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Letter
H5N1 Influenza A Virus and
Infected Human Plasma
Salin Chutinimitkul,* Parvapan Bhattarakosol,* Surangrat Srisuratanon,†
Atthapon Eiamudomkan,† Kittipong Kongsomboon,† Sudarat Damrongwatanapokin,‡
Arunee Chaisingh,‡ Kamol Suwannakarn,* Thaweesak Chieochansin,* Apiradee
Theamboonlers,* and Yong Poovorawan*![Comments](https://webarchive.library.unt.edu/eot2008/20090117112018im_/http://www.cdc.gov/ncidod/eid/images/email.gif)
*Chulalongkorn University Bangkok, Bangkok, Thailand; †Srinakharinwirot
University, Nakhon Nayok, Thailand; and ‡National Institute of
Animal Health, Bangkok, Thailand
Suggested
citation for this article
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).
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.
We express our thanks
to the Thailand Research Fund (Senior Research Scholar), Royal Golden
Jubilee PhD Program and Center of Excellence in Viral Hepatitis Research,
and Prasert Auewarakul for their generous support of our study.
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Suggested citation
for this article:
Chutinimitkul S, Bhattarakosol
P, Srisuratanon S, Eiamudomkan A, Kongsomboon K, Damrongwatanapokin S,
et al. H5N1 influenza A virus and infected human plasma [letter]. Emerg
Infect Dis [serial on the Internet]. 2006 Jun [date cited]. Available
from http://www.cdc.gov/ncidod/EID/vol12no06/06-0227.htm
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