Archives of Virology (2019) 164:2131–2135

https://doi.org/10.1007/s00705-019-04266-1

BRIEF REPORT

The analysis of clinical and laboratory data: a large outbreak

of dengue fever in Chaozhou, Guangdong province, China
Fen Lin1 · Hui Yang2 · Lin Zhang1 · Sen‑Hai Fang1 · Xiao‑Fen Zhan1 · Li‑Ye Yang1

Received: 12 November 2018 / Accepted: 13 February 2019 / Published online: 17 May 2019
© The Author(s) 2019

Abstract
A large-scale dengue fever (DF) outbreak occurred in Chaozhou, Guangdong province, China 2015. In our study, 528 dengue-
positive patient samples were collected for clinical and laboratory data analysis. 491 cases (93.0%) were primary dengue
fever (PDF), 22 cases (4.2%) were dengue hemorrhagic fever (DHF) and 15 cases (2.8%) were diagnosed with severe dengue
fever (SDF). All cases were infected by dengue virus serotype 2 (DENV-2), and the isolated strains belonged to cosmopolitan
genotype, which were grouped closely with Malaysia strains from 2010 to 2014. Moreover, the study showed that laboratory
indices have significantly difference in PDF, DHF and SDF patients. A comprehensive analysis of these data could assist and
guide the clinical diagnosis for DF, which has an important significance for the control of dengue virus infection.

Introduction all year round. Dengue epidemics in Chaozhou have been

reported sequentially since 1978, but there has been no
DF known as break borne fever, is a mosquito-borne infec- massive outbreak, and only sporadic cases were reported in
tious tropical disease caused by DENV and the infection the past decade [5, 6]. In 2015, a large-scale DF outbreak
in humans results in different clinical manifestation, rang- occurred with 1372 confirmed DENV infections. The aim
ing from mild symptoms such as fever to life-threatening of the present study was to describe the clinical and labo-
dengue hemorrhagic fever (DHF) and dengue shock syn- ratory data of hospitalized DF patients, and to determine
drome (DSS). It occurs primarily in the equatorial regions the source of the virus causing this outbreak. Moreover, our
of Africa, Latin Americas, South-East Asia, and the Western study analysis highlighted the difference of laboratory indi-
Pacific [1]. The incidence of DENV infection has increased ces in PDF, DHF and SDF patients.
30-fold since the 1960s [2]. World Health Organization
(WHO) estimates that approximately 3900 million people
in 128 countries are at risk of infection with DENV [1, 3]. Materials and methods
Guangdong was the most severe DF epidemic region in
mainland China [4]. Chaozhou is an eastern city of Guang- A total of 528 dengue cases were collected from Chaozhou
dong province, bordering Fujian province and the South Central Hospital during September to October 2015. These
China Sea, characterized by a humid subtropical climate, subjects included 260 males and 268 females with a mean
where the Aedes mosquitos are widely distributed and breed age of 44.17 years (range: 7 months to 95 years). DF diag-
nosis and classification of dengue severity was based on
Handling Editor: Tim Skern. “national diagnostic criteria and principle of management
of dengue fever (WS 216–2008)” [7] and the “Guangdong
Fen Lin and Hui Yang contributed to the work equally. Province guidelines for dengue fever diagnosis and treat-
ment (2014)”, patients were defined as DHF if they had
* Li‑Ye Yang
[email protected] fever, thrombocytopenia, bleeding, and evidence of plasma
leakage, SDF patients were defined by evidence of plasma
1
Central Laboratory, Chaozhou Central Hospital Affiliated leakage associated with shock or respiratory distress,
to Southern Medical University, Chaozhou 521021, severe bleeding, or severe organ involvement. Clinical
Guangdong, China
information (age, gender, sign, symptom) and laboratory
2
Department of Laboratory Medicine, School of Medicine, data (complete blood counts, blood chemistry, virology) of
Yangtze University, Jingzhou, Hubei, China

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2132 F. Lin et al.

DF patients were obtained by hospital information system Results

LIS. All laboratory-confirmed dengue infected sera were
stored at −80 °C for molecular analysis. Based on the data from Chaozhou Center for Diseases
Viral RNA was extracted directly from the patients’ Control (CDC), the first dengue case was confirmed in
serum using QIAamp Viral RNA Mini kit (Qiagen) fol- June 20, and all dengue cases were reported in Chaozhou
lowing the manufacturer’s instructions and stored at -70 until November 18, 2015. The majority of cases of DENV
°C for DENV typing and sequencing. infection occurred in September and October, with daily
The serotypes including DENV-1, DENV-2, DENV-3 newly emerged cases peaking in mid-September and more
and DENV-4 were identified by reverse transcription than 50 newly cases reported each day.
polymerase chain reaction (RT-PCR) using the primers 528 (male: 260, female: 268) clinically diagnosed
as described previously [8]. Briefly, a cDNA copy of a and laboratory-confirmed DF cases were sampled from
portion of the viral genome was produced in a reverse Chaozhou Central Hospital. Of these, 491 (93.0%) mani-
transcriptase reaction using SuperScript II Reverse Tran- fested PDF, 22 (4.2%) DHF, and 15 (2.8%) were SDF.
scriptase (Invitrogen, USA). Then, consensus primers The symptom composition was as follows: myalgia (362,
were used to amplify a 511-bp product with 2×Taq PCR 68.6%), headache (272, 51.5%), rash (141, 26.7%), bleed-
MasterMix kit (Aidlab, Beijing, China) in the first round ing (22, 4.2%), vomiting (41, 7.8%), diarrhea (38, 7.2%),
PCR. Nested PCR was performed with type-specific prim- ecchymosis (12, 2.3%). In addition, 25 (4.7%) cases
ers in the second round PCR. required blood transfusion. 9 of the 15 SDF patients were
40 serum samples were randomly selected for phyloge- elderly (>75 years) with underlying diseases-diabetes,
netic and evolutionary analysis from antigen positive serums. coronary heart disease, and pulmonary tuberculosis. One
cDNA was synthesized by reverse transcription with Super- case evolved to death and the other 14 cases eventually
Script II Reverse Transcriptase (Invitrogen, USA). Two pairs recovered and were discharged from hospital.
of primers were designed to amplify the full length E gene: Among the laboratory-confirmed cases, approximately
D2E1F:(5’-GAGC ​ CCT​ GAT​ TTT
​ CAT​ CTT
​ ACT-3’), D2E1R (433) 82% cases presented thrombocytopenia, (216)
(5’-GCA​CTC​TGA​TAA​CTA​TTG​TTC​CAT​-3’), D2E2F (5’- 40.9% exhibited leucopenia, (267) 50.6% and (229) 43.7%
GCG​AAG​AAA​CAG​GAT​GTT​GTTG-3) and D2E2R (5’- showed serum AST and LDH elevation, respectively.
TTG​AAG​GGG​ATT​CTG​GTT​GGA-3’). PCR products were Compared to the PDF, PLT in patients with SDF was sig-
confirmed by electrophoresis and purified using an Agarose nificantly lower than that of PDF patients. The labora-
Gel DNA Extraction kit (TaKaRa). Sequencing was then tory indices also showed that the levels of WBC, ALT,
performed by the Invitrogen Company (Guangzhou, China). AST, LDH, CK, CK-MB were significantly higher in SDF
The nucleotide sequences of the viruses were analyzed to patients than those of PDF and PHF patients (P<0.05)
determine the sequence identity using the Lasergene soft- (Table 1).
ware package (DNASTAR, Madison, WI). The E gene from the serum samples of 40 patients with
The multiple nucleotide sequence alignments were DENV-2 virus infection were amplified and sequenced. All
performed using CLUSTAL W software (http://www.ebi. the 40 DENV-2 isolates sequenced in this study revealed
ac.uk/clust​alw/). A total of 40 reference sequences of the 96.4–98.2% nucleotide sequence similarity among them.
DENV envelope gene with sampling dates were retrieved Phylogenetic analysis was performed using sequences of
from GenBank. These sequences represented different geo- DENV-2 E gene and 40 reference strains from global avail-
graphical areas. Phylogenetic analyses was performed using able in Genbank. As shown in Figure 1, the phylogenetic
MEGA 6.0 software. For the construction of phylogenetic tree demonstrated that all the strains from this study were
trees, Tajima-Nei model with the maximum likelihood (ML) clustered into cosmopolitan genotype, which were closely
method was used. The statistical robustness and reliability related to DENV-2 isolates previously circulating in south-
of the branching order within each phylogenetic tree were east Asia. Sequence alignment showed that the E protein of
evaluated by a bootstrap analysis with 1000 replications. D1/ newly isolated DENV-2 strain had 100% amino acid homol-
USA/Hawaii /1945 strain (AF425619) was used as out-group ogy to strains isolated from Malaysia in 2012, in contrast to
to root the tree. the strains observed in 2014 Guangdong epidemic.
Statistical analysis was conducted with SPSS 17.0 statisti- Compared to other strains isolated in Guangdong prov-
cal software. Chi-square test was used for counting material, ince of China, the E protein of the strains isolates in this
and t-test was used for measurement material, laboratory outbreak share above 99.8% amino acid homology to recent
data were compared between the groups using single fac- Guangdong DENV-2 isolates since 2001. This result dem-
tor ANOVA analysis, P<0.05 was considered statistically onstrates that the newly isolated DENV is not “complete
different. novel”, the virus was present for a long time.

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Clinical and molecular characteristics of dengue fever in Chaozhou 2133

Table 1  Comparsion of laboratory indices among SDF and PDF, can cause acute damage to these organs and raised AST
DHF groups levels may not only be due to liver involvement. One study
Index PDF DHF SDF in Thailand found DF may be an important cause of acute
liver failure in children [11]. Since the majority of our
WBC ­(109/L) 3.72±0.091 3.22±0.241 5.41±1.100★▲
patients were adults, additional studies in pediatric popula-
PLT (g/L) 81±2.307 39±6.584 19±5.037 ★
tions will be useful for confirming this observation
HCT (male) 0.385±0.003 0.397±0.007 0.380±0.022
Previously, the study by van Gorp et al. reported higher
HCT (female) 0.386±0.003 0.386±0.010 0.409±0.016
serum TG levels and lower serum CHO, HDL and LDL
CRP (mg/L) 16±1.248 11±2.325 22±1.170
levels in SDF disease, which suggested that these levels
ALT (U/L) 39±1.745 43±11.921 85±28.648★▲
could be used as prognostic markers to predict clinical
AST (U/L) 52±2.439 59±16.950 188±65.812★▲
outcome [12]. The reason for these lipoproteins changes in
TG (mmol/L) 1.46±0.052 1.67±0.291 1.82±0.360
dengue infection is unclear. In vitro studies of the patho-
CHO (mmol/L) 3.83±0.046 3.76±0.191 3.93±0.217
physiology of DENV and other flavivirus infections sug-
HDLC (mmol/L) 1.17±0.015 1.18±0.072 1.09±0.077
gest that lipids and lipoproteins may play a role in modi-
LDLC (mmol/L) 2.04±0.038 1.89±0.180 2.02±0.214
fying virus infectivity in target cells [13]. In our study,
CK (U/L) 262±26.165 128±20.057 800±248.577★▲
we found no association between CHO level and dengue
CK-MB (U/L) 16.89±0.378 16.48±1.466 22.79±3.22★▲
severity. In the two previous studies that used multivari-
LDH (U/L) 268±6.273 276±23.117 485±126.123★▲
able models for examining the relationship between CHO
★: severe dengue versus primary dengue, P<0.05 and SDF, HDL-C, LDL-C and SDF outcome, a link was
▲: severe dengue versus dengue hemorrhagic fever, P<0.05 observed in one of these studies, but not in the other [14,
15]. The relationship between development of SDF and
serum lipoproteins components such as CHO, LDL, HDL,
Discussion VLDL and TG in the pathogenesis of dengue infection,
and its underlying mechanism requires further studies.
Historically, DF has re-emerged in the Southern prov- Notably, DEVN-2 was the cause of the 2015 epidemic
inces of China. Chaozhou, belonging to Chaoshan region while previously only DEVN-1 had been reported in this
of Guangdong, it is well known as the hometown of Chi- region. Earlier studies have shown that secondary infec-
nese and Ceramic Capital of China. Chaozhou has fre- tion with different DENV serotypes increases the risk of
quent interactions with many East Asia countries that have developing SDF [16, 17]. In the case of a new circulating
DENV epidemics. In 2015, hot weather and rain prevailed serotype, individuals may be more susceptible to infec-
in Chaozhou, and mosquitoes multiplied wildly, which con- tion and may progess to a serious condition that requires
tributed to the spread of dengue virus. During September to further public health attention.
October, 1372 dengue cases were reported in this city, and Our molecular epidemiological study showed that the
528 confirmed DENV infections were collected from our DENV-2 isolated in Chaozhou belongs to the cosmopoli-
hospital for clinical and laboratory data analysis. tan genotype, clustering with those from Southeast Asia
Majority of the patients admitted to our hospital showed countries. These findings indicate that southern China
typical but mild dengue fever. There were 15 patients who is closely linked to Southeast Asia DENV transmis-
developed SDF, most of them were elderly (>75 years) and sion. In fact, there are frequent occurrences of DENV-1
had underlying diseases like diabetes. The reason why only and DENV-2 in Southeast Asian countries [4] whereas
few DENV infected individuals progressed to severe dengue the DENV-2 cosmopolitan genotype has also spread to
disease is poorly understood. the Indian subcontinent, the South Pacific islands, Latin
As expected, PLT in patients with SDF was significantly America, and Somalia [18]. Previous studies have shown
lower than that of PDF and DHF patients. Moreover, DHF/ that separate groups exist within the Cosmopolitan Geno-
DSS with prolong and profound shock and organ failure, type [19]. Future phylogenetic analyses based on entire
especially liver failure were commonly observed. This was genome sequences are needed to provide more robust data
confirmed in this study by higher degree of ALT, AST on the nature of Chinese endemicity.
elevation and CK, LDH values in DF patients, and was Interestingly, we observed that the strain from this out-
pronounced in SDF where it reached a 3~4-fold increase. break were closely related to the strains observed in Malay-
This observation was in accordance with many previous sia from 2010 and 2014, but not to the strains from China in
studies [9, 10]. Our data indicated that elevated AST and the same years. The phylogenetic analysis was supported by
ALT occurred in 50.6% and 29.9% of DF patients, respec- the finding that the first confirmed dengue infected patient in
tively. AST is not only expressed in liver but also in the Chaozhou had visited Malaysia early that year. It is possible,
heart, skeletal muscle, brain, and kidneys. DF infection therefore, that this year epidemic in Chaozhou may have

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2134 F. Lin et al.

Fig. 1  Phylogenetic tree of
DENV-2. The DENV-2 isolated
in this study were marked with
asterisk (*)

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Clinical and molecular characteristics of dengue fever in Chaozhou 2135

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