Japanese encephalitis outbreak, Yuncheng, China, 2006.

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LETTERS
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 7, July 2007 1123
Yohei Doi,* Jennifer Adams,*
Alexandra O’Keefe,* Zubair
Quereshi,* Lindsay Ewan,* and
David L. Paterson*1
*University of Pittsburgh School of Medi-
cine, Pittsburgh, Pennsylvania, USA
References
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spectrum beta-lactamases: a clinical
update. Clin Microbiol Rev. 2005;18:
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Escherichia
Address
Paterson, Infectious Diseases Unit, University
of Queensland, Royal Brisbane and Women’s
Hospital, Butterfi eld St, Herston, Queensland,
QLD 4029 Australia; email: david.antibiotics@
gmail.com
for correspondence: David L.
J apanese
Enc ephalitis
Outbreak,
Yuncheng, China,
2006
To the Editor: Japanese encepha-
litis (JE) epidemics have occurred only
in Asia. More than 50,000 cases of JE
with ≈10,000 deaths have been report-
ed since 1998 (1,2). The People’s Re-
public of China reported 5,104 cases
and 214 deaths in 2005. Most of these
deaths occurred in infants (3,4).
During July and August 2006,
an outbreak of viral encephalitis oc-
curred in Yuncheng, Shanxi Province,
People’s Republic of China. A total of
66 cases (1.32/100,000 population)
were reported, including 19 deaths
(case-fatality rate 28.8%). The cases
had a widespread distribution over 9
counties and involved 37 towns and
61 administrative villages. The ratio
of male-to-female patients was 1:0.89.
A distinct clinical feature of this out-
break was the age distribution. More
than 86% of the patients were >30
years of age, with only 10% of pa-
tients <7 years of age; ≈95% of the
deaths occurred in patients >50 years
of age (5).
We report serologic and virologic
fi ndings for the 2006 outbreak of viral
encephalitis. Forty-six clinical speci-
mens collected from 34 patients who
had a diagnosis of viral encephalitis,
including 33 serum samples and 13
cerebrospinal fl uid (CSF) samples,
were studied. All serum samples were
screened for immunoglobulin M (IgM)
to West Nile virus (WNV) by using the
WNV IgM-capture ELISA kit (PanBio,
Brisbane, Queensland, Australia) and
for IgM to dengue virus or Japanese
encephalitis virus (JEV) by using the
JE-Dengue IgM Combination ELISA
kit (PanBio). Results for JEV were con-
fi rmed by using the JE Virus IgM-Cap-
ture ELISA kit (Shanghai B & C Enter-
prise Development Co. Ltd, Shanghai,
People’s Republic of China).
WNV-specifi c or dengue vi-
rus–specifi c IgM was not detected in
any samples. JEV-specifi c IgM was
detected in 27 (80%) patients, which
indicated recent JEV infections. The
other 7 patients were negative for JEV
by ELISA and reverse transcription–
PCR (RT-PCR). Increases >4-fold in
neutralizing antibodies were detected
in acute- and convalescent-phase se-
rum samples from 9 patients (10 se-
rum pairs were collected during the
outbreak).
Attempts were made to detect
virus in CSF of patients and in 2,400
mosquitoes. Mosquitoes (mainly Cu-
lex spp.) were collected in cow sheds
and hog pens around houses and pro-
cessed into pools of 100. Total RNA
was extracted from CSF or mosquito
homogenate by using the QIAamp
viral RNA extraction kit (QIAGEN,
Valencia, CA, USA) according to the
manufacturer’s specifi cations. RT was
performed by using Ready-To-Go-You
Prime First Strand Beads (Amersham
Pharmacia Biotech, Piscatawy, NJ,
USA) and a seminested PCR to ampli-
fy 492-bp gene fragments of the pre-
membrane (PrM) sequence of JEV by
using the Takara LA Taq PCR kit (Ta-
kara Bio Inc., Shiga, Japan). The prim-
ers were derived from Ishikawa strain
genome sequences (GenBank acces-
sion no. AB051292). Primers PrMF:
5′-CGT TCT TCA AGT TTA CAG
CAT TAG C-3′ (251–275), PrMR1:
5′-CGY TTG GAA TGY CTR GTC
CG-3′ (724–743), and PrMR2: 5′-
CCY RTG TTY CTG CCA AGC ATC
CAM CC-3′ (901–925) were used.
JEV PrM gene was amplifi ed from
CSF of 6 (46%) of 13 patients and 10
of 24 pools of mosquitoes by using the
same seminested RT-PCR. To identify
JEV genotype(s) involved in this out-
break, PCR products were sequenced.
Eleven sequences (GenBank acces-
sion nos. EF434264–EF434274) were
obtained from 6 patients and 5 pools
of mosquitoes. The 11 sequences were
compared phylogenetically with17
known JEV strains of the 4 recognized
1Current affi liation: University of Queens-
land, Royal Brisbane and Women’s
Hospital, Herston, Queensland, Australia

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LETTERS
1124 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 7, July 2007
genotypes (classifi ed on the basis of
a 240-nt region of the prM gene). As
shown in the Figure, the 11 sequences
were those of JEV.
Further analysis showed that these
11 sequences can be grouped into gen-
otypes I and III. Both genotypes were
found in patient and mosquito sam-
ples, indicating that these genotypes
co-circulated during this JE outbreak.
JE has been endemic in Yuncheng
for many years (6). A vaccine against
JE (SA14–14–2) has been used in this
area in infants, but not in adults. This
might be 1 reason why a higher adult
incidence was found in this outbreak.
JEV genotype III had been the pre-
dominant genotype in previous years,
but genotype I has been recently de-
tected at increased frequencies (7–10).
Detection of 2 JEV genotypes in 1 epi-
demic has not been reported. Whether
simultaneous circulation of >1 geno-
type during an outbreak indicates a
new type of emergence of JEV or that
this has occurred and not been detect-
ed is unknown.
Acknowledgments
We thank Charles H. Calisher, Zhen
F. Fu, and Ichiro Kurane for assistance
with preparation of this article.
This study was supported by
grants from the National Science and
Technology Department of China (no.
2003BA12A08-01) and the Japan Health
Science Foundation.
Li-Hua Wang,* Shi-Hong Fu,*
Huan-Yu Wang,*
Xiao-Feng Liang,†
Jing-Xia Cheng,‡
Hong-Mei Jing,§ Gen-Lao Cai,¶
Xing-Wang Li,# Wen-Yuan Ze,**
Xin-Jun Lv,* Hua-Qing Wang,†
Ding-Lin Zhang,¶ Yun Feng,*
Zun-Dong Yin,†
Xiao-Hong Sun,* Tie-Jun Shui,†
Ming-Hua Li,* Yi-Xing Li,†
and Guo-Dong Liang*
*Institute for Viral Disease Control and Pre-
vention, Beijing, People’s Republic of Chi-
na; †Chinese Center for Disease Control
and Prevention, Beijing, People’s Republic
of China; ‡Shanxi Center for Disease Con-
trol and Prevention, Taiyuan, People’s Re-
public of China; §Yuncheng Center for Dis-
ease Control and Prevention, Yuncheng,
People’s Republic of China; ¶Yuncheng
Infectious Diseases Hospital, Yuncheng,
People’s Republic of China. #Beijing Di-
tan Hospital, Beijing, People’s Republic of
China; and **Beijing Institute of Biological
Products, Beijing, People’s Republic of
China
References
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Sannarangalah S, Saxena P, et al. Japanese
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Figure. Phylogenetic analysis of Japanese encephalitis virus strains predicted from
premembrane gene sequences. Neighbor-joining tree was generated by using MEGA
3.1 software (www.megasoftware.net) and rooted with Murray Valley encephalitis (MVE)
virus sequence information. Bootstrap confi dence limits for 1,000 replicates are indicated
above each branch. Horizontal branch lengths are proportional to genetic distance; vertical
branch lengths have no signifi cance. Scale bar indicates no. nucleotide substitutions
per site. All sequences from this study are in boldface. Genotypes are indicated on the
right. Designations are listed fi rst, followed by country, source, and year of isolation. CSF,
cerebrospinal fl uid.

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LETTERS
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 13, No. 7, July 2007 1125
Address
Liang, State Key Laboratory for Infectious
Disease Control and Prevention, Institute for
Viral Disease Control and Prevention, Chinese
Center for Disease Control and Prevention, 100
Yingxin St, Xuanwu District, Beijing 100052,
People’s Republic of China, email: gdliang@
hotmail.com
for correspondence: Guo-Dong
Chloroquine-
Resistant
Plasmodium vivax,
Brazilian Amazon
To the Editor: Plasmodium vivax
is the protozoan that causes the second
most common form of malaria. Some
resistant strains to chloroquine (CQ)
occur in a few places in Asia and the
Indo-Pacifi c Region (1–4). Although
resistance of P. vivax to CQ has al-
ready been described in South Ameri-
ca (5–7), there are limited data regard-
ing this issue.
CQ plus primaquine is the stan-
dard treatment for vivax malaria
worldwide. Presently, this drug regi-
men exhibits satisfactory effi cacy in
the Brazilian Amazon. However, in
recent years several treatment fail-
ures presumably related to CQ resis-
tance, have been reported in the city
of Manaus (Amazonas) where vivax
malaria predominates (7). This obser-
vation warrants local attention despite
these cases having no confi rmation
of CQ blood levels on the basis of
the appearance of asexual parasites
against CQ plus desethylchloroquine
levels exceeding the minimally effec-
tive plasma concentration proposed for
sensitive parasite strains (>10 ng/mL)
(8), according to Pan American Health
Organization recommendations (9).
From September 2004 to February
2005, a 28-day in vivo test was con-
ducted at the Foundation for Tropical
Medicine of Amazonas (FMTAM) in
Manaus, Brazil, to assess the effi cacy
of standard supervised CQ therapy.
The test involved 166 volunteers with
uncomplicated vivax malaria. Each
volunteer was administered uncoated,
scored, 150-mg CQ tablets (10 + 7.5
+ 7.5 mg/kg at 24-hour intervals) (9).
Primaquine was withheld until day
28 (dose regimen of 30 mg/day for 7
days). Among the 109 volunteers who
completed the in vivo test, 19 had pos-
itive blood smears within the 28-day
follow-up (1 on day 14, 3 on day 21,
and 15 on day 28). All were required
to undergo alternative therapy (mefl o-
quine). Adequate CQ absorption was
confi rmed in these cases on day 2 with
a mean ± SD CQ plasma concentration
of 785.4 ± 800.1 ng/mL) (10) Suspect-
ed therapeutic failure (P. vivax CQ re-
sistance) was confi rmed in 11 (10.1%)
of 109 persons with a mean isolated
choloroquine plasma concentration
>10 ng/mL (356.6 ± 296.1 ng/mL) (9).
Desethylchloroquine levels in plasma
were not measured.
Previously, a CQ effi cacy study
demonstrated that 4.4% of those test-
ed had CQ-resistant P. vivax (7). In
comparison, the proportion of failures
(10.1%) in the current study seems to
be relevant; even though most of the
P. vivax infections (98, 89.9%) were
successfully evaluated and adequate
clinical and parasitologic responses
were obtained. Currently, the FMTAM
Manaus Outpatient Clinic is detecting
patients from different areas of the city
who show parasitologic recurrences
after correct treatment within 28 days
of the routine clinical follow-up. This
observation is an indirect indicator of
the possible regional spread of P. vivax
CQ-resistant strains (unpub. data).
We believe our fi ndings are im-
portant and merit the attention of
local public health authorities. Con-
sidering the possibility of emerging
underestimated P. vivax CQ resis-
tance in Manaus, we feel it is essen-
tial to quickly clarify whether such
documented resistance can copromote
vivax malaria outbreaks in malaria-
endemic areas within the Amazon.
This study was supported by the Bra-
zilian Ministry of Health and the US Agen-
cy for International Development as part
of the scientifi c program of the Amazonian
Surveillance Network for Antimalarial
Drugs Resistance (RAVREDA).
Franklin Simoes
de Santana Filho,*
Ana Ruth de Lima Arcanjo,*
Yonne Melo Chehuan,*
Monica Regina Costa,*
Flor Ernestina Martinez-
Espinosa,*† Jose Luis Vieira,‡
Maria das Graças
Vale Barbosa,*§
Wilson Duarte Alecrim,*¶
and Maria das Graças Costa
Alecrim*§¶
*Foundation for Tropical Medicine of Ama-
zonas, Manaus, Amazonas, Brazil; †Foun-
dation for Research Support of Amazonas,
Manaus, Amazonas, Brazil; ‡Federal Uni-
versity of Pará, Belém, Pará, Brazil; §Ama-
zonas State University, Manaus, Amazonas,
Brazil; and ¶Nilton Lins University, Manaus,
Amazonas, Brazil
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1. Marlar-Than, Myat-Phone-Kyaw, Aye-
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