provided bedside care in hospital Sat and slept on index patient’s bed, hugged him, wiped 20 sputum from his mouth, helped to change bedsheets and clothes exposed to faeces, washed toilet bowl after diarrhoea, and washed spittoon with bare hands 3 Fever (40·0ºC), chills, cough, frequent sputum production, extensive watery diarrhoea 5 None Nov 28 Provided bedside care in hospital Sat on index case’s bed, fed him 0·5 4 Fever (38·3ºC), cough, frequent sputum production, tachypnoea 4 None Nov 29 Provided bedside care in hospital Close physical contact 0·5 5 Fever (38·3ºC), cough, frequent sputum production, restlessness, dyspnoea 3 Surgical mask and long-sleeved gown Dec 2 Funeral Close physical contact with index patient’s body 0·33 8 NA 1 Gloves, gown, hair cover, N95 respirator, eye protection Table 2: Detailed exposure of case two to the index case before the onset of illness 4 www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6 Articles Index case 11/11 Ate poultry Travel Visited market Ate poultry Ate poultry Onset 16/11 17/11 18/11 19/11 23/11 24/11 Dinner together Hospital- Intensive Confirmed isation H5N1 care 27/11 29/11 1/12 Died 2/12 Dinner together, ate poultry Case two 11/11 17/11 24/11 27–29/11 Visited market Visited market Bedside care 2/12 3/12 4/12 5/12 26/12 Attended Onset Hospital- Confirmed Discharge funeral H5N1 isation Figure 2: Timeline of pertinent exposures and dates of onset of illness Paired acute and convalescent sera were collected from 30 (33%) close contacts (table 3), including from the index case’s girlfriend. A convalescent serum specimen was collected from 61 (67%) other close contacts (table 3), including the ill doctor. All serum tested negative for H5N1 antibodies (ie, all H5N1 neutralising antibody titres <1:10) by microneutralisation and horse red-blood-cell haemagglutinin inhibition assays. Discussion In this family cluster of confirmed cases of infection with highly pathogenic avian influenza A (H5N1) virus in mainland China, we believe that the index case transmitted H5N1 virus to his father while his father cared for him in the hospital. The index case had high fever, cough, extensive sputum production, and watery diarrhoea while his father had prolonged, direct, and close unprotected contact with him. His father did not have any known exposure to poultry or to any other ill person before the onset of his illness. Possible transmission sources include inhalation of droplets expelled by the index case through coughing or via contact with the index case’s clothes that were contaminated with diarrhoeal stool and subsequent inoculation of mucous membranes or the respiratory tract. Unlike in previous reports,6,12 viral isolates were available from all cases. Our epidemiological findings are supported by genetic sequencing data that indicate that both case’s H5N1 viral isolates were virtually identical. We cannot ascertain when person-to-person transmission of H5N1 virus occurred, but the most likely incubation period after the father’s unprotected exposure to his severely ill son is 4–5 days (range 3–6). 08TL948_2 Urgent Keys Transmission is unlikely to have occurred during exposure to the index case at a restaurant on the day RB Text typed Keyof 1 Key 2 fever onset, since the index case had yet to start Key 3 Author Image redrawn coughing, or during a final 30-min hospital visit 3 days Key 4 Key 5 before the father’s illness Ella Kilgour NSBC started. Although the index Key 6 case’s respiratory tract H5N1 viral load was probably Key 7 XX/XX/08 Checked by high during his hospitalisation, his father wore a surgical mask during his last visit, but did not wear protective equipment during earlier hospital exposures. Flexi-shapes and other vectors We doubt that transmission occurred at the funeral of the index case, a day before the onset of the father’s illness, when personal protective equipment was used. The only potential place of exposure to H5N1 virus that we identified for the index case was a market visit 6 days before the onset of illness. Visiting a wet poultry market, where avian influenza A viruses can be maintained and amplified,21 has been identified as a risk factor for H5N1 infection in Hong Kong SAR22 and urban China.23 No outbreaks of H5N1 in poultry were identified in Nanjing before or after the two human cases were detected, and no sick or dead poultry were seen at markets in Nanjing during our investigations. Only H5-vaccinated poultry were sold in the markets visited by the cases. In parallel with our investigations, 540 cloacal, faecal, and environmental specimens were collected from live poultry, wild birds, and surfaces at 23 poultry markets, restaurants (including markets and restaurants visited by both cases), and parks in Nanjing City between Dec 2 and Dec 9. All specimens tested negative by real-time RT PCR for H5N1 and by virus culture at the Jiangsu Provincial Centre for Animal Disease Control and Prevention (Yaoxing Liu, personal communication). With regard to the father, the estimated H5N1 incubation period of 3–6 days after exposure to his son is more plausible2 than the 8 days that had passed since his last visit to the market. However, we cannot exclude completely the possibility that both cases were infected with virtually the same H5N1 viral strain through exposures to different live poultry markets located 10 km apart with different sources of poultry. The index case is unlikely to have acquired H5N1 virus infection through consumption of cooked poultry or Measuring bars Graph marks Arrows Labels poultry products, including boiled duck blood. There is 1·3 2 5 no Key 1epidemiological evidence to date that H5N1 viruses Key 2 be transmitted A B to human beings by consumption of can Key 3 24 properly cooked Key 4 C D poultry. H5N1 virus infection has been Key 5 detected in the gastrointestinal tract, but whether this is Tick Marks Key 6 E F infection is currently unknown. 25 the site of initial We do Error bar Key 7 Axis breakH5N1 virus not believeG that the index case acquired H infection from contact with his pet dog, although infection of dogs with H5N1 virus has been documented.26 1A serum Text in the first box is centred specimen collected from the dog on Jan 7, 2008, www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6 Font reference and special characters €$£¥∆Ωµ∏π∑Ωαβχδεγηκλμτ†‡ ∞�婧¶√+−±×÷≈<>≤≥↔←↑→↓ 5 Articles tested negative for H5N1 neutralising antibodies (unpublished data). The index patient’s positive bacterial blood culture result on Nov 28 delayed a diagnosis of H5N1 until he deteriorated while receiving broad-spectrum antibiotics. The effect of S choleraesuis infection on susceptibility to infection with H5N1 virus—or the severity of such infection—is unknown, especially since only one colony was found on blood culture, and contamination or partial pretreatment as an outpatient cannot be ruled out. S choleraesuis causes swine paratyphoid, and occasionally infects human beings and can cause bacteraemia.27,28 Transient bacteraemia could explain the occasional febrile episodes he experienced in the 3 months before his H5N1 illness, although the timing of his bacterial infection is unknown. Since this pathogen is usually acquired from pigs, this infection could indicate food or water contamination.27 However, the index case was reported to have eaten only properly cooked meat. We found no evidence of transmission of H5N1 virus from the cases to any other close contacts, including the index case’s mother and girlfriend, both of whom had prolonged, direct, and close unprotected exposure to both patients. A convalescent serum specimen was available for more than 90% of contacts—including two who had subsequently reported illness—and all were negative for H5N1 antibodies. All 34 individuals who reported visiting a live poultry market in the 2 weeks before the onset of Only exposed to index case Only exposed to case two Household (n=0) Social (n=0) Health-care workers (n=64) Household (n=4) Social (n=3) Health-care workers (n=12) Household (n=5) Exposed to both cases Social (n=2) Health-care workers (n=1) Total (N=91) Age (years) .. .. 31 (25·25–39·75) 51 (31·5–75) 43 (32–50) 32 (30–38·75) 52 (37–54) 38·5 (25–52) 47 Sex (male) .. .. 18 (28%) 3 (75%) 2 (67%) 6 (50%) 2 (40%) 0 (0%) 0 (0%) 31 (34%) Contact with well-appearing poultry* .. .. 59 (92%) 4 (100%) 2 (67%) 10 (83%) 3 (60%) 2 (100%) 0 (0%) 80 (88%) Contact with sick or dead poultry .. .. 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) Visited wet poultry market .. .. 20 (31%) 3 (75%) 2 (67%) 6 (50%) 2 (40%) 1 (50%) 0 (0%) 34 (37) 32 (26–44) Exposure Type of contact with H5N1 cases Provided direct care .. .. 51 (80%) 0 (0%) 0 (0%) 11 (92%) 2 (40%) 1 (50%) 1 (100%) 66 (73%) Close physical contact .. .. 51 (80%) 0 (0%) 0 (0%) 11 (92%) 2 (40%) 1 (50%) 1 (100%) 66 (73%) Exposed to case <1 m .. .. 60 (94%) 4 (100%) 3 (100%) 12 (100%) 5 (100%) 2 (100%) 1 (100%) 87 (96%) Recalled case coughing or sneezing .. .. 54 (84%) 3 (75%) 1 (33%) 12 (100%) 5 (100%) 2 (100%) 1 (100%) 78 (86%) Contact with respiratory or .. fecal secretions .. 17 (27%) 0 (0%) 0 (0%) 0 (0%) 1 (20%) 0 (0%) 0 (0%) 18 (20%) Duration of exposure to cases (h) .. .. Oseltamivir used for chemoprophylaxis .. .. 6 (2–14·88) 1·5 (0·63–2·38) 0·25 (0·16–1) 66 (61·5–66) 72·08 (19·52–156·04) 51 (48–54) 55 (86) 3 (75%) 2 (67%) 12 (100%) 5 (100%) 1 (50%) 1·5 0 (0%) 7 (2–36) 78 (86%) Personal protection equipment N95 respirator .. .. 0 (0%) 0 (0%) 0 (0%) 12 (100%) 0 (0%) 0 (0%) 1 (100%) 13 (14%) Surgical mask .. .. 53 (83%) 0 (0%) 0 (0%) 0 (0%) 2 (40%) 1 (50%) 0 (0%) 56 (62%) Glasses .. .. 0 (0%) 0 (0%) 0 (0%) 8 (67%) 0 (0%) 0 (0%) 0 (0%) 8 (9%) Face shield .. .. 0 (0%) 0 (0%) 0 (0%) 11 (92%) 0 (0%) 0 (0%) 0 (0%) 11 (12%) 46 (51%) Gloves .. .. 34 (53%) 0 (0%) 0 (0%) 12 (100%) 0 (0%) 0 (0%) 0 (0%) Gowns .. .. 4 (6%) 0 (0%) 0 (0%) 3 (25%) 0 (0%) 0 (0%) 1 (100%) Febrile respiratory symptoms .. .. 1 (2%) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 1 (50%) 0 (0%) 2 (2%) Time from last exposure to acute serum collection for paired sera (days) .. .. 4 (2·5–7) 4† 4† 5 (3–6) 3 (3–3) 1† .. 4 (3–6) Time from last exposure to .. convalescent serum collection for paired sera (days) .. 27 (25·5–32) 24† 25† 23 (22–23) 23 (21–23) 25† .. 24 (23–27) Time from last exposure to only convalescent serum collection (days) .. 26 (25–27) 22 (18–35) 24·5 (24–25) 23† 21·5 (21–22) 26† 22 26 (25–27) .. 8 (9%) Data are median (IQR) or n (%). *Including direct contact (touching), preparation, cooking, and consumption of well-appearing poultry. †For each of these analyses, data available for one participant only. Table 3: Type of exposure and sera collection of 91 close contacts 6 www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6 Articles illness in the two cases were also negative for antibodies against the virus. In 1997, one social contact,29 six (12%) household contacts,29 and eight (4%) health-care workers30 exposed to cases had H5N1 neutralising antibodies. Our findings are consistent with studies done among health-care workers exposed to H5N1 cases in Vietnam31,32 and Thailand33 during 2004. More than 90% of H5N1 case clusters have occurred in blood-related family members.2 Although exposures must always be assessed among non-blood-relatives, this observation suggests that research is needed into potential genetic susceptibility to H5N1 virus infection.34 The major challenge of such a study is the small number of case clusters that have occurred in different countries, which would require close international collaborations. Unlike the index case, who was not diagnosed with H5N1 until just before death and did not receive oseltamivir, case two received early treatment with oseltamivir and rimantadine. On the fifth day of illness, he was also given post-vaccination plasma from a participant in a clade 1 H5N1 vaccine clinical trial.20 The plasma showed fairly low cross-reactivity with the clade 2.3.4 H5N1 virus strain isolated from this case. Although case two’s treatment with post-vaccination plasma was uncontrolled, as it was when another H5N1 patient also received convalescent H5N1 plasma,35 survival of both cases warrants research into antiviral treatment combined with passive immunotherapy for individuals infected with H5N1 virus. Corticosteroids were given to both cases here, although WHO recommends against routine corticosteroid treatment for such patients.36 Our investigations had several limitations. We could not elicit a complete exposure history from the index case before his death, but we interviewed his contacts, including his girlfriend, to verify his activities as much as possible. We also believe that it is difficult to trace all contacts who came within 1 m of the cases. We might have missed seroconversion in the nine contacts that remained healthy during the 10-day surveillance period, but who had declined serum collection. However, we believe that our serological findings from the other contacts, including all household and family contacts, suggest that it is unlikely that any asymptomatic or subclinical H5N1 virus infections were missed. We did not isolate any more than one H5N1 virus from either case to compare isolates during the case’s clinical course. Stool specimens were not collected from the index case for H5N1 testing, but H5N1 virus has been detected or isolated from rectal swab and stool specimens from other patients infected with H5N1,37 including the index case’s father. Lastly, we were unable to assess H5N1 viral load in serial clinical specimens from case two because the quality of some of the specimens was suboptimal. Our conclusion that limited, non-sustained person-to-person transmission of H5N1 virus probably occurred in this family cluster does not imply that H5N1 viruses have attained the ability to transmit more efficiently in human beings. A switch in receptor binding affinity has been suggested as a necessary factor,38,39 but human infection with H5N1 virus that binds to receptors with α2,6-sialic acid linkages has occurred without further spread.40 Viral characteristics required for sustained person-to-person H5N1 virus transmission remain unknown. H5N1 clusters require urgent investigation because of the possibility that a change in the epidemiology of H5N1 cases could indicate that H5N1 viruses have acquired the ability to spread more easily among people. Contributors Yu Wang, Hongjie Yu, Hua Wang, and Zijian Feng designed the protocol of investigation, set up the field epidemiology and clinical investigation, contacted all investigators; Hongjie Yu draft the manuscript; Dexin Li, Yuelong Shu, Jie Dong, Leying Wen, Libo Dong, Min Wang, Zhiyang Shi, and Ling Gu were responsible for virus isolation, microneutralisation, haemagglutination inhibition assay, RT PCR and real-time RT PCR testing, including the experimental design and analysis of data; Timothy Uyeki provided technical assistance for the epidemiological investigations, helped to review the data, and contributed to revising the manuscript. All other co-authors participated in collection and management of data. Conflict of interest statement We declare that we have no conflict of interest. Acknowledgments We thank the local Centres for Disease Control and Prevention of Xuanwu District and Gulou District in Najing City for assistance in coordinating field investigations and provision logistics support. The views expressed in this study are those of the authors and do not represent the policy of China CDC or the US CDC. This study was supported by grants (2004BA519A17, 2004BA519A71, and 2006BAD06A02) from the Chinese Ministry of Science and Technology, the US National Institute of Allergy and Infectious Diseases, National Institutes of Health (CIPRA grant U19 AI51915), and the China-US Collaborative Program on Emerging and Re-emerging Infectious Diseases. References 1 WHO. 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