healthmedicinet

The case under consideration in this article is a 31-year-old male surgeon working
in the emergency department (ED) of a Pudong hospital in Shanghai (SH-PDH), China.
He was obese (BMI: 29.39, ?28 in China 3]), with a five-year history of hypertension and suspected diabetes, and was a non-smoker.
There was no history of previous drug or food allergies or blood transfusions.

Clinical history

On January 11
^th
2014, the patient showed symptoms of an influenza-like illness (ILI) (fever, cough,
sore throat, dizziness, headache and myalgia) and self-treated with Analginum (Fig. 1). Four days later, the surgeon sought medical care and took mezlocillin only. From
January 11
^th
to January 16
^th
, he continued working (~8 hours a day) at the hospital until he developed dyspnea.
He was not treated with oseltamivir prior to his admission into SH-PDH intensive-care-unit
(ICU) on January 17
^th
. His illness rapidly progressed with bilateral pulmonary infiltration, hypoxia and
lymphopenia. Oxygen therapy and mechanical ventilation were started. Additionally,
oseltamivir (75 mg orally and 150 mg intra-gastrically), glucocorticoid, immunoglobulin
and broad-spectrum antibiotics therapy (imipenem and vancomycin, 1 g intravenously
every 12 hours) were administered. At 8:00 AM on January 17
^th
, he had a fever (39 °C), productive cough, chest tightness and shortness of breath.
The white blood cell count was 6.20?×?10
⁹
/L with 83.4 % neutrophils and 14.5 % lymphocytes (Table 1). A computed tomography chest scan showed consolidation in both lungs (Fig. 2). At 8:47 AM, the patient was given non-invasive ventilation but he continued to
suffer from hypoxaemia. As his condition worsened, he was started on invasive ventilation
with positive end-expiratory pressure at 11:28 AM. The patient died of acute respiratory
distress syndrome, severe pneumonia, and type I respiratory failure at 4:59 AM on
January 18
^th
.

Fig. 1. Timeline of the H7N9 patient’s illness, treatment, death and his close contacts. PPE:
personal protective equipment

Table 1. Clinical characteristics of the 31-year-old-case

Fig. 2. Representative radiographic findings of the laboratory-confirmed 31-year-old Shanghai
surgeon infected with H7N9 influenza. Chest radiograph of this patient was taken at
7 days after onset of symptoms, showing bilateral pulmonary infiltrates of airspace
consolidation and severe consolidation in the left lobe

Field epidemiology investigation

Retrospective investigation showed that X lived with his six family members (including
his 7-month-pregnant wife) in a 112-m
²
-sized house without birds or other animals. Although he had not visited LPMs, he
usually rode a bicycle to work and passed by the LPM -A where bird feces were spread
on the road near the market gate. His mother-in-law did grocery shopping at the LPM-B
which is 2.5 miles from X’s home almost every day. One of his neighbors owned about
14 pigeons. On January 19
^th
-20
^th
, 2014, six pigeon-related samples (2 poultry feces, 1 throat swab, 1 drinking water,
1 polluted water, and 1 swab of cage) from X’s neighbor and 68 LPM-associated samples
collected from LPM-related samples [including throat/anal swabs, and feces of poultry
and environmental specimens (chicken cages, cutting boards, water from washing poultry
flowing on the floor)] were tested by real-time reverse-transcriptase-polymerase-chain-reaction
(rRT-PCR) 4]. The results showed that the positive rates of H7N9 were 14.6 % (6/41), 18.5 % (5/27),
and 0 % (0/6) from LPM-B-, LPM-A- and pigeon-related specimens, respectively. Ten
days before the onset of X’s illness, all six family members were asymptomatic and
no guests had visited.

At the hospital (SH-PDH), the ED is a 400-m
²
U-shaped emergency room, including six 10-m
²
-sized neighboring rooms, where neither windows nor systematic regular ventilation
system were installed. The ER-A and ER-C (Fig. 3) consultation rooms were used by the Departments of Surgery and Internal Medicine,
respectively. In the 10 days before disease onset, X took care of 187 surgical patients
in ER-A1. Three of X’s patients who developed febrile illness and recovered completely
were migrants lost to follow-up. Further investigations revealed that two other patients
who had sought medical care in ER-C during January 1
^st
-10
^th
2014 had also died. One was a ninety-seven-year-old man with chronic obstructive pulmonary
disease (COPD-1) who arrived at ER-C on January 7
^th
, talked with an ED-internist (Dr.-A1), was immediately hospitalized [cared for by
3 doctors (Drs.-A2, A3, and A4) and one nurse (N-A1)], and died the next day. The
other was an eighty-eight-year-old woman with severe pneumonia (SP-1) who went to
ER-C on January 5
^th
, consulted an ED-internist Dr.-B1, was subsequently hospitalized (cared for by Drs.-B2,
B3, and B4, and N-B1), and died on January 8
^th
from an infection that was not identified. Specimens from 10 persons [COPD-1, SP-1,
8 HCWs except Dr.-A1 andDr.-B1] were not available for H7N9 tests. Follow-up of all
10 HCWs who had direct contact with COPD-1 and SP-1 showed that none of them became
ill (no fever/cough/pneumonia) between January 10
^th
2014 and January 20
^th
2014. In addition, both the throat and blood specimens collected on January 20
^th
from Dr.-A1 and Dr.-B1 in ER-C, who shared the same space or air with X in ER-A1,
were H7N9-negative.

Fig. 3. Spatial distribution of the six consultation rooms in the emergency department of
the Pudong Hospital. ””: door of consultation room; ER: emergency room; H7N9-(+) patient
X and his close contact Y worked in ER-A1. In addition, X and another close contact
Z worked in ER-C1. The COPD-1 visited ER-C on January 7 and SP-1 visited ER-C on January
5. ER-B1 is used for emergency handling and case management of the patients from ER-A
and ER-C, shared by the two departments of Surgery and Internal Medicine. ER-B2 was
used for cleaning trauma by ED-surgeons. Surgeons in ER-A generally did not wear oral
masks

Identification and Tracing of close contacts

In our investigation of human-to-human transmission, we defined close contacts of
X as those who had direct contact but without taking any personal protective equipment
(PPE) between January 11
^th
and January 18
^th
. In total, 35 close contacts of X were identified for daily monitoring, including
9 family members (3 visited him at the hospital but lived separately) and 26 HCWs
[11, 10, 2, and 3 from ED, ICU, radiology, and supporting staff, respectively]. None
had respiratory or other H7N9-related symptoms within 10 days of their last exposure
to X. On January 20th, we collected throat swabs for rRT-PCR, and all samples were
H7N9-negative. Since serological surveillance provides more information on total infection,
we then tested serum samples collected from all 35 close contacts by haemagglutination
inhibition (HAI) assay 5], using influenza A/Anhui/1/2013 (H7N9) virus as a viral antigen and horse red blood
cells. Furthermore, the two anti-H7N9-positive HCWs were investigated in detail. The
HCW with a serotiter of 1:40 against the H7N9 virus was a 37-year-old male ED surgeon
(Y), and the other one with a serotiter of 1:20 was a 30-year-old male ED internist
(Z). These three HCWs (X, Y, and Z) did not care for the same patient from January
1
^st
to January 17
^th
. They had never worked in the ICU and did not have any overlapping encounters with
patients of COPD-1 and SP-1. X and Y had worked together but had not worn masks in
ER-A1 for two days (8 hours/day) on January 3
^rd
and 15
^th
January (4 days after the onset of X’s illness). In addition, X talked with Z in ER-B1
(for emergency handling of patients) for 10 minutes without their masks at 3:00 AM
on January 17
^th
before being admitted to the ICU. Sixteen environmental samples were collected from
ER-A1 (the surface of telephone receiver, printer, work table, and medical waste),
ER-B2 (rails of hospital beds, oxygen humidifier, sputum aspirator surface, ECG monitor
wall, outer surface of IV stand), and ICU (ECG monitor wall, surface of blood pressure
meter, outer surface of sputum aspirator, Infusion pump surface, stethoscope surface
and rails of hospital beds). They were H7N9-negative by rRT-PCR.

Virology and associated molecular investigations

On January 18
^th
2014, the throat swab of X collected on the day of death was sent to Shanghai Municipal
Center for Disease Control and Prevention, and confirmed as H7N9 by rRT-PCR on January
19
^th
. Phylogenetic analysis for all eight H7N9 viral genomic segments was performed by
both neighbour-joining (NJ) and maximum-likelihood (ML) methods with MEGA version
5.10. The reliability of the unrooted tree was assessed by bootstrap with 1000 replications.
Bootstrap values greater than 60 % are shown for selected nodes. All the nucleotide
sequences of the eight viral gene segments of X’s H7N9 virus (A/Shanghai/PD-02/2014,
PD-2) are available at GenBank with the following accession numbers: KJ549801 (PB2),
KJ549802 (PB1), KJ549803 (PA), KJ195797 (HA), KJ195798 (NP), KJ195799 (NA), KJ195800
(M), KJ195801 (NS).

The eight viral genes shared between X’s H7N9 virus (A/Shanghai/PD-02/2014, PD-2)
and the avian-origin H7N9 virus isolates from the two LPMs in this study (A/Chicken/Shanghai/PD-CN-02/2014,
CN-2 and A/Environment/Shanghai/PD-JZ-01/2014, JZ-1) revealed nucleic acid sequence
identity percentages ranging from 97.8 % to 99.5 % (Table 2), in which the percentages of the PA gene ranked lowest (97.9 %, 97.8 %). However,
the overall viral sequence identity was higher (ranging 98.1 %?~?99.7 %) between the
two Shanghai human cases, PD-2 and PD-1 (A/Shanghai/PD-01/2014, PD-1), while PD-1
was another non-epidemiologically linked H7N9 human case that was also confirmed in
January in SH-PD. Similarly, viral sequence patterns for all the eight individual
genes were also closer between PD-2 and PD-1 than those between PD-2 and CN-2 or JZ-1.
GenBank accession numbers of PD-2, JZ-1, and CN-2 are as follows: PB2 (KJ549799, KJ549791,
KJ549783), PB1 (KJ195791, KJ549792, KJ549784), PA (KJ549800, KJ549793, KJ549785),
HA (KJ195792, KJ549794, KJ549786), NP (KJ195793, KJ549795, KJ549787), NA (KJ195794,
KJ549796, KJ549788), M (KJ195795, KJ549797, KJ549789), NS (KJ195796, KJ549798, KJ549790).

Table 2. The nucleic acid identity percentages among four H7N9 isolates

The phylogenetic tree topology was the same, using ML and NJ methods; hence the results
of MJ tree are displayed in Fig. 4. The results showed that all the four genes (HA, NA, M and NS) of PD-2 clustered
with PD-1 (i.e. the same sub-lineage) whereas the two LPM-derived H7N9 isolates belonged
to another sub-lineage. HA, PA, PB2, and NP of JZ-1 are at the same branch as those
of PD-2. The PD-2 had more amino acid changes from avian to human signatures, with
markers of mammalian-host adaptation 1], such as HA-Q226L HA-G228S (H3 numbering), and PB2-E627K. In contrast, CN-2 and JZ-1
isolates kept one avian-associated molecular signature with no change at PB2-E627
among these three amino acids. For clinical concern, the PD-2 virus was still oseltamivir-sensitive
(NA-E119 6], R292 5], and N294 1]) but Amantadine-resistant (M2-S31N) 1], 7]. Most importantly, of these four SH-H7N9 viruses, only the PD-2 possessed PB2-D701N
(aerosol transmissibility in ferrets) 8], 9], which has public health significance.

Fig. 4. Phylogenetic relationships of the eight full-length genes of A/Shanghai/PD-02/2014.
Horizontal distances are proportional to the genetic distance. Three colors represent
the three different types of the sources of H7N9 viruses: (1) the A/Shanghai/PD-02/2014
(H7N9) virus was isolated from this young surgeon (X) is shown in red, (2) the A/Shanghai/PD-01/2014
(H7N9) virus was isolated from another PD patient in January without epidemiological
linkage is shown in green, and (3) H7N9 viruses from the two LPMs are shown in blue.
A/Chicken/Shanghai/PD-CN-02/2014 virus was isolated from the LPM-A near H7N9-(+) patient
X’s hospital. A/Environment/ Shanghai/ PD-JZ-01/2014) was isolated from the LPM-B
closer to H7N9-(+) patient X’s home

The study was approved by the Ethical Committee of the Shanghai Municipal Center for
Disease Control and Prevention.