H5N1 Influenza A Virus and Infected Human Plasma

Posted 05/08/2006
Salin Chutinimitkul; Parvapan Bhattarakosol; Surangrat Srisuratanon; Atthapon
Eiamudomkan; Kittipong Kongsomboon; Sudarat Damrongwatanapokin; Arunee
Chaisingh; Kamol Suwannakarn; Thaweesak Chieochansin; Apiradee Theamboonlers;
Yong Poovorawan
To the Editor Since January 2004, a total of 22 persons have been confirmed
infected with avian influenza A virus (H5N1) in Thailand; 14 of these patients
died. Three waves of outbreaks occurred during the past 2 years. The last
patient of the third wave was a 5-year-old boy whose symptoms developed on
November 28, 2005; he was hospitalized on December 5 and died 2 days later. The
child resided in the Ongkharak District, Nakhon Nayok Province, ≈70 km
northeast of Bangkok. Villagers informed the Department of Livestock after the
patient's illness was diagnosed. Five dead chickens had been reported in this
area from November 28 to December 1, 2005. Samples from these chickens could not
be obtained, thus, no H5N1 testing was performed. The boy had fever, headache,
and productive cough for 7 days before he was admitted to the Her Royal Highness
Princess Maha Chakri Sirindhorn Medical Center. Clinical examination and chest
radiograph showed evidence of lobar
pneumonia. He was treated with antimicrobial drugs (midecamycin and penicillin
G) and supportive care, including oxygen therapy. On December 7, the patient's
condition worsened, and severe pneumonia with adult respiratory distress
syndrome developed. Laboratory tests showed leukopenia (2,300 cells/mm3),
acidosis, and low blood oxygen saturation by cutaneous pulse oximetry (81.6%).
Oseltamivir was administered after his parents informed hospital staff about the
boy's contact with the dead chicken. However, the boy died the same day; no
autopsy was performed. On December 9, the cause of death was declared by the
Ministry of Public Health to be H5N1 influenza virus.
A blood sample was collected from the patient on December 7; anticoagulation
was accomplished with ethylenediaminetetraacetic acid (EDTA) for repeated
biochemistry analysis and complete blood count. The plasma from the EDTA blood
sample was separated 2 days later and stored at –20°C for 12 days. The sample
was subsequently given to the Center of Excellence in Viral Hepatitis, Faculty
of Medicine, Chulalongkorn University, for molecular diagnosis and then stored
at –70°C, where specific precautions implemented for handling highly infectious
disease specimens such as H5N1 influenza virus were observed. Plasma was
examined by multiplex reverse transcription–polymerase chain reaction
(RT-PCR)[1] and multiplex real-time RT-PCR,[2] both of which showed positive
results for H5N1 virus. The virus titer obtained from the plasma was 3.08 × 103
copies/mL. The plasma specimen was processed for virus isolation by embryonated
egg injection, according to the standard protocol described
by Harmon.[3] Briefly, 100 µL 1:2 diluted plasma was injected into the
allantoic cavity of a 9-day-old embryonated egg and incubated at 37°C. The
infected embryo died within 48 hours, and the allantoic fluid was shown to
contain 2,048 hemagglutinin (HA) units; also, subtype H5N1 was confirmed.[1,2]
Whole genome sequencing was performed and submitted to the GenBank database
under the strain A/Thailand/NK165/05 accession no. DQ 372591-8. The phylogenetic
trees of the HA and neuraminidase (NA) genes were constructed by using MEGA 3[4]
for comparison with H5N1 viruses isolated from humans, tigers, and chickens from
previous outbreaks in 2004 and 2005 (Figure). The sequence analyses of the
viruses showed that the HA cleavage site contained SPQREKRRKKR, which differed
from the 2004 H5N1 virus by an arginine-to-lysine substitution at position 341.
That finding had also been observed in wild bird species during earlier
outbreaks in Thailand in 2004.[5] Similar to the 2004–2005
H5N1 isolates from Thailand, a 20–amino acid deletion at the NA stalk region
was observed. Moreover, the amino acid residues (E119, H274, R292, and N294) of
the NA active site were conserved, which suggests that the virus was sensitive
to oseltamivir. In addition, a single amino acid substitution from glutamic acid
to lysine at position 627 of PB2 showed increased virus replication efficiency
in mammals.[6]


Figure 1. (click image to zoom) Phylogenetic analysis of the
hemagglutinin and neuraminidase genes of H5N1 from study patient compared with
sequences from previous outbreaks (2004–2005).

Observing live influenza virus in human serum or plasma is unusual.
However, in 1963, low quantities of virus were isolated from blood of a patient
on day 4 of illness,[7] and in 1970, the virus was cultivated from blood
specimens from 2 patients.[8] Recently, a fatal case of avian influenza A (H5N1)
in a Vietnamese child was reported. The diagnosis was determined by isolating
the virus from cerebrospinal fluid, fecal, throat, and serum specimens;[9] viral
RNA was found in 6 of 7 serum specimens 4–9 days after the onset of illness.[10]
In this case, the H5N1 virus could be isolated from plasma on day 10 after
symptoms developed. This case showed the virus in the patient's blood, which
raises concern about transmission among humans. Because probable H5N1 avian
influenza transmission among humans has been reported,[11] this case should be a
reminder of the necessity to carefully handle and transport serum or plasma
samples suspected to be infected with H5N1 avian
influenza. Because viable virus has been detected in blood samples, handling,
transportation, and testing of blood samples should be performed in a biosafety
(category III) containment laboratory to prevent the spread of the virus to
healthcare and laboratory workers.



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