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. Cumulative number of confirmed human cases of avian
influenza A/(H5N1) reported to WHO. http://www.who.int/csr/
disease/avian_influenza/country/cases_table_2008_04_02/en/
(accessed March 19, 2008).
2
Writing Committee of the Second WHO Consultation on Clinical
Aspects of Human Infection with Avian Influenza A (H5N1) Virus.
Update on avian influenza A (H5N1) virus infection in humans.
N Engl J Med 2008; 358: 261–73.
3
WHO. WHO guidelines for investigation of human cases of avian
influenza A. http://www.who.int/csr/resources/publications/
influenza/WHO_CDS_EPR_GIP_2006_4r1.pdf (accessed
March 3, 2008).
4
Yuen KY, Chan PK, Peiris M, et al. Clinical features and rapid viral
diagnosis of human disease associated with avian influenza A
H5N1 virus. Lancet 1998; 351: 467–71.
5
Peiris JS, Yu WC, Leung CW, et al. Re-emergence of fatal human
influenza A subtype H5N1 disease. Lancet 2004; 363: 617–19.
6
WHO. Avian influenza—situation in Indonesia—update 16.
http://www.who.int/csr/don/2006_05_31/en/ (accessed
March 3, 2008).
7
Kandun IN, Wibisono H, Sedyaningsih ER, et al. Three Indonesian
clusters of H5N1 virus infection in 2005. N Engl J Med 2006;
355: 2186–94.
8
Sedyaningsih ER, Isfandari S, Setiawaty V, et al. Epidemiology of
cases of H5N1 virus infection in Indonesia, July 2005–June 2006.
J Infect Dis 2007; 196: 522–27.
9
Oner AF, Bay A, Arslan S, et al. Avian influenza A (H5N1) infection
in eastern Turkey in 2006. N Engl J Med 2006; 355: 2179–85.
10 Anon. Human avian influenza in Azerbaijan, February–March
2006. Wkly Epidemiol Rec 2006; 81: 183–88.
www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6
7
Articles
11
12
13
14
15
16
17
18
19
20
21
22
23
24
8
Olsen S, Ungchusak K, Sovann L, et al. Family clustering of avian
influenza A (H5N1). Emerg Infect Dis 2005; 11: 1799–801.
Ungchusak K, Auewarakul P, Dowell SF, et al. Probable
person-to-person transmission of avian influenza A (H5N1).
N Engl J Med 2005; 352: 333–40.
WHO. Collecting, preserving and shipping specimens for the
diagnosis of avian influenza A(H5N1) virus infection. Guide for
field operations. www.who.int/csr/resources/publications/
surveillance/WHO_CDS_EPR_ARO_2006_1/en/ (accessed
March 3, 2008).
WHO. Recommendations and laboratory procedures for detection
of avian influenza A(H5N1) virus in specimens from suspected
human cases. http://www.who.int/csr/disease/avian_
influenza/guidelines/labtests/en/ (accessed March 3, 2008).
Spackman E, Senne DA, Myers TJ, et al. Development of a real-time
reverse transcriptase PCR assay for type A influenza virus and the
avian H5 and H7 hemagglutinin subtypes. J Clin Microbiol 2002;
40: 3256–60.
WHO. WHO manual on animal influenza diagnosis and
surveillance. http://www.who.int/vaccine_research/
diseases/influenza/WHO_manual_on_animal-diagnosis_and_
surveillance_2002_5.pdf (accessed March 3, 2008).
Rowe T, Abernathy RA, Hu-Primmer J, et al. Detection of antibody
to avian influenza A (H5N1) virus in human serum by using a
combination of serologic assays. J Clin Microbiol 1999; 37: 937–43.
Stepheson I, Wood JM, Nicholson KG, Zambon MC. Sialic acid
receptor specificity on erythrocytes affects detection of antibody to
avian influenza haemagglutinin. J Med Virol 2003; 70: 391–98.
WHO. WHO case definitions for human infections with
influenza A (H5N1) virus. http://www.who.int/csr/disease/avian_
influenza/guidelines/case_definition2006_08_29/en/ (accessed
March 3, 2008).
Lin J, Zhang J, Dong X et al. Safety and immunogenicity of an
inactivated adjuvanted whole-virion influenza A (H5N1) vaccine:
a phase I randomised controlled trial. Lancet 2006; 368: 991–97.
Webster RG. Wet markets—a continuing source of severe acute
respiratory syndrome and influenza? Lancet 2004; 363: 234–36.
Mounts AW, Kwong H, Izurieta HS, et al. Case-control study of risk
factors for avian influenza A (H5N1) disease, Hong Kong, 1997.
J Infect Dis 1999; 180: 505–08.
Yu H, Feng Z, Zhang X, et al. Human influenza A(H5N1) cases,
urban areas of People’s Republic of China, 2005–2006.
Emerg Infect Dis 2007; 13: 1061–64.
Scientific Report of the Scientific Panel on Biological Hazards.
Food as a possible source of infection with highly pathogenic avian
influenza viruses for humans and other mammals.
Eur Food Safety Authority J 2006; 74: 1–29.
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
Gu J, Xie Z, Gao Z, et al. H5N1 infection of the respiratory tract and
beyond: a molecular pathology study. Lancet 2007; 370: 1137–45.
Songserm T, Amonsin A, Jam-on R, et al. Fatal avian influenza A
H5N1 in a dog. Emerg Infect Dis 2006; 12: 1744–47.
Chiu CH, Su LH, Chu C. Salmonella enterica serotype Choleraesuis:
epidemiology, pathogenesis, clinical disease, and treatment.
Clin Microbiol Rev 2004; 17: 311–22.
Chassagne P, Perol MB, Doucet J, et al. Is presentation of
bacteremia in the elderly the same as in younger patients?
Am J Med 1996; 100: 65–70.
Katz JM, Lim W, Bridges CB, et al. Antibody response in individuals
infected with avian influenza A (H5N1) viruses and detection of
anti-H5 antibody among household and social contacts. J Infect Dis
1999; 180: 1763–70.
Buxton Bridges C, Katz JM, Seto WH, et al. Risk of influenza A
(H5N1) infection among health care workers exposed to patients
with influenza A (H5N1), Hong Kong. J Infect Dis 2000; 181: 344–48.
Liem NT, Lim W. Lack of H5N1 avian influenza transmission to
hospital employees, Hanoi, 2004. Emerg Infect Dis 2005; 11: 210–15.
Schultsz C, Dong VC, Chau NV, et al. Avian influenza H5N1 and
healthcare workers. Emerg Infect Dis 2005; 11: 1158–59.
Apisarnthanarak A, Erb S, Stephenson I, et al. Seroprevalence of
anti-H5 antibody among Thai health care workers after exposure to
avian influenza (H5N1) in a tertiary care center. Clin Infect Dis 2005;
40: e16–18.
Pitzer VE, Olsen SJ, Bergstrom CT, Dowell SF, Lipsitch M. Little
evidence for genetic susceptibility to influenza A (H5N1) from
family clustering data. Emerg Infect Dis 2007; 13: 1074–76.
Zhou B, Zhong N, Guan Y. Treatment with convalescent plasma for
influenza A (H5N1) Infection. N Engl J Med 2007; 357: 1450–51.
WHO. Clinical management of human infection with avian
influenza A (H5N1) virus. http://www.who.int/csr/disease/avian_
influenza/guidelines/clinicalmanage07/en/index.html (accessed
March 3, 2008).
de Jong MD, Simmons CP, Thanh TT, et al. Fatal outcome of
human influenza A (H5N1) is associated with high viral load and
hypercytokinemia. Nat Med 2006; 12: 1203–07.
Stevens J, Blixt O, Tumpey TM, et al. Structure and receptor
specificity of the hemagglutinin from an H5N1 influenza virus.
Science 2006; 312: 404–10.
Yamada S, Suzuki Y, Suzuki T, et al. Haemagglutinin mutations
responsible for the binding of H5N1 influenza A viruses to
human-type receptors. Nature 2006; 444: 378–82.
Auewarakul P, Suptawiwat O, Kongchanagul A, et al. An avian
influenza H5N1 virus that binds to a human-type receptor. J Virol
2007; 81: 9950–55.
www.thelancet.com Published online April 8, 2008 DOI:10.1016/S0140-6736(08)60493-6