Phommanivong et al. Tropical Medicine and Health _#####################_
DOI 10.1186/s41182-016-0020-y

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Tropical Medicine
and Health

RESEARCH

Open Access

Co-circulation of the dengue withchikungunya virus during the 2013
outbreak in the southern part of Lao PDR
Viengvaly Phommanivong1, Seiji Kanda1*, Takaki Shimono1, Pheophet Lamaningao1, Andrew Waleluma Darcy1,
Nobuyuki Mishima1, Bounthanh Phaytanavanh2 and Toshimasa Nishiyama1

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Abstract

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Background: During the 2013 outbreak, 4638 infection cases and 32 deaths have been recorded in the southernpart of Laos. In recent years, the chikungunya virus (CHIKV) emerged in the part of the country bordering
Cambodia. Dengue virus (DENV) and CHIKV are transmitted by common mosquito vectors. Both diseases have
similar clinical presentations; therefore, CHIKV infections might go undiagnosed in DENV-endemic areas. Thus, rapid
detection and accurate diagnosis are crucial for differentiating between the two viruses (DENV and CHIKV). In thisstudy, we demonstrated that CHIKV and two serotypes of DENV are circulating in Laos. In addition, we encounteredpatients that had been concurrently infected with multiple DENV serotypes or DENV and CHIKV.

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Methods: Plasma samples were collected from 40 patients with suspected DENV infections during an outbreak
between July and August 2013. The reverse transcription polymerase chain reaction was performed to detect thefour DENV serotypes and CHIKV using specific primers. Specifically, the complete envelope gene sequences of theviruses were sequenced and subjected to phylogenetic analysis.
Results: Forty acute-phase plasma samples from patients with suspected dengue infections were tested for thepresence of DENV viral RNA using molecular methods. Among the 40 samples, 14 samples were positive for DENV,
2 samples were positive for both viruses (DENV-2 and DENV-3), whereas DENV-1 and DENV-4 were not detectedduring the study period. We also encountered 10 samples that were positive for CHIKV. Of the 10 CHIKV-positive
samples, 3 samples were co-infected by DENV-2, and 2 samples were co-infected by DENV-3. Phylogenetic analysis
revealed that the 2013 dengue outbreak in Laos involved DENV-2 genotype Asian I and DENV-3 genotype II.
Moreover, the Laotian CHIKV strains grouped together with those isolated during outbreaks on the Indian Ocean
Islands within the East Central South African genotype.
Conclusions: These findings revealed that two serotypes (DENV-2 and DENV-3) and CHIKV were detected.
Furthermore, infection of multiple DENV serotypes and CHIKV was also observed in the 2013 dengue outbreak. Thisis the first documented evidence of co-infection with CHIKV and one of two DENV serotypes.
Keywords: Dengue virus, Chikungunya virus, RT-PCR, Co-infection, Outbreak, Phylogenetic analysis, Co-circulation* Correspondence: kandas@hirakata.kmu.ac.jp
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Department of Public Health, Kansai Medical University, 2-5-1, Shinmachi,
Hirakata-shi, Osaka 573-1010, Japan
Full list of author information is available at the end of the article© 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Phommanivong et al. Tropical Medicine and Health _#####################_

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Background
DF (dengue fever) is a mosquito-borne viral diseasecaused by the dengue virus (DENV), which belongs to
the Flavivirus genus, Flaviviridae family, and has been
categorized into four different serotypes (DENV-1 to
DENV-4). It commonly occurs in tropical and subtropical regions [1]. The World Health Organization (WHO2009) estimates that more than 50 million dengue infections occur yearly, resulting in half a million cases ofdengue hemorrhagic fever (DHF) and 22,000 deaths,
mainly among children. DENV is endemic in Southeast
Asia, the Pacific, and the Americas [2]. However, in recent years, the hyperendemic circulation of all four dengue serotypes has been detected in Southeast Asian
countries [3]. Other Flavivirus such as Japanese encephalitis (JE) is also endemic, occurring in Laos [4].
In Laos, dengue infections exhibit a cyclical pattern, i.e.,they occur approximately every 2–5 years [5]. DENV serotypes responsible for such infections in Laos were first
confirmed in 1994, and a case involving co-infection with
two DENV serotypes was reported [6]. Since then, larger
epidemics caused by all four serotypes have occurred [7,8]. DENV-1 has emerged in several provinces and caused
sporadic clinical cases in different areas of Laos between
2010 and 2011 [8]. The dominant circulating serotype
subsequently switched from DENV-1 to DENV-3, and
DENV-3 virus was the predominant DENV circulating in
Laos at the end of June 2012 [7]. However, while somesuspected cases of DENV infection were confirmed using
laboratory detection, other cases of dengue infection werediagnosed based on clinical symptoms [9].
Chikungunya has been identified in more than 60 countries in Asia, Africa, Europe, the Americas, the Indian
Ocean, and Pacific Islands [10]. In 2012, in a community
survey, 31 % (16 of 52) cases of chikungunya virus(CHIKV) infection was recorded in the southern part ofLaos [11]. The CHIKV is a member of the Alphavirus
genus, which belongs to the Togaviridae family. Infectionof CHIKV has similar clinical presentations with DENV
and co-circulates in overlapping geographic regions;hence, it can be underdiagnosed in areas where the
DENV-endemic occurs [10]. Few studies of the molecularepidemiology of serotypes/or genotypes of DENV andCHIKV were reported in Laos [7, 8, 11].
In the present study, the specimens were screened forthe presence of DENV and CHIKV using the reverse
transcription polymerase chain reaction (RT-PCR) during the 2013 outbreak of DF in southern Laos. Our
results highlight that CHIKV and two serotypes of
DENV are circulating in the southern part of Laos,
which shares borders with Cambodia and Thailand. In
addition, we encountered patients that had been concurrently co-infected with multiple DENV serotypes or
DENV and CHIKV.

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Methods
Study sites

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88Champasak province (CPS) (610 km south of Vientiane
capital) lies to the southwest in Laos (Fig. 1). It shares aborder with Thailand to the west, Salavan, Sekong, andAttapeu provinces to the north and east, and Cambodia
to the south. The Champasak hospital, a provincial hospital, is arranged in the third level of health services at
the national level where there is inadequate laboratory
facilities to diagnosis of infectious diseases.

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Clinical characterization of patients and sample collection

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Forty hospitalized patients and 3 additional cases (1 casefrom Oudomsay province and 2 cases from Vientiane
capital) were investigated during the outbreak of DF/
DHF from the end of July to the beginning of August
2013. Forty patients, aged 5 to 65 years presented withacute DENV infection at days 1–6 after the onset offever with two more of the following symptoms: headache, myalgia, arthralgia, skin rash, and hemorrhage. Allof these 40 patients were diagnosed with DENV infection. The history of their illness and complete bloodcounts: white blood cells (WBC), platelet counts (PLT),
and hematocrit (HCT), were obtained from a physician
at the Champasak hospital.
A total of 8–10 ml of whole blood samples are collected in tubes that contained EDTA as an anticoagulant.
Plasma samples were separated and preserved in an
RNA Shield™ reagent (Zymo Research) that could protect from RNA degradation. These specimens were thentransferred to the Laboratory of Public Health department, Kansai Medical University, Japan.

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Laboratory procedures

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The plasma samples were separated from the patients’
whole blood by centrifugation at 1000×g for 5 min at 4 °C. A total of 200–500 μl of plasma samples were directly
used for the viral RNA extraction and RT-PCR. The
remaining plasma specimens were kept at −20 °C prior
to testing and were stored at −80 °C until further use.

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RNA extraction and PCR

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Total RNA was extracted from patient’s plasma sample
using TRIzol® reagent (Invitrogen Inc.), according to the
manufacturer’s protocol with the following modifications. Then, the extracted RNA was used to synthesize
first-strand cDNA with random primers and reverse
transcriptase (ReverTra Ace®: Toyobo) for 1 h at 42 °C[12]. In the PCR analysis, the cDNA was used as a template and amplified using serotype-specific primers forserotypes D1 to D4 of DENV according to the methodof Lanciotti et al. [13] or a specific primer for CHIKV[14]. The general PCR conditions were as follows: 94 °C
for 2 min, 98 °C for 10 s, and 54–62 °C for 30 s for 35–

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Phommanivong et al. Tropical Medicine and Health _#####################_

Q3f1:1

Fig. 1 A map of the study area (Champasak Province, Laos)

40 cycles. After their amplification, the PCR products
were electrophoresed and visualized by staining 1.5 %140 agarose gel with ethidium bromide, and specific bands
141 were visualized with an ultraviolet transilluminator.
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142 Sequencing of the envelope (E) gene and E1 geneIn order to identify the genotypes of DENV and CHIKV,
we tried to analyze the sequences of the DENV-2, DENV145 3, and CHIKV isolates detected during the screening
146 process described above. PCR was performed by using
147 cDNA derived from the DENV-2-, DENV-3-, or CHIKV148 positive patients’ samples as a template and a primer pair
149 for each target region to amplify the complete envelope
150 (E) gene of DENV and E1 envelope glycoprotein gene of
151 CHIKV. The following sets of specific primers for DENV152 2 (Den2-911F 5′-TGACRG CTGTCGCTCCTTCA-3′,
153 Den2-2444R 5′-CARCTCACAAYGCAACCACTATC-3′,
154 1485 bp), DENV-3 (Den3-815F 5′-GCCCTTAGGCACCC
155 AGGGTT-3′, Den3-1752R 5′-CCCGCGAAAATGCTTG
156 TGC-3′, Den3-1398F 5′-CGCAAGGAG TCACGGCT
157 GAG-3′, Den3-2539R 5′-GCCTGCAATGGCTGTTGC
158 C-3′, 1479 bp) [7], and CHIKV (Chik E1Fseq1 5′-GCT
159 CCGCGTCCTTTACC-3′, Chik E1RSeq1 5′-ATGGCG
160 ACGCCCCCAAAGTC, 540 bp) were used for the PCR
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amplification. The PCR amplicons were directly sequenced using the BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems). The sequencing was
performed using the following conditions: 96 °C for 1 min
followed by 35 cycles of 96 °C for 10 s, 50 °C for 5 s, and
60 °C for 4 min. Sequence chromatograms for bothstrands were obtained using an ABI3730XL automated sequence analyzer (Applied Biosystems).

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Phylogenetic analysis of DENV and CHIKV

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The complete nucleotide sequences of the E gene of theLaotian DENV-2 (1485 bp) and DENV-3 (1479 bp)
strains, and the partial nucleotide sequences of the E1gene of CHIKV (540 bp) were aligned using ClustalW[15]. A phylogenetic tree was constructed using the
maximum likelihood (ML) method. The ML analysis
was performed using the General Time Reversible(GTR) model with a gamma distribution, and the proportion of invariable sites (I) was estimated by MEGA
v5.2 (http//www.megasoftware.net) [16]. The reliabilityof the analysis was evaluated in a bootstrap test with
10,000 replications. Representative strains of the DENV1 and DENV-3 serotypes were used as the outgrouptaxon for the DENV-3 and DENV-2 tree, respectively.

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Phommanivong et al. Tropical Medicine and Health _#####################_The sequence of the O’nyong-nyong virus, strain IPD
A234 (GenBank accession number: NC001512 and
186 AF192890), was used as an outgroup for the CHIKV tree
187 [17]. Sequences of all Laotian DENV and CHIKV are de188 posited in the DNA Data Bank of Japan (DDBJ) under
189 accession number LC147056-LC147057 for DENV-2,
190 LC147058-LC147061 for DENV-3, and LC147062191 LC147064 for CHIKV, respectively.
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192 Ethics statement
193 This study was approved (No. 276/NECHR) by the Na-tional Ethics Committee for Health Research, Ministryof Health, Lao PDR, and the Institutional Review Boardof Kansai Medical University (reference no.1430). In197 formed consent was obtained from each participant, as
198 well as parental permission for children involved in the
199 research.
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200 Results
201 Clinical features

All of the plasma samples were collected from patients
203 with suspected DENV infections that were treated at the
204 Champasak hospital during an outbreak of DF. Forty
205 subjects were enrolled (13 in the 5–15 years age group,
206 23 in the 16–45 years age group, and 4 in the 46–
207 65 years age group), and 22 (55 %) of them were female.
208 The median age of the patients was 20.50 years (range
209 5–65 ).
T1 210As shown in Table 1, all of the patients developed a
211 fever (days 1–6) and produced positive results in the
212 tourniquet test. Nearly all of the patients (97.5 %) experi213 enced headaches during their hospitalization. Muscle
214 pain was present in 87.5 % of patients, and joint pain
215 (70 %) and retro-orbital pain (72.5 %) were also com216 mon. Digestive problems were observed in 17 (42.5 %)217 patients. The patients’ other symptoms included chills
218 (17.5 %), skin rash (15 %), bleeding from the nose or
219 gums (5 %), petechiae (5 %), and bleeding that occurred
220 within 8 days of onset (2.5 %). Seventy-nine percent of
221 the patients exhibited lower white blood cell counts3
222 (leukopenia <5000/mm ). Thrombocytopenia (<100,000/
3
223 mm ) was observed in 34 % of cases, and 23 % of pa224 tients were presented with increases in their HCT levels
225 of >20 % compared with the baseline. There were no
226 deaths during the study period.
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Table 1 Clinical features of hospitalized patients (N = 40)
No. of patients

%

t1:1
t1:2
t1:3

Fever

40

100

t1:4

Headache39

97.5 t1:5

Retro-orbital pain (eye pain)29

72.5 t1:6

Digestive problems (nausea/vomiting)17

42.5 t1:7

Muscle pain (myalgia)

3587.5 t1:8

Join pain (arthralgia)

2870

Chills717.5 t1:10

Skin rash

6

15

t1:11

Petechiae

2

5

t1:12

Bleeding nose or gum

2

5

t1:13

Bleeding within 8 days

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2.5

t1:14

Tourniquet test

40

100

Leukopenia (<5000/mm3)3078.9 t1:17

Thrombocytopenia (<100,000/mm3)

1334.2 t1:18

Elevated hematocrit (>20 % increased)

923.1 t1:19

Symptoms and clinical tests
Symptoms

t1:9

t1:15

Clinical tests

t1:16

DENV-2 and DENV-3 co-infection was detected in 2
(5 %) samples. Moreover, CHIKV was also detected in
10 samples (25 %). Of the 10 CHIKV-positive cases, 3
samples were co-infected by DENV-2 and 3 samples coinfected by DENV-3, respectively. The sequences ofthese PCR products from all positive samples were also
confirmed by sequencing analysis.

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DNA sequencing analysis

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Serotypes/genotypes were determined by PCR and/or sequencing analysis using forward and reverse primers of
the complete envelope gene of DENV-2 (Den2-911F and
Den2-2444R, 1485 bp) and DENV-3 (Den3-815F and
Den3-1752R; Den3-1398F and Den3-2539R, 1479 bp),
and partial E1 gene of CHIKV (Chik E1Fseq1 and Chik
E1RSeq1, 540 bp). Entire gene sequences of two DENV2, four DENV-3, and partial gene sequences of three
CHIKV were then analyzed by phylogenetic analysis.
The results showed that the percentage of similar among
the two DENV-2 was 99 %, four DENV-3 ranged from90 to 97 %, CHIKV ranged from 62 to 67 % when thosecompared to each other and to strains representative ofthe different serotypes/genotypes available on GenBank.

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Phylogenetic analysis of DENV-2

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The complete E gene sequences of two distinct DENV
isolates (LAO13VTE582 and LAOCPS13C33) from the2013 outbreak were determined and compared with sequences of 37 representative DENV-2 strains of eachgenotype published in GenBank. Two strains of DENV-2
from Laos viruses were closely related each other and

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227 Screening of clinical samples by PCR

Detection and typing of the four DENV serotypes andCHIKV in plasma samples by PCR assay using specificprimers for DENV serotypes 1 to 4 and CHIKV.
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In the results of the 40 specimens, 7 (17.5 %) and 5
232 (12.5 %) were found to be positive for DENV-2 and233 DENV-3, respectively. However, DENV-1 and DENV-4
234 were not detected in the present study. Furthermore,

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belonged to genotype Asian I (Fig. 2). The genotype
Asian I consists of viruses mainly from Southeast Asia,
266 including Thailand, Cambodia, Vietnam, China, and
267 Myanmar. No Asian II genotype and Asian/America
268 genotype strains were found during dengue outbreak in
269 Laos 2013.
264265

270 Phylogenetic analysis of DENV-3

The DENV-3 strains isolated in the current study andpreviously isolated DENV-3 strains from other provincesof Laos (Laungprabang, Oudomsay, and Champasak)
274 and Vientiane were compared with sequences of 34 rep275 resentative DENV-3 strains of each genotype obtained
276 from GenBank database. Sequences of four strains for277 DENV-3 from Laos were grouped together within genoF3 278 type II (Fig. 3). The genotype II of DENV-3 is common
279 in Southeast Asian countries and clusters within the
280 viral strains from China, Myanmar, the Philippines,
281 Bangladesh, Thailand, Cambodia, and Vietnam. The add282 itional DENV-3 isolated in Vientiane in 2013 (LAOV283 TE13LN680428 and LAOVTE13LN680428) [7] belong
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to genotype III (Fig. 3). No genotype I and genotypes III 284
strains were found in the study period.
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Phylogenetic analysis of CHIKV

Analysis of the partial E1 gene sequences of 19 representative strains of each genotype of CHIKV published in
GenBank, including sequences of three representative
strains of CHIKV from Laos demonstrated that all
CHIKV strains from the present study were closely related to each other and other viruses from Cambodia(isolated in 2011) [18]. All study sequences clustered together with the causative CHIKV strains isolated during
an epidemic in the Indian Ocean Islands and belonged
to the East Central South African genotype (ECSA). TheECSA genotype consists of viral strains from Southeast
Asia and other countries, including Reunion Island and
Kenya (Fig. 4).

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Discussion
300
In 2013, Laos experienced a major DF/DHF outbreak 301
presented with nearly 50,000 dengue cases and 92 deaths 302

Fig. 2 A maximum likelihood tree constructed based on the complete envelope gene sequence of DENV-2. Each DENV-2 isolate is shown
together with its country of origin followed by two digits, which indicate the year in which it was isolated, and its GenBank accession number.
Two representative strains that were isolated in the present study are indicated by filled triangles. Bootstrap values of <60 % are not shown. The
scale bar indicates the mean number of nucleotide substitutions per site

Phommanivong et al. Tropical Medicine and Health _#####################_

f3:1
f3:2
f3:3
f3:4

Fig. 3 A maximum likelihood tree constructed based on the complete envelope gene sequence of DENV-3. Each DENV-3 isolate is shown
together with its country of origin followed by two digits, which indicate the year in which it was isolated and its GenBank accession number.
Four representative strains that were isolated in the present study are indicated by filled triangles. Bootstrap values of <60 % are not shown. The
scale bar indicates the mean number of nucleotide substitutions per site

(MOH, 2013). Because CHIKV infection has similar clinical features with DENV infection and co-circulates inoverlapping geographic distributions, therefore, CHIKV
306 may be misdiagnosed in areas where DENV endemic
307 occur [10]. During dengue fever outbreak, the Lao med308 ical doctor only diagnosed the dengue infections among309 patients. Consequently, we want to identify that these
310 patients are really infected by dengue virus or other in311 fectious disease. The present study showed that fever,
312 headache, retro-orbital pain, a positive tourniquet test,
313 and body and joint pain are common symptoms in pa314 tients that have been infected with DENV. Additionally,
315 our data also revealed that arthralgia (joint pain) and
316 skin rash were the most common symptoms found in
317 CHIKV-infected patients (data not shown), and similar
318 findings were reported by Ali et al.[19].
319
In our study, a molecular screening specific for both
320 DENV and CHIKV infections was performed on 40
321 acute-phase plasma samples collected from patients with
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suspected dengue infection in southern Laos during an
outbreak between July and August 2013. DENV was detected by PCR in 30 % and CHIKV in 12.5 % of samples.
Two samples (5 %) were co-infected by both viruses(DENV-2 and DENV-3), and five samples (12.5 %) wereco-infected by DENV and CHIKV, respectively. Although
the enrolled patients included five cases that were suffering from DHF, none of the patients died, and no cases of
DENV-1 or DENV-4 were found during the study
period. In our analysis of 40 samples, 52.5 % were foundto be dengue-negative by RT-PCR. These samples might
not have been collected during the acute phase of the infection (plasma viremia reduction) [20].
In Laos, the dominance serotype changes from year toyear since 2010. DENV-1 was dominant in 2010 and2011; DENV-3 was dominant in 2012 followed by
DENV-2, according to the National Dengue surveillance,
Lao PDR [8]. Our findings indicated that both DENV-2(17.5 %) and DENV-3 (12.5 %) were dominant serotypes

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f4:1
f4:2
f4:3
f4:4

Fig. 4 A maximum likelihood tree constructed based on the partial envelope (E1) gene sequence of CHIKV. Each CHIKV isolate is shown together
with its country of origin followed by two digits, which indicate the year in which it was isolated and its GenBank accession number. Three
representative strains that were isolated in the present study are indicated by filled triangles. Bootstrap values of <60 % are not shown. The scalebar indicates the mean number of nucleotide substitutions per site

circulating in southern Laos in 2013. In addition, otherresearchers reported that DENV-3 (94 %) was dominant,followed by DENV-2 (3 %) circulating virus in Vientiane
344 capital, whereas few cases of DENV-1 and DENV-4
345 (ranged from <1 to <6 %) have been recorded from May
346 2012 to December 2013 [7]. That corresponds with our
347 data; DENV-1 and DENV-4 were not detected. Concur348 rent infection by multiple DENV serotypes (DENV-2
349 and DENV-3) was identified during the 2013 dengue
350 outbreak in Laos. Furthermore, co-circulation of DENV351 2 (38.7 %) and DENV-3 (29.3 %) were also reported in
352 Thailand during dengue outbreak from 2004 to 2010353 [21]. These findings suggested that DENV serotype 2
354 and 3 may have remained viruses in the circulation in
355 these areas for a long time or they may have been intro356 duced from a neighboring country such as Thailand.
357 Geographically, Laos is located nearby Thailand com358 pared with other countries in Southeast Asia. With the359 increased movement and/or migration of infected people
360 within and between countries, hyperendemicity (the co361 circulation of multiple DENV serotypes) may be oc362 curred [22].
363
The first case of dual infection with DENV-1 and
364 DENV-2 was a resident in Vientiane, the capital of Laos,
365 who was presented with mild symptoms of dengue,
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which were not severe enough to require admission [6].
Since then, there have been no further reports of dual
DENV infections in Laos. According to the data obtained in the present study, we also found that the coinfected patients were more likely to present the DHF
including, fever, digestive trouble, skin rash, a positive
tourniquet test, leukopenia, and bleeding; these patientsneeded admission to hospital during their illness.
We determined the genotypes of the isolated DENV-2
and DENV-3 viruses via phylogenetic analyses of theircomplete E gene sequences. DENV-2 is categorized into
five genotypes: cosmopolitan, Asian-I, Asian-II, AsianAmerican, and American [23].
Based on complete E gene sequences, DENV-2 hasbeen divided into five genotypes: Cosmopolitan, Asian-I,
Asian-II, Asian-American, and American [23]. The Laotian DENV-2 were collected in the 2013 outbreak fromdifferent localities in Laos (610 km South (Champasak
province)–Central (Vientiane capital)). Sequences ofthese two viruses strains of DENV-2 were closely related
within genotype Asian I (Fig. 2). The genotype Asian I
of DENV-2 isolates from Laos in 21010 and 2013grouped together with viruses from Southeast Asian
countries, including Cambodia (2009), Thailand (2010),
Vietnam (2010 and 2011), Myanmar (2010), China

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Phommanivong et al. Tropical Medicine and Health _#####################_(2009), Southeast Asia (2010), and Laos (2010) [24, 25].
The genotype Asian I found in the current study and393 those from Southeast Asian countries formed a mono394 phyletic relationship with very high support values
395 greater than 98 % are shown. We suggested that the
396 genotype Asian I of DENV-2 has remained in dominant
397 circulation in Laos for a long time since 2010 until an
398 outbreak in 2013. The genotype Asian I of DENV-2 is399 also the predominant genotype circulating in many parts
400 of Southeast Asia, except Malaysia, Singapore, Indonesia,
401 and the Philippines [23].
402
Among the five genotypes of DENV-3 (I–V) [26], se403 quences of DENV-3 strains from the 2013 outbreak, to404 gether with other Laotian sequences collected from
405 Laungprabang, Oudomsay, and Champasak provinces
406 and Vientiane capital were grouped into the same clus407 ter within genotype II (Fig. 3). The Laotian DENV-3
408 genotype II isolates were most closely related to those409 isolated
from China (2013), Myanmar (2009),
410 Bangladesh (2008), the Philippines (2010), Thailand
411 (2010), Cambodia (2008), and Vietnam (2010) [25, 27].
412 All of the Laotian DENV-3 genotype II viruses obtained
413 in this study and sequences from other Southeast Asian
414 countries formed a monophyletic relationship with very
415 high values bootstrap support (>98 %). We suggested
416 that they had a single origin and have been circulating in
417 Lao PDR for a long time. Two different genotypes of
418 DENV-3 (genotype II and III) have been reported to419 have co-circulated in Laos in 2013 [7]. Even though two
420 studies have been implemented in the same year, the421 findings are not the same. Although our sample size is422 small, the analysis presented in this study suggested that
423 DENV-3 genotype II is circulating in the southern parts
424 of Laos and has also invaded other parts of the country.
425 Moreover, DENV-3 genotype II is the dominant circulat426 ing genotype in many countries in Southeast Asia [25].
427
Despite the small number of reported cases at the Na428 tional dengue surveillance in the Lao PDR, and the fact
429 that our study could only identify that two (5 %) cases of430 concurrent co-infection of DENV serotypes 2 and 3 were
431 observed, Lardo et al. reported that concurrent infec432 tions of dengue viruses 2 and 3 have been proposed as
433 one of contributing factors to severe dengue [28]. In the
434 present study, it is difficult to conclude that a co435 infected patient with two serotypes (i.e., DENV-2 and436 DENV-3) became afflicted with a more severe form of
437 dengue (DHF/DSS) because of only two cases were ex438 perienced. Moreover, we did not have enough informa439 tion about their clinical symptoms during hospital
440 admission. In addition, the relationship between concur441 rent infections and severe forms of dengue (DHF/DSS)
442 requires further study.
443
On the other hand, the current chikungunya epidemic
444 in Southeast Asia is being driven by the appearance of a391
392

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strain of CHIKV that originated in Africa [29] andspread to Asian countries such as Cambodia [18] andThailand [30]. At present, CHIKV is known to be circulating in southern Laos [11] and is currently spreading
to other regions of the country. During the 2013 outbreak of DENV in Laos examined in this study, we also
found patients that had been infected with CHIKV. In
fact, CHIKV-positive patients accounted for 25 % (10/40) of patients and 12.5 % (5/40) of the patients wereco-infected with DENV-2 or DENV-3. Other studies
have already recorded a high proportion of double infected cases with CHIKV and DENV (29 % from New
Delhi, India, 12.4 % from West Bengal, India, and 37
cases from Gabon) [31, 32]. Detection of double infection of CHIKV and DENV in this study demonstrated
the probability that many chikungunya cases may go
misdiagnosed in areas where two viruses coexist [10]. In
Laos, a diagnosis of dengue and chikungunya infection
was based on patient’s clinical symptoms and in general
samples were not checked by serological test such as a
rapid test. In this study, we did not perform the virus
isolation from samples.
Phylogenetic analysis divided CHIKV isolates into
three distinct genotypes based on their geographic origins: the West African (WAf ) genotype, East/Central/
South African (ECSA) genotype, and Asian genotype[33]. Our findings demonstrated that the partial E1 genesequences of the Laotian CHIKV strains clustered together with homologous strains from Indian Ocean
CHIKV outbreaks within the ECSA genotype. All ofthese Laotian CHIKV strains were closely related to the
CHIIKV strains that caused outbreaks in Cambodia, but
not high bootstrap support values below 70 (Fig. 4) [18]and clustered together with other isolates from recent
outbreaks in Asian countries (Thailand, Myanmar,
China, Cambodia, Malaysia, Sri Lanka, and India) [18,30, 34]. A high degree of sequence similarity between
the Laotian and Cambodian strains and the fact that the
Cambodian CHIKV outbreak occurred in 2011 wheresharing borders with southern Laos and data from community survey [11], we suggested that CHIKV ECSA
genotype is still endemic or is continuously reintroduced
to the area and has invaded various regions of Laos.

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Conclusions
488
Dengue is still a prevalent mosquito-borne disease in 489
Laos. Molecular detection and serotyping of dengue and 490
chikungunya were carried out on acute-phase plasma 491
samples that were collected during the 2013 dengue 492
fever outbreak from Laos. Our data suggested that the 493
identification of concurrent infection with two serotypes 494(DENV-2 and DENV-3) and co-infections with CHIKV 495
and two DENV serotypes have been confirmed during 496
the 2013 outbreak. Furthermore, our study indicated 497

Phommanivong et al. Tropical Medicine and Health _#####################_

that the occurrence of DENV and CHIKV co-infections
occurred in areas where these two viruses co-circulated.
500 This is the first study to report on patients that had been
501 co-infected with CHIKV and one of two DENV serotypes
502 in Laos. These findings from our study will be helpful in
503 the mitigation of priority actions such as improving sur504 veillance and timely intervention to present and future
505 outbreak threats.
498
499

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515

Acknowledgements
We would like to thank all the individuals who participated in the study,
especially the Champasak hospital, Oudomsay hospital, and Mahosot
hospital laboratory staffs for their hard work and collaboration and Dr.
Sengthong Bilangkoun and Dr. Khampho Chaleuvong for their help and kind
support during this study. We are grateful to Phonepasong Ayé
Soukhathammavong and Jordan Donaldson for their kind assistance. We also
thank Yasuko Nishimura for her valuable secretary assistance. This research
was partially supported by the research grant D2 from Kansai Medical
University.

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519

Funding
This research was partially funded by the Kansai Medical University. The
funders had no role in the study design, data collection and analysis,
decision to publish, or preparation of the manuscript.

520 Availability of data and materials
521 Not applicable.

9.

10.

11.

12.

13.

14.

15.

16.

17.

18.

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Authors’ contributions
TN conceived the idea for the study. VP, TS, PL, AWD, and BP have been
involved in collecting data. VP and AWD performed the laboratory testing.
SK, TS, and PL provided the technical supervision. VP Analyzed and drafted
the manuscript. SK and PL revised the manuscript for significant intellectual
contribution. All authors read and approved the final manuscript.

19.
20.

21.

528 Competing interests
529 The authors declare that they have no competing interests.
22.

530 Author details
531 1Department of Public Health, Kansai Medical University, 2-5-1, Shinmachi,
Q2 532 Hirakata-shi, Osaka 573-1010, Japan. 2Champasak Provincial Health
533 Department, Ministry of Health, Champasak, Lao PDR.
534 Received: 8 March 2016 Accepted: 4 July 2016
535

23.

24.
25.

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