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Table of Contents
SHORT RESEARCH COMMUNICATION
Year : 2018  |  Volume : 55  |  Issue : 2  |  Page : 168-171

Circulation of all dengue virus serotypes during dengue outbreak in Sandakan, Sabah, Malaysia (2016)


Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, Jalan UMS, Sabah, Malaysia

Date of Submission05-Oct-2017
Date of Acceptance02-Feb-2018
Date of Web Publication1-Oct-2018

Correspondence Address:
Tin Sabai Aung
Department of Pathobiology and Medical Diagnostics, Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, Jalan UMS, 88400, Kota Kinabalu, Sabah
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.242566

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  Abstract 


Keywords: Dengue virus, Malaysia, serotypes, cocirculation


How to cite this article:
Gintarong TJ, Emran A, Sherin A, Thein TT, Aung TS. Circulation of all dengue virus serotypes during dengue outbreak in Sandakan, Sabah, Malaysia (2016). J Vector Borne Dis 2018;55:168-71

How to cite this URL:
Gintarong TJ, Emran A, Sherin A, Thein TT, Aung TS. Circulation of all dengue virus serotypes during dengue outbreak in Sandakan, Sabah, Malaysia (2016). J Vector Borne Dis [serial online] 2018 [cited 2018 Oct 21];55:168-71. Available from: http://www.jvbd.org/text.asp?2018/55/2/168/242566



Dengue is a mosquito-borne viral infection caused by dengue virus (DENV). It consists of four serotypes, namely DENV 1, DENV 2, DENV 3 and DENV 4. It is a single-stranded RNA virus belonging to the genus Flavivirus of Flaviviridae family. Infection with one serotype does not protect against the other serotypes, and subsequent infections make people prone to dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS)[1]. Dengue is transmitted from one person to another by the bite of infected female Aedes aegypti and Ae. albopictus mosquitoes, which are prevalent throughout the world.

In Malaysia, the incidence[2] rate of dengue has increased from 69.6 per 100,000 population in the year 2011 to 328.3 per 100,000 in the year 2016. In the year 2015, about 111, 285 cases of dengue were reported which included 301 deaths. The number of cases (95, 693) were 16.3% higher[3] as compared to the previous year (2014). All four dengue serotypes (DENV 1–4) are prevalent in Malaysia with the changing predominant dengue virus serotypes[4].

Sandakan is a city situated at the east coast of Sabah State, East Malaysia in the Island of Borneo. Two dengue outbreaks[5] occurred in Sandakan from January to February, and July to September in 2016. The temperature in Sandakan varies from 76 to 90 °F (24.4 to 32.2 °C). Highest temperature is experienced during July to October. Heavy rainfall with an average total accumulation of 12.6 inches (320.04 mm) is observed from December to February. However, comparatively lower rainfall is observed during April to June with an average total accumulation of 3.8 inches (96.52 mm)[6].

This study is a cross sectional descriptive study, conducted during the second dengue outbreak in Sandakan to identify the circulating dengue virus serotypes and to know the age and sex distribution of dengue cases. The study period was between July and October 2016. Serum samples were collected from 81 clinically suspected dengue patient's attending the outpatient department of Hospital Duchess of Kent, Sandakan, Sabah, Malaysia and tested for dengue NS1 antigen by rapid test (Dengue Early Rapid Test, PanBio, US) at the hospital's pathology laboratory. In total 31 samples were NS1 antigen positive, which were collected for dengue serotyping by polymerase chain reaction (PCR) at the Department of Pathobiology and Medical Diagnostics of Faculty of Medicine and Health Sciences, Universiti Malaysia Sabah, Malaysia.

RNA extraction was done from 140 μ1 of serum using QIAamp RNA Blood Mini Kit (QIAGEN, Inc., Germany). One-step RT-PCR was performed using one-step RT-PCR kit (QIAGEN) followed by nested PCR on the RT-PCR product using HotStarTaq master mix kit (QIAGEN). Primers used were C-prM amplimers designed by Lanciotti et al[7] and redesigned by Chien et al[8] and Chew et al[9] [Table 1].
Table 1: Primer sequences for dengue and dengue serotypes with respective product sizes[9]

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RT-PCR amplification was done using the one-step RT-PCR (QIAGEN) kit reagents and 5 μ1 of extracted RNA, 25 pmol each of mD1 and D2 primers [Table 1] with the total reaction mixture of 25 μ1. The reaction included reverse transcription at 50°C for 30 min, initial denaturation and activation of Taq polymerase at 94°C for 15 min, 55°C for 15 sec and 72°C for 30 sec, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 56°C for 30 sec and extension at 72°C for 1 min. Final extension was at 72°C for 10 min and hold at 4°C. Nested PCR was done on above RT-PCR product using HotStarTaq master mix kit (QIAGEN). The mD1 and type specific primers rTS1, mTS2, TS3, and rTS4 [Table 1] were added respectively into four separate reaction mixture tubes with a total reaction mixture of 25 μ1. Amplification included polymerase activation at 95°C for 15 min followed by 25 cycles of denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec and extension at 72°C for 30 sec. Final extension was at 72°C for 1 min and hold at 4°C. Negative control was included for each and every PCR run. The PCR product obtained was subjected to 2% agarose gel electrophoresis. The gel was stained with ethidium bromide solution for 15 min. Gel documentation was done after rinsing the gel with distilled water. The amplicon sizes were 208, 119, 288, and 260 bp for DENV 1, DENV 2, DENV 3 and DENV 4 respectively [Figure 1].
Figure 1: Agarose gel electrophoresis of products obtained from dengue nested PCR—(a) DENV 1 (208 bp), DENV 3 (288 bp) and DENV 4 (260 bp) positive sample; (b) DENV 1 (208 bp) and DENV 4 (260 bp) positive sample; (c) DENV 2 (119 bp) and DENV 3 (288 bp) positive sample; (d) DENV 3 (288 bp) positive sample; and (e) DENV 1 (208 bp) positive sample; Lane 1—50 bp DNA ladder Figure 1]a, 100 bp DNA ladder [Figure 1]b, [Figure 1]c, [Figure 1]d and [Figure 1]e; Lanes 2, 3, 4, and 5—Dengue nested PCR products with dengue 511 bp products from one-step RT-PCR with dengue nested PCR product/products of the samples; and Lane 6—Negative control.

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Out of 31, dengue NS1 positive sera tested for dengue serotypes, five samples were PCR negative and 26 were positive. DENV1 positives were 4 (15.4%), DENV 3 pos itive was 1 (3.85%), DENV 4 positives were 5 (19.2%), DENV 1+2 positives were 6 (23.1%), DENV 1+4 positives were 6 (23.1%), DENV 2+3 positives were 3 (11.5%), and DENV 1+3+4 positive was 1 (3.85%). Agarose gel electrophoresis of PCR products from representative samples are shown in [Figure 1]. Overall dengue serotypes detected were 17 (39.53%), 9 (20.93%), 5 (11.63%), and 12 (27.91%) for DENV 1, 2, 3 and 4 respectively.

Age distribution of cases are shown in [Figure 2]. The youngest patient detected positive for PCR was 14-yr-old and the oldest was of 66 yr. Positive male patients were more than females (17 vs 8) with one unknown gender.
Figure 2: Age distribution of dengue PCR positive samples (n = 26).

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There were 10 (38.46%) patients infected with single DENV serotype, 15 (57.70%) with two serotypes and one with three serotypes (3.84%). According to Centre for Disease Control and Prevention, infection by one dengue serotype produces long-term immunity to that serotype but not to the other serotypes[10]. Therefore, in dengue endemic countries, people can become infected more than once by different serotypes over their lifetime[10]. In this study, DENV 1 was the most common serotype detected, which was followed by DENV 4, DENV 2 and DENV 3. In a similar study carried out in Negeri Sembilan state of Malaysia in 2012, Ahmad Nizal et al[4] have also reported DENV 1 (43.9%) as the commonest dengue serotype, though the prevalence of other serotypes varied with DENV 3 (31.1%) as 2nd-most prevalence serotype followed by DENV 2 (20%) and DENV 4 (5%)[4]. According to Ng et al[11], the predominant dengue virus serotype in Sabah state was DENV 4 in the year 2013. This indicates that the predominant serotype might have changed from DENV 4 to DENV 1 in the year 2016 in Sabah leading to dengue outbreak. The most affected age group in this study was 21-30 yr group, which is quite similar to the study of Ahmad-Nizal et al[4] wherein the young adults aging ≥15 yr were more affected with dengue. Only two dengue NS1 positive cases required hospital admission in the month of July for DHF and the rest 29 NS1 positive cases were treated as outpatients.

There was no mortality among NS1 positive cases in this study. There were five dengue NS1 positive samples that were negative in the PCR, which might be due to relatively late collection of samples. During this period, there was reduction in dengue RNA which codes for dengue NS1, while NS1 protein was still present. According to Alcon et al[12] NS1 protein could be detected in dengue patients with negative dengue RT-PCR. Singh et al[13] have also stated that PCR tests are more sensitive at Day 1 which gradually decrease by Day 5.

In conclusion, this study showed that in dengue endemic areas, cocirculation of all four DENV serotypes is possible during dengue outbreak, as most of the cases in this study were found to be coinfected with more than one DENV serotype. DENV 1 is the predominant serotype and young adults, specially males are prone to the disease in the study area.

Ethical approval

The study was approved on 15 June 2016, by Medical Research & Ethics Committee, Malaysia (NMRR-15-2378-26766 (IIR)).

Conflict of interest: None to declare.


  Acknowledgements Top


The research grant (Grant Code: SBK0216-SKK-2015) provided by the Universiti Malaysia Sabah, Sabah for this study is gratefully acknowledged. The authors are also thankful to the Director of Hospital Duchess of Kent, Sandakan, Sabah, Malaysia for allowing us to collect the samples for this study.



 
  References Top

1.
Dengue. Atlanta, USA: Centre for Disease Control and Prevention (CDC). [Updated June 19, 2014]. Available from: http://www.cdc.gov/dengue/epidemiology/ (Accessed on February 24, 2015).  Back to cited text no. 1
    
2.
Malaysia dengue incidence rate and case fatality rate for year 2000–2016. Available from: http://idengue.remotesensing.gov.my/idengue/content/statistik.pdf (page 5) (Accessed on January 14, 2018).  Back to cited text no. 2
    
3.
Emerging disease surveillance and report, dengue situation updates. Dengue situation updates. Philippines: World Health Organization, Western Pacific Region. Available from: http://www.wpro.who.int/emerging_diseases/DengueSituationUpdates (Accessed on May 29, 2016).  Back to cited text no. 3
    
4.
Ahmad-Nizal MG, Rozita H, Mazrura S, Zainudin MA, Hidayatulfathi O, Faridah MA, et al. Dengue infections and circulating serotypes in Negeri Sembilan. MJPJM 2012; 12(1): 21-30.  Back to cited text no. 4
    
5.
Jabatan Kesihatan Negeri Sabah. Buletin Epidemiologi Sabah 2016; 52:1-21. Available from: http://www.jknsabah.gov.my/v2/muatturun/lainlain/epidjkns/2016/Buletin%20 Epid%20Negeri%20Sabah%20ME52%20%282016%29.pdf (Accessed on February 14, 2017).  Back to cited text no. 5
    
6.
Average weather in Sandakan Malaysia. Available from: https://weatherspark.com/y/132128/Average-Weather-in-Sandakan-Malaysia-Year-Round (Accessed on January 9, 2018).  Back to cited text no. 6
    
7.
Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ and Vorndamt AV. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 1992; 30 (3): 545-51.  Back to cited text no. 7
    
8.
Chien LJ, Liao TL, Shu PY, Huang JH, Gubler DJ, and Chang GJJ. Development of real-time reverse transcriptase PCR assays to detect and serotype dengue viruses. J Clin Microbiol 2006; 44(4): 1295-304.  Back to cited text no. 8
    
9.
Chew MH, Rahman MM, Jelip J, Hassan MR and Isahak I. All Serotypes of dengue viruses circulating in Kuala Lumpur, Malaysia. Curr Res J Biol Sci 2012; 4(2): 229-34.  Back to cited text no. 9
    
10.
CDC DENV-1-4 real-time RT-PCR assay for detection and serotype identification of dengue virus. USA: Centre for Disease Control and Prevention (CDC). [Updated Dec 4, 2013]. Available from: http://www.cdc.gov/dengue/resources/rt_pcr/CDC-PackageInsert.pdf (Accessed on February 24, 2015).  Back to cited text no. 10
    
11.
Ng LC, Chem YK, Koo C, Mudin RN, Amin FM, Lee K-S, et al. Dengue outbreaks in Singapore and Malaysia caused by different viral strains. Am J Trop Med Hyg 2015; 92(6): 1150-5.  Back to cited text no. 11
    
12.
Alcon S, Talarmin A, Debruyne M, Falconar A, Deubel V, Flamand M. Enzyme-linked immunosorbent assay specific to dengue virus type 1 nonstructural protein NS1 reveals circulation of the antigen in the blood during the acute phase of disease in patients experiencing primary or secondary infections. J Clin Microbiol 2002; 40(2): 376-81.  Back to cited text no. 12
    
13.
Singh MP, Majumdar M, Singh G, Goyal K, Preet K, Sarwal A, et al. NS1 antigen as an early diagnostic marker in dengue: Report from India. Diagn Microbiol Infect Dis 2010; 68(1): 50-4.  Back to cited text no. 13
    


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