Molecular epidemiology of dengue viruses in three provinces of Lao PDR, 2006-2010

Abstract

Few data on dengue epidemiology are available for Lao PDR. Here, we provide information on the complexity of dengue epidemiology in the country, demonstrating dynamic circulation that varies over space and time, according to serotype. We recruited 1,912 consenting patients presenting with WHO dengue criteria at Mahosot Hospital, Vientiane (central Laos), between 2006 and 2010. Between 2008 and 2010, 1,413 patients with undifferentiated fever were also recruited at Luang Namtha (LNT) Provincial Hospital (northern Laos) and 555 at Salavan (SV) Provincial Hospital (southern Laos). We report significant variations in Dengue virus (DENV) circulation between the three sites. Peaks of DENV infection were observed in the rainy seasons, although 11% of confirmed cases in the provinces and 4.6% in the capital were detected during the dry and cool seasons (between December and February). Four DENV serotypes were detected among the 867 RT-PCR positive patients: 76.9% DENV-1, 9.6% DENV-2, 7.7% DENV-4 and 5.3% DENV-3. DENV-1 was the predominant serotype throughout the study except in LNT in 2008 and 2009 when it was DENV-2. Before July 2009, DENV-2 was not detected in SV and only rarely detected in Vientiane. DENV-3 and DENV-4 were commonly detected in Vientiane, before 2008 for DENV-4 and after 2009 for DENV-3. The phylogenetic analyses of DENV envelope sequences suggest concurrent multiple introductions of new strains as well as active DENV circulation throughout Laos and with neighboring countries. It is therefore of great importance to develop and strengthen a year-round nation-wide surveillance network in order to collect data that would allow anticipation of public health issues caused by the occurrence of large dengue outbreaks.

Fig 1. Map of the Lao PDR with Vientiane, Luang Namtha and Salavan study sites.

Fig 1 is adapted from [23].

Fig 2. Seasonality of recruited patients and confirmed dengue cases at the three sites.

Total: recruited patients. Conf dengue: confirmed dengue cases, Average Temp: monthly mean temperature.

Fig 3. Dengue virus serotypes distribution over time at the three sites.

Available sequences for each site are indicated with circles for complete CDS and with crosses for envelope on the top of the histograms, including sequences previously published from same locations [23].

Fig 4. Positions of the 10 clusters containing Lao strains on phylogenetic tree built with DENV-1 envelop sequences.

3,108 DENV-1 envelope sequences, obtained from the European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database in April 2014, were aligned with the DENV-1 sequences obtained in this study. A tree was built with this dataset using a maximum-likelihood (ML) method (General Time Reversible Model with invariant sites and a gamma distribution of rates across sites) and bootstrap resampling with 500 replicates. 81 Lao strains are dispersed in 10 clusters (bootstrap >70) shaded here in separate colors. The bootstrap values are displayed on the tree only for those clusters. The list of the strains grouped in the different clusters is provided in S6 Table. Two strains from this study do not belong to any of those clusters and are displayed in the tree. The Lao strain from 1996 is not shown on the figure and belongs to Asia 2 clade.

Fig 5. Temporal and geographical distribution of the strains from the ten DENV-1 clusters.

3,108 DENV-1 envelope sequences, obtained from the European Molecular Biology Laboratory (EMBL) Nucleotide Sequence Database in April 2014, were aligned with the DENV-1 Lao sequences obtained in this study. A tree was built (Fig 4) and ten clusters (bootstrap >70) containing the strains from this study were identified. In order to visualize the circulation of all the strains from these different clusters, we plotted each strain according to time of collection and location. Each of the 10 clusters is displayed in a different color: yellow for the strains from cluster # 1, light green for cluster #2, red for cluster #3, blue for cluster #4, dark green for cluster #5, medium green for cluster # 6, pink for cluster #7, grey for cluster #8, light orange for cluster #9 and dark orange for cluster #10. The list of all the strains included in each cluster is provided as S6 Table.