Dear Editor,
West Nile virus (WNV) is a mosquito-borne virus.
1
Infection with WNV generally causes a self-limiting disease, with central nervous
system symptoms in severe cases. As the discovery of encephalitis caused by WNV in
the United States in 1999, the virus has become a serious public health concern and
a global emerging infectious disease.
2, 3
However, most WNV-infected patients have only a transient fever with mild clinical
manifestations, especially during the early stages of infection, which can easily
be overlooked by clinicians and patients.
4
Typhoid fever is an infectious intestinal disease caused by the bacterium Salmonella
typhi. Severe cases may show numerous clinical manifestations, including rose-colored
spots on the abdomen and hepatosplenomegaly. As with other acute infectious diseases,
a high-grade fever may occur during the early stage of the disease; this can easily
lead to misdiagnoses of these acute infectious diseases.
5, 6
WNV was first isolated in mainland China from mosquito specimens in Jiashi County,
Kashi Region, southern Xinjiang, following local outbreaks of viral meningitis and
encephalitis caused by WNV.
7, 8
Although WNV is an emerging pathogen in China and East Asia,
9
there have been no recent reports of WNV encephalitis cases or epidemics in the region.
Determining the incidence of WNV infection has become an important public health concern.
During the investigation of WNV infections in the Kashi Region, Xinjiang, China, we
found a high incidence of typhoid fever in the area, with large numbers of outpatients
with fevers during the summer and autumn.
10
Accurate clinical diagnosis is difficult without laboratory-specific detection because
WNV infection and typhoid fever occur frequently during the summer and autumn, and
both pathogens can cause a fever during the initial stages of infection. Therefore,
in this study, serum was collected from suspected typhoid fever patients at Jiashi
County Hospital, Xinjiang, China, during the acute phase of infection. The presence
of WNV-specific immunoglobulin M (IgM) antibodies and neutralizing antibodies was
examined, and tests were performed for typhoid fever. The results indicate that there
were WNV-infected patients among those with typhoid fever.
Acute-phase serum was collected from 124 cases of suspected typhoid fever within 7
days at the Typhoid Fever Outpatient Department during the summer and autumn from
July to September 2011, at Jiashi County Hospital, Xinjiang, China. Aliquots of serum
were stored at –70 °C until use. The collected sera were examined for Salmonella infection
and WNV infection simultaneously.
7
Salmonella infection was detected by the Widal reaction (WR). Briefly, the agglutination
test was performed against the H (flagellum) and O (cell) antigens of Salmonella typhi
and ‘H’ antigens of Salmonella paratyphi A and Salmonella paratyphi B, as described
previously.
11
An acute serum sample was deemed positive when the antibody agglutination test reached
O≥1:80, H≥1:160. WNV- and Japanese encephalitis virus (JEV)-specific IgM antibodies
were detected by IgM-ELISAs (WNV IgM Capture DxSelect (Focus Diagnostics, Inc., Cypress,
CA, USA) and JEV IgM Capture ELISA Kit (Panbio, Sinnamon Park, Queensland, Australia).
7
For anti-WNV IgM antibody-positive patients, sera were collected during both the acute
and recovery phases, in which neutralizing antibodies against WNV and JEV were detected
by the 90% plaque-reduction neutralization test (PRNT90), respectively. Patients with
a ≥4-fold difference in the WNV-neutralizing antibody titers of the convalescent serum/acute
period serum samples were identified as positive for WNV infection.
12
The results of the WR indicated that 81% (101/124) of the acute-phase serum samples
were positive (that is, patients with typhoid fever), with WR serum geometric mean
titers (95% confidence interval) of O 125 (110–142) and H 170.2 (160–186). At the
same time, 34 samples were positive for anti-WNV IgM antibodies among 124 serum samples,
suggesting the presence of WNV infection in patients with typhoid fever. To understand
the levels of neutralizing antibodies against WNV in the acute and convalescent sera
of WNV IgM-seropositive patients, we retrospectively investigated the WNV IgM-seropositive
patients and collected the corresponding convalescent serum samples. In total, 21
convalescent serum samples were collected. In this study, paired acute-phase/convalescent
serum samples from 21 patients were tested for WNV-neutralizing antibodies using the
PRNT90. The results indicate that the WNV-neutralizing antibody titer during the acute
phase of WNV infection was between 1:10 and 1:20, whereas that in the convalescent
sera ranged from 1:40 to 1:320. From 21 paired serum samples, there were ≥4-fold differences
in the WNV-neutralizing antibody titer between acute and convalescent serum samples
from 11 patients. Those 11 patients who were positive for anti-WNV IgM antibodies
and had a ≥4-fold difference in WNV-neutralizing antibody titer between the convalescent/acute
serum samples were identified as being positive for WNV infection (Table 1). To eliminate
cross-neutralization reactions between JEV and WNV, the same samples were examined
for JEV-neutralizing antibodies at the same time. As we know, the existence of cross-reactions
to one virus is usually affected by the antibody level against another virus. The
results indicate that the JEV-neutralizing antibody results were negative (PRNT90<1:10),
which were hypothesized due to low antibody levels against WNV, although some of the
acute-phase serum samples were weakly positive for JEV-specific IgM antibodies. The
detection of WNV nucleic acid by PCR and cell culture was performed using acute-phase
serum samples from the 11 WNV-infected patients, all of which yielded negative results.
The 11 WNV-infected patients were between the ages of eight and 88 years old. Among
them, seven patients were ≥60 years old, three were 26–47 years old and one patient
was eight years old. Elderly individuals were more susceptible, consistent with the
observed age distribution of the WNV infection.
4, 13
Overall, five of the 11 WNV patients were found to be seropositive by WR (clinical
manifestations of fever, headache, sore throat, limb weakness and hepatosplenomegaly).
Laboratory testing indicated that these five patients were co-infected with Salmonella
typhi and WNV, whereas the remaining six patients were negative for Salmonella typhi
and had only the WNV infection (Table 1).
Jiashi County (in the Kashi Region of western China) is an economically underdeveloped
area. The natural environment is mainly desolate beach and desert with perennial drought;
ponds have been built to collect rainwater and snowmelt for the irrigation of farmland
and livelihood is suitable for mosquito-breeding areas and habitats for WNV-competent
mosquitoes (for example, Culex pipiens). Typhoid is a common infectious disease in
the area.
14
To facilitate the timely treatment of typhoid patients, the Jiashi County Public Health
Administrative Department set up a ‘Typhoid Fever Outpatient Service’ at the county
hospital to provide specialized treatment for patients with typhoid fever. Patients
with confirmed or suspected typhoid fever in the area during the summer were asked
to first visit the Typhoid Fever Outpatient Service. The hospital conducts epidemiological
investigations, provides medical examinations for patients, and collects acute-phase
serum samples from suspected cases for laboratory testing. To prevent the spread and
decrease the prevalence of typhoid fever, serum-positive patients are admitted to
the hospital for treatment. Patients with a fever but a negative Salmonella typhi
test have yet to be investigated. Patients with mild symptoms are suggested to undergo
home observation, whereas severe cases are admitted to the hospital for further observation
and treatment. The Typhoid Fever Outpatient Service of Jiashi County Hospital made
it possible to identify patients with subclinical WNV infections in the present study.
In this study, 124 acute-phase serum samples were collected from patients with suspected
typhoid fever at the Typhoid Fever Outpatient Service of Jiashi County Hospital. The
laboratory test results indicated that 27% (34/124) were positive for anti-WNV IgM
antibodies, of which 52% (11/21) showed a ≥4-fold difference in the WNV-neutralizing
antibody titers of convalescent/acute serum samples, suggesting a higher incidence
of WNV infection in patients with fever in Jiashi, Xinjiang, China.
Among the 11 patients with a WNV infection, 5 were positive for typhoid fever, indicating
that they were co-infected with Salmonella typhi and WNV (Table 1). The clinical manifestations
of Salmonella typhi and WNV co-infected patients were fever accompanied by a loss
of appetite, nausea, vomiting, abdominal distension, abdominal pain and other digestive
tract symptoms, whereas six patients with only a WNV infection exhibited either fever
or fever with generalized symptoms. On the basis of early clinical manifestations
of both groups, a WNV infection is accompanied by relatively simple clinical symptoms
of fever and discomfort, whereas patients co-infected with Salmonella typhi and WNV
show more complex symptoms, usually with gastrointestinal discomfort. This follow-up
survey indicates that regardless of the patients’ infection status (Salmonella typhi,
WNV or both), the patients were all alive two to three months after infection with
no aggravation of their illness and no occurrence of viral encephalitis, suggesting
that these patients, and especially those with WNV infection, showed only a transient
fever and a mild clinical presentation.
This study revealed that WNV infection occurred sporadically in Jiashi County, with
few cases clustered in the same village, suggesting a broad distribution for WNV in
the area. Although the clinical symptoms of WNV infection were relatively mild and
easily ignored, changes in the local climate (such as increased temperature and rainfall)
would increase the density of local mosquitos (the vector), resulting in a greater
prevalence and spread of WNV. Therefore, the disease spread and risk of epidemics
would be increased. The detection and monitoring of WNV infection, especially in cases
of unexplained fever, are important to accurately assess the burden of WNV infection
and to prevent epidemics.