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. Shading indicates the 7 cluster areas where entomologic investigation was carried out. Data include cases reported through September 2008. The...

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.

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.

Virologic Investigation

Figure 2

Figure 2. Phylogenetic analysis of the chikungunya virus (CHIKV) envelope 1 (E1) gene. The maximum-likelihood method was used to construct the phylogenetic tree by using 1,002 nucleotides of the sequence of the E1...

The E1 gene of CHIKV from 85 imported and locally acquired infections was analyzed. Because there were several groups of similar sequences, the phylogenetic tree was constructed by using only 17 sequences that represented all imported as well as locally acquired strains at different time points. The tree also included 5 CHIKV from Sri Lanka sequenced at the EHI and 17 global sequences retrieved from the GenBank database (Figure 2). Phylogenetic analysis showed that all viruses reported in Singapore after January 2008, except 1, were related to the ECSA genotype. CHIKV isolated from the remaining infection was of Asian lineage and was imported from Indonesia (Figure 2). All ECSA-type viruses formed a distinct clade, together with isolates from India, Sri Lanka, Italy, and the Indian Ocean Islands (Figure 2). In the phylogenetic tree, the viruses isolated during the first outbreak in the Little India area clustered closely with those reported in India in 2006. One isolate from an imported case from Maldives also clustered within this group. In contrast, viruses isolated during the second local episode in the Teachers’ Estate in May 2008 and in all other areas from July 2008 grouped with those imported from Malaysia. Similarly, CHIKV isolated during the third local episode in the Farrer Road area in June 2008 clustered separately with isolates from Sri Lanka (Figure 2).

CHIKV isolated during the first local outbreak was wild-type (alanine) at aa residue 226 (A226) of the E1 gene, whereas, those detected during the second, third, and subsequent local episodes contained valine (A226V). Besides A226V, CHIKV isolated during the second local outbreak showed 2 synonymous mutations at nucleotide positions 300 (C300T) and 363 (A363G) of the E1 gene, which were not present in viruses involved in the first and third outbreaks. Of these isolates, C300T was unique to CHIKV strains imported from Malaysia. C300T and A363G were also found in all viruses detected in imported cases from Malaysia after June 2008. Similarly, CHIKV isolated in the third local episode was unique because it showed 2 synonymous mutations at nucleotide positions 105 (A105G), 1308 (C1308T) and a nonsynonymous mutation at nucleotide position 633 (A633C [K211N]) of the E1 gene, the combination of which was unique to CHIKV isolates from Sri Lanka. Therefore, we defined the combinations of C300T + A363G and A105G + A633C + C1308T as genetic signatures of isolates from Malaysia and Sri Lanka, respectively. These observations demonstrated that the first 3 episodes of chikungunya transmission in Singapore were most likely due to independent importations of distinct viruses from different geographic locations.

Entomologic Investigation

Aedes larval collection data showed that Ae. albopictus was the predominant species in all cluster areas, except Little India, an urban area where the first outbreak occurred (Table 2). In the Little India cluster, larval abundance index in the Clive Street area (2.14:1) was even higher than the generalized ratio for the whole cluster (1.77:1). The Clive Street area is a highly urbanized area and reported the highest number of chikungunya cases (n = 10) within the Little India cluster. This observation was further strengthened by adult mosquito surveillance, which yielded only Ae. aegypti in the Little India cluster. In contrast, Ae. albopictus (n = 164) was the only Aedes sp. caught in other cluster areas (Table 2). Adult Ae. albopictus mosquitoes from the Kranji Way and Bah Soon Pah Road areas were positive for CHIKV by RT-PCR. In Kranji Way, 7 (9.1%) of 77 female Ae. albopictus mosquitoes were positive for CHIKV, whereas 6 (13.5%) of 45 mosquitoes were positive in the Bah Soon Pah Road area. The E1 gene sequences of those 13 Ae. albopitcus-borne CHIKV were identical to sequences of strains imported from Malaysia. All mosquito-borne viruses possessed the A226V substitution.