Reemergence of Chikungunya virus in Cambodia.
Disease transmission (Health aspects)
Roces, Maria Concepcion
Char, Meng Chuor
|Publication:||Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040|
|Issue:||Date: Dec, 2012 Source Volume: 18 Source Issue: 12|
Chikungunya virus (CHIKV; family Togaviridae; genus Alphavirus) is
an arthropod-borne virus transmitted to humans by Aedes spp. mosquitoes
(1). It is an enveloped, positive-sense, single-stranded RNA virus with
a genome of -11.8 kb (2). Three genotypes have been identified: Western
African, East Central South African (ECSA), and Asian (3).
First identified in Tanzania in the mid-1950s, CHIKV circulated in the 1960s in sub-Saharan Africa and several Asian countries (4,5). Reemergence of CHIKV (ECSA genotype) was reported in Democratic Republic of Congo in 1999-2000 (6,7) and in Kenya in 2004 (4). This genotype emerged in Comoros followed by Reunion Island, the Seychelles, Mauritius, Mayotte, and India in 2005 (the Indian Ocean outbreak); in Sri Lanka and Malaysia in 2006; in Singapore and Thailand in 2008; and in China in 2010 (8).
In Cambodia, CHIKV was first detected in 1961, probably the Asian genotype that was circulating in the region at that time (9). Since 2000, all blood specimens collected by the National Dengue Control Program, Ministry of Health Cambodia, during hospital-based surveillance of dengue and investigation of suspected dengue cases have been screened for IgM against CHIKV, dengue virus (DENV), Japanese encephalitis virus (JEV), and other arboviruses. Despite this testing, no evidence of acute or recent CHIKV infections has been found. To confirm CHIKV infection in samples positive by serologic testing, we conducted realtime reverse transcription PCR (RT-PCR) and complete genome sequencing of the samples. In 2011, we detected CHIKV ECSA genotype in patients in Cambodia and analyzed the phylogenetic origin of the strains.
We obtained samples from 3 sources: national dengue-like surveillance, an encephalitis study, and the outbreak investigation. From surveillance, during 2000-2011, an average of >700 paired serum samples were collected annually from patients admitted to sentinel hospitals for dengue-like syndrome (Battambang, Siem Reap, Kampong Cham, Takeo, Phnom Penh; Figure 1) in the National Dengue Control Program (10). From the encephalitis study, conducted July 2010 through July 2011, samples from 196 patients were collected as part of a surveillance study of central nervous system infections in Jayavarman VII hospital in Siem Reap (with written consent from patients or legal guardians and study approval by the National Ethics Committee in Cambodia). During the outbreak investigation, serum was collected during investigations by National Health authorities in Preah Vihear Province on August 16 (n = 9) and December 9-10, 2011 (n = 8), of outbreaks of suspected measles-like or dengue-like illnesses (Table).
All serum was tested at the Institut Pasteur in Cambodia. Acute-phase and/or convalescent-phase specimens were tested for IgM against flaviviruses (DENV, JEV, and Langat virus) and alphaviruses (CHIKV Ross C 347 strain and Sindbis virus). We used in-house IgM-capture ELISA as described by Vong et al. (11) with JEV, Langat, Sindbis, DENV, and CHIKV antigens; CHIKV was isolated by use of a mosquito cell line (clone C6/36 of Aedes albopictus cells) (11). Viral RNA was extracted from 140 [micro]L of serum by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany) according to manufacturer's recommendations. The presence of CHIKV RNA was determined by real-time RT-PCR selective for the E1 gene, according to a protocol adapted from Pastorino et al. (12) for a different Taq polymerase kit (SuperScript III Platinum One-Step Quantitative RT-PCR kit; Invitrogen, Carlsbad, CA, USA). Each series of tests included a negative control.
[FIGURE 1 OMITTED]
Among 19 samples positive for CHIKV by real-time RT-PCR, 8 were selected for complete genome sequencing. A total of 22 overlapped PCR products were obtained by using primers published by Schuffenecker et al. (13) and sent to Macrogen (Seoul, South Korea) for sequencing.
Sequence assembly and alignment were performed by using the CLC Main Workbench 5.5 package (CLC bio A/S, Aarhus, Denmark). The complete coding region (11,319 nt) of 8 CHIKV isolates from Cambodia were aligned with 64 reference strains available in GenBank. Phylogenetic analysis was performed by using the maximum-likelihood approach incorporating the GTR+r4 model of nucleotide substitution with a bootstrap resampling process of 1,000 replications.
During this reemergence of CHIKV in 2011, a total of 24 patients with fever, sometimes associated with acute arthritis or encephalitis (suggesting that many classical infections were not reported because encephalitis is a rare complication of chikungunya), had positive RT-PCR and/ or IgM-capture ELISA results for CHIKV. The first 2 cases were identified in May 2011 by the National Dengue Control Program in Battambang Province (eastern Cambodia) near the Thailand border (Figure 1). These 2 cases were in children hospitalized for suspected dengue. Subsequent cases were reported the same year in Siem Reap (2 cases, June and July), Kampong Thom (1 case, July), Kampong Cham (1 case, October), and Kandal (1 case, December) Provinces. Two other outbreaks were documented in villages in Preah Vihear Province (northern Cambodia) in August (9 cases) and December (8 cases) 2011 (Table). The sequence of outbreaks, in time and space, suggests that the virus was introduced to areas bordering Thailand and progressed through Cambodia, affecting city and villages along major northwest to southeast routes (Figure 1). Average patient age was 20 years (range 2-56 years); cases were distributed equally among male and female patients.
The alignment of the E1 gene sequence of CHIKV showed that all 8 strains carried the same amino acid substitution in the E1 protein (E1-A226V) as did the strains that were naturally selected by the mosquito vector a few months after the beginning of the Indian Ocean outbreak (13). Phylogenetic analysis of the complete genome sequence revealed that all viruses from Cambodia clustered with those isolated during the Indian Ocean outbreak and within the ECSA phylogenetic group (Figure 2). These isolates from Cambodia were closely related to the viruses isolated from southern Thailand during the 2008-2009 outbreak with bootstrap values <70 (data not shown) and to other isolates from the recent outbreaks in Asia (Singapore, Malaysia, Indonesia, and China). The pairwise nucleotide comparison of the complete coding region showed a high average percentage of similarity (>99.50%) with the recent isolates from Thailand, Malaysia, Singapore, and China. The identity between the strains from Cambodia ranged from 99.89% to 99.93% at the nucleotide level. Of note, the Cambodian strains can be separated into 2 groups supported by a bootstrap value of 100, suggesting that the viruses isolated in Battambang and Preah Vihea Provinces, which each border Thailand, could have been introduced separately, although we cannot exclude the possibility of introduction from other Asian countries as well.
As numbers of reported cases, numbers of provinces affected, and abundance of mosquito vectors (Ae. aegypti and Ae. albopictus) increase, the risk for local transmission will probably increase in the next few years, and levels of CHIKV infection could reach those of DENV infection. The ECSA genotype could then become endemic to Cambodia, which could face the same situation as in the 1960s, when a number of chikungunya cases were reported in Cambodia, although the 1960s epidemic did not last long (according to data available) and was not followed by continuous virus circulation leading to successive outbreaks. As CHIKV reemerges after 50 years of absence or low-level transmission, cocirculation with DENV might cause substantial challenges for public health, especially hospital overloading and increased needs for case management. This outbreak of CHIKV ECSA genotype spread rapidly in Cambodia over a short 7-month period. The outbreak should serve as a warning for health authorities to prepare, not only in Cambodia, but also in other areas where, to our knowledge, this genotype has not been reported, such as Vietnam and Lao People's Democratic Republic.
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We thank Mao Sok, Ky Santy, Yun Saophondara, Deng Ankirboss, and Eng Chaya, and the staff of the Technical Bureau of Preah Vihear Provincial Health Department, for providing samples, and we thank Ung Sam An and the staff from National Institute of Public Health Cambodia for their fruitful collaboration.
This work was supported by French Ministry of Research. The serosurveillance studies and outbreak response were funded by a World Health Organization grant to the Ministry of Health and Institut Pasteur in Cambodia in 2011. The World Health Organization acknowledges the Korea International Cooperation Agency and the World Bank for their generous support for these activities.
Dr Duong is a senior virologist at the Institut Pasteur in Cambodia. His research interests include arboviruses and emerging zoonotic viruses.
Author affiliations: Institut Pasteur in Cambodia, Phnom Penh, Cambodia (V. Duong, A.-C. Andries, S. Ong, V. Deubel, A. Tarantola, P. Buchy); Ministry of Health, Phnom Penh (C. Ngan, T. Sok, R. Huy, S. Ly, M.C. Char,); Jayavarman VII Hospital, Siem Reap, Cambodia (B. Richner, D. Laurent); World Health Organization, Phnom Penh (N. Asgari-Jirhandeh, S. Bjorge, M.C. Roces); and Preah Vihear Provincial Health Department, Preah Vihear, Cambodia (B. Hok)
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Address for correspondence: Philippe Buchy, Institut Pasteur in Cambodia, Virology Unit, No. 5, PO Box 983, Monivong Blvd, Phnom Penh, Cambodia; email: firstname.lastname@example.org
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Table. Characteristics of 24 patients with positive results for chikungunya virus, Cambodia, 2011 * Age, Patient Project y/sex no. V0603308 NDCP 2/M V0603310 NDCP 4/F V0705309 CNS 5/M V0719309 CNS 14/M V0719310 CNS 7/M V1005304 NDCP 15/M V1024306 CDC 11/F V1024307 CDC 11/F V1024308 CDC 28/F V1024309 CDC 30/F V1024310 CDC 17/M V1024311 CDC 2.5/F V1024312 CDC 13/F V1024313 CDC 10/F V1024314 CDC 31/F V1214306 NMC 49/M V1214307 NMC 56/M V1214308 NMC 29/M V1214309 NMC 39/F V1214310 NMC 17/F V1214311 NMC 27/M V1214312 NMC 16/M V1214333 NMC 30/F V1207304 NDCP 9/F Date of symptom Date Patient onset sampled no. V0603308 May 26 May 28 V0603310 May 26 May 28 V0705309 Jun 23 Jun 30 V0719309 Jul 9 Jul 17 V0719310 Jul 10 Jul 13 V1005304 Sep 30 Oct 4 V1024306 Aug 10 Aug 16 V1024307 Aug 15 Aug 16 V1024308 Aug 8 Aug 16 V1024309 Aug 8 Aug 16 V1024310 Aug 13 Aug 16 V1024311 Aug 16 Aug 16 V1024312 Aug 16 Aug 16 V1024313 Aug 14 Aug 16 V1024314 Aug 15 Aug 16 V1214306 Dec 9 Dec 9 V1214307 Dec 6 Dec 9 V1214308 Dec 5 Dec 9 V1214309 Dec 9 Dec 10 V1214310 Dec 9 Dec 10 V1214311 Dec 7 Dec 10 V1214312 Dec 9 Dec 10 V1214333 NA Dec 10 V1207304 Dec 1 Dec 6 Initial syndrome Patient Province reported no. V0603308 BTB Dengue ([dagger]) V0603310 BTB Dengue V0705309 SRP Encephalitis ([double dagger]) V0719309 SRP Encephalitis V0719310 KTH Encephalitis V1005304 KCH Dengue V1024306 PVH Measles ([section]) V1024307 PVH Measles V1024308 PVH Measles V1024309 PVH Measles V1024310 PVH Measles V1024311 PVH Measles V1024312 PVH Measles V1024313 PVH Measles V1024314 PVH Measles V1214306 PVH Dengue V1214307 PVH Dengue V1214308 PVH Dengue V1214309 PVH Dengue V1214310 PVH Dengue V1214311 PVH Dengue V1214312 PVH Dengue V1214333 PVH Dengue V1207304 KAN Dengue IgM-capture ELISA Patient 1st 2nd no. V0603308 Neg Pos V0603310 Neg Pos V0705309 Neg Pos V0719309 Pos Pos V0719310 Pos Pos V1005304 Neg Pos V1024306 Neg NA V1024307 Pos NA V1024308 Neg NA V1024309 Pos NA V1024310 Neg NA V1024311 Neg NA V1024312 Neg NA V1024313 Neg NA V1024314 Neg NA V1214306 Neg NA V1214307 Neg NA V1214308 Neg NA V1214309 Neg NA V1214310 Neg NA V1214311 Pos NA V1214312 Neg NA V1214333 Neg NA V1207304 Pos Pos RT-PCR Patient Serum SN no. V0603308 Pos Pos V0603310 Pos Pos V0705309 Neg Neg V0719309 Neg Neg V0719310 Pos Neg V1005304 Pos Pos V1024306 Pos Neg V1024307 Neg Neg V1024308 Pos Neg V1024309 Pos Pos V1024310 Pos Pos V1024311 Pos Pos V1024312 Pos Pos V1024313 Pos Pos V1024314 Pos Pos V1214306 Pos Neg V1214307 Pos Neg V1214308 Pos Pos V1214309 Pos Pos V1214310 Pos Pos V1214311 Neg Neg V1214312 Pos Pos V1214333 Pos Pos V1207304 Neg Neg Full-genome Patient sequencing no. V0603308 Partial V0603310 Complete V0705309 ND V0719309 ND V0719310 ND V1005304 ND V1024306 Complete V1024307 ND V1024308 Complete V1024309 ND V1024310 Complete V1024311 Complete V1024312 ND V1024313 Complete V1024314 Complete V1214306 ND V1214307 ND V1214308 ND V1214309 ND V1214310 ND V1214311 ND V1214312 ND V1214333 ND V1207304 ND * All patients had negative IgM-capture ELISA results for flaviviruses (dengue 1-4, Japanese encephalitis, Langat) and other alphaviruses (Sindbis). RT-PCR, real-time reverse transcription PCR; 1st, acute-phase serum sample; 2nd, convalescent-phase serum sample; SN, supernatant; NDCP: National Dengue Control Program, National Malaria Center, Ministry of Health; BTB, Battambang; Neg, negative; Pos, positive; CNS, central nervous system infection (encephalitis) study; SRP: Siem Reap; ND, not done; KTH: Kampong Thom; KCH: Kampong Cham; CDC, Communicable Disease Control Department, Ministry of Health, Preah Vihear outbreak investigation in August 2011; PVH: Preah Vihear; NA, not available; NMC, National Malaria Center, Ministry of Health, Preah Vihear outbreak investigation in December 2011; KAN, Kandal. ([dagger]) Dengue-like: fever with headache, retro-orbital pain, myalgia, joint pain, and/or hemorrhage. ([double dagger]) Encephalitis: fever with convulsion, vomiting, and/or hemiplegia. ([section])Measle-like: fever with rash, cough, or join pain.
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