Chikungunya, Singapore | CDC EID

Volume 15, Number 8–August 2009
Research
Entomologic and Virologic Investigation of Chikungunya, Singapore
Lee-Ching Ng, Li-Kiang Tan, Cheong-Huat Tan, Sharon S.Y. Tan, Hapuarachchige C. Hapuarachchi, Kwoon-Yong Pok, Yee-Ling Lai, Sai-Gek Lam-Phua, Göran Bucht, Raymond T.P. Lin, Yee-Sin Leo, Boon-Hian Tan, Hwi-Kwang Han, Peng-Lim S Ooi, Lyn James, and Seow-Poh Khoo
Author affiliations: National Environment Agency, Singapore (L.-C. Ng, L.-K. Tan, C.-H. Tan, S.S.Y. Tan, H.C. Hapuarachchi, K.-Y. Pok, Y.-L. Lai, S.-G. Lam-Phua, G. Bucht, S.-P. Khoo); Tan Tock Seng Hospital, Singapore (R.T.P. Lin, Y.-S. Leo); and Ministry of Health, Singapore (B.-H. Tan, H.-K. Han, P.-L. S. Ooi, L. James)

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Abstract
Local transmission of chikungunya, a debilitating mosquito-borne viral disease, was first reported in Singapore in January 2008. After 3 months of absence, locally acquired Chikungunya cases resurfaced in May 2008, causing an outbreak that resulted in a total of 231 cases by September 2008. The circulating viruses were related to East, Central, and South African genotypes that emerged in the Indian Ocean region in 2005. The first local outbreak was due to a wild-type virus (alanine at codon 226 of the envelope 1 gene) and occurred in an area where Aedes aegypti mosquitoes were the primary vector. Strains isolated during subsequent outbreaks showed alanine to valine substitution (A226V) and largely spread in areas predominated by Ae. albopictus mosquitoes. These findings led to a revision of the current vector control strategy in Singapore. This report highlights the use of entomologic and virologic data to assist in the control of chikungunya in disease-endemic areas.

Chikungunya is a mosquito-borne infectious disease caused by chikungunya virus (CHIKV), which belongs to the family Togaviridae and genus Alphavirus. CHIKV causes a nonfatal, self-limiting disease characterized by abrupt onset of high fever, severe arthralgia, or arthritis, often associated with skin rash.

CHIKV was first isolated during an outbreak in Tanganyika (now Tanzania) in 1952–1953 (1). The virus is believed to have originated in Africa and subsequently was introduced into many regions of Asia (2). The first CHIKV isolation in Asia was in Thailand in 1958 (3), followed by India in 1963 (4). A 2002–2003 serosurvey on 531 healthy young adults in Singapore showed a low prevalence (0.3%) of chikungunya antibodies (5). Although CHIKV has caused several large-scale epidemics in Asia and the Pacific region, it largely was neglected until its reemergence in the Indian Ocean Islands in early 2005 (6). Since then, CHIKV has caused outbreaks in India (7), Sri Lanka (8), Singapore (9), Malaysia (10), and Italy (11), focusing global attention on this newly emerging disease.

CHIKV is an enveloped, positive strand RNA virus with a genome of ≈11.8 kb (12). Phylogenetic analysis of the CHIKV genome has identified 3 lineages; West African, Asian and East, and Central and South African (ECSA) (13). The Asian lineage circulated in Asia until it was replaced by the ECSA type, which emerged during the 2005–2006 outbreaks in the Indian Ocean Islands and India (6).

Unlike in Africa, where the virus is maintained in a sylvatic cycle, chikungunya in Asia has been an urban disease, typically found in dengue-endemic areas and transmitted largely by Aedes aegypti mosquitoes. However, the predominant Aedes sp. in locations such as Réunion Island, where chikungunya emerged in 2005, was Ae. albopictus (14). The spread of chikungunya into rural areas during the later stages of outbreaks in India further confirmed the potential of Ae. albopictus mosquitoes in transmitting CHIKV (15). These changes were concurrent with the emergence of a strain having an alanine to valine substitution at codon 226 (A226V) of the envelope 1 (E1) gene in Réunion Island (16) and India (17). This mutation is known to increase the transmissibility of the virus by Ae. albopictus mosquitoes (18).

Because there is no licensed vaccine or specific drug therapy available to cure the illness, intervention relies upon vector control and minimizing mosquito-human contact. The first chikungunya outbreak in Singapore during January 2008 was successfully contained by combining aggressive vector control operations with active case detection and isolation of patients (9). On February 21, 2008, 24 days (2 incubation periods) after the last reported case, the outbreak was declared closed (9). After 3 months of no cases, local chikungunya cases resurfaced in May 2008 causing an outbreak that is yet to be resolved. This outbreak coincided with a rise in chikungunya incidence in Malaysia (10). In this report, we focus on the virologic and entomologic investigations carried out in Singapore, which assisted in the effort against the emergence of chikungunya in 2008.

Methods
Case Surveillance
Singapore initiated a chikungunya surveillance system in late 2006. The medical community was apprised by the Ministry of Health to look out for chikungunya cases among febrile patients, especially when associated with symptoms and signs (e.g., arthralgia, rash) suggestive of chikungunya (9). At the Environmental Health Institute (EHI), a national public health laboratory, an active laboratory-based surveillance was set up among a network of general practitioners. Confirmed cases were categorized as imported or local based on detailed travel history. Virologic analysis described in this study was performed on samples received by the EHI as part of the national public health surveillance program designed for chikungunya in Singapore.

Laboratory Diagnosis
Diagnosis of chikungunya was confirmed by detection of a fragment of the nonstructural protein 1 gene of CHIKV by a real-time reverse transcription–PCR (RT-PCR) protocol described previously (19). CHIKV RNA was extracted from serum by using QIAamp viral RNA mini kit (QIAGEN, Hilden, Germany), and the amplification was performed in a LightCycler 2.0 system by using LightCycler RNA Master SYBR Green I kit (Roche Diagnostics GMbH, Mannheim, Germany) according to manufacturers' instructions. All tests included 2 negative controls: a PCR control and a negative extraction control of DNAse/RNAse-free water. The positive control was RNA extracted from a CHIKV culture with a known PFU titer determined by plaque assay. The presence of CHIKV was determined based on the melting peaks (83.07°C–84.17°C) of the positive control amplifications.

Design of Specific Primers for Sequencing
All primers were essentially constructed towards strains of the Indian Ocean and Central African origin using Gene Runner 3.05 (Hastings Software, Inc., Hastings, NY, USA) and Primer Select 5.03 (DNASTAR Inc., Madison, WI, USA) software. The primer sequences used are listed in Table 1.

Sequencing of the E1 Gene
Complimentary DNA was synthesized as described in SuperScript III First-Strand synthesis system for RT-PCR (Invitrogen Corp., Carlsbad, CA , USA). All templates were purified with the QIAquick PCR purification kit (QIAGEN) before sequencing. Sequencing was performed using BigDye Terminator Cycle Sequencing kit, according to manufacturer's instructions (Applied Biosystems, Foster City, CA, USA).

Phylogenetic Analysis
The nucleotide sequences were assembled using the SeqMan II version 5.03 (DNASTAR) and aligned using Clustal W multiple alignment tool in the BioEdit Sequence Alignment Editor version 7.0.9.0 (20). The phylogenetic tree was inferred based on the 1,002-nt sequence of the E1 gene from aa residues 91 to 424, using the maximum-likelihood (ML) method as implemented in PAUP* version 4.0b10 (21). Bootstrapping to access the robustness of the ML tree topology was performed using the neighbor-joining method under the ML criterion based on 1,000 replicates.

Entomologic Surveillance
Figure 1

Figure 1. Geographic and temporal distribution of 123 indigenous chikungunya cases in Singapore...


Figure 2

Figure 2. Phylogenetic analysis of the chikungunya virus (CHIKV) envelope 1 (E1) gene...

Seven local transmission clusters representing major local outbreaks were selected for entomologic investigation: Little India (1°18´24´´N, 103°50´57´´E), Queen Street (1°17´ 52´´N, 103°51´05´´E), Teachers' Estate (1°23´0´´N, 103° 49´43´´E), Kranji (1°25´30´´N, 103°45´43´´E), Sungei Kadut (1°25´1´´N, 103°45´2´´E), Mandai Estate (1°24´31´´N, 103°45´34´´E), and Bah Soon Pah Road (1° 24´45´´N, 103°49´E) (Figure 1). These areas were classified naturally into urban (Little India and Queen Street), suburban (Teachers' Estate) and rural (Kranji, Sungei Kadut, Mandai Estate and Bah Soon Pah Road). The georeferenced Aedes larvae collection data from the chikungunya clusters were extracted from the Geographic Information System (ArcGIS) database of the National Environmental Agency, Singapore. The database, which is a part of the national vector control program, was assembled based on routine vector surveillance data obtained daily through area-wide inspection for mosquito breeding by ≈500 vector control officers.

The ultimate objective of this routine exercise was to identify as many active breeding places as possible in all residential and nonresidential premises within each cluster area. The collected larvae were separated into species based on morphologic identification before their numbers were counted. For this study, larval surveillance data were expressed as the larval abundance index, the ratio between the numbers of Ae. aegypti and Ae. albopictus larvae collected. For a single case, the number of larvae found within a 200-m radius of the case was used to calculate the larval abundance index, whereas the number of larvae found within the boundary of the cluster area was used in widespread clusters. Larval data collected 3 months before and after the first case reported from each cluster were used to calculate the index.

In each cluster area selected, adult mosquito surveillance was also conducted to determine the Aedes spp. composition and to confirm the presence of CHIKV in identified mosquitoes. Adult mosquitoes were collected using the sweep-net method, the Biogents (BG) Sentinel Trap (Biogents AG, Regensburg, Germany) or both. In each area, adult mosquito surveillance was conducted within 1-week from the beginning of the outbreaks, usually at the location from where the highest number of cases was reported. The survey was conducted once in all areas, except for Kranji Way, where it was carried out twice with a gap of 1 week between each collection. The number of locations surveyed ranged from 1 to 25 premises in each area, with higher number of premises in urban areas and lower numbers in rural areas in general. However, if a single case was reported from a cluster area, the adult mosquito survey was conducted in a few randomly selected premises within the neighboring area of the index case, even if it was an urban area. The sweep net method was performed in Little India and Teachers' Estate areas. The BG Sentinel traps were deployed in Queen Street, Sungei Kadut, Mandai Estate, and Bah Soon Pah Road areas. The number of traps deployed in each area ranged from 4 to 15 traps, with a trapping duration of 12 to 24 hours on each occasion. The sweep net method and BG Sentinel traps were used in Kranji Way. Adult Aedes mosquitoes were crushed individually in minimum essential medium before RT-PCR was performed as for serum samples. The isolated viruses were sequenced and analyzed as described above.

Results
Chikungunya Cases
From December 2006 through December 2007, a total of 1,375 samples were tested at the EHI for chikungunya; 10 of these cases were positive by PCR or immunoglobulin M testing. Epidemiologic investigation showed that all these cases were imported from India, Maldives, Sri Lanka, and Indonesia, which generally reflected the regional distribution of chikungunya during that time.

More than 7,000 samples from general practitioners, hospitals, and active case detection were tested from January through September 2008. In January 2008, the first locally acquired case of chikungunya was detected in the Little India area by a general practitioner involved in the chikungunya surveillance network (Figure 1). A total of 13 locally acquired chikungunya cases were confirmed by PCR before the outbreak was finally brought under control.

Between the first episode of transmission and May 2008, 6 cases imported from Sri Lanka (n = 2), Indonesia (n = 3), and Malaysia (n = 1) were diagnosed. By June, the number of imported cases increased, and the local scene remained relatively quiet with only 2 episodes of local transmission in Teachers' Estate area in late May (2 cases) and Farrer Road area in early June (1 case) (Figure 1). Both of these episodes were in suburban residential areas. Active case detection did not show any additional cases associated with those 2 episodes. Locally acquired cases occurred again in July 2008 coinciding with a rise in imported cases from Malaysia. By the end of September 2008, there was a cumulative total of 231 cases comprising 108 imported and 123 locally acquired infections. Of the imported cases, 92% (n = 99) had travel history to Malaysia, largely to the state of Johor, whereas the 123 local cases were distributed across 25 different locations.

After July 2008, transmission was more active in rural industrial and farming areas of Singapore, with the biggest clusters being in Kranji, Sungei Kadut, and Bah Soon Pah Road (Figure 1). Notably, during the active case surveillance using PCR, 2 viremic cases were found 1 day before the onset of clinical manifestations, with viral loads of 750 pfu/mL and 40 pfu/mL of blood, determined by using an external standard curve generated by plotting 10-fold serially diluted virus from a concentration of 108 pfu/mL, against respective crossing-point values of real-time PCRs.

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