LETTERS

Author affiliations: Lao-Oxford-Mahosot Bartonella and Rickettsia in arthropods West African, and East/Central/South Hospital-Wellcome Trust Research Unit, from the Lao PDR and from Borneo, African (ECSA) genotypes (2). Malaysia. Comp Immunol Microbiol Mahosot Hospital, , (S. Dit- Infect Dis. 2012;35:51–7. http://dx.doi. In , the first report of trich, K. Phommasone, P. Panyanivong, org/10.1016/j.cimid.2011.10.003 CHIKV infection occurred in Bang- S.D. Blacksell, A. Dubot-Pérès, J. Caston- 7. Joshi N, Caputo GM, Weitekamp MR, kok in 1958 (3); later, sporadic cases guay-Vanier, P.N. Newton, D.H. Paris); Nuff- Karchmer AW. Infections in patients of chikungunya fever occurred in with diabetes mellitus. N Engl J Med. ield Department of Medicine, University of 1999;341:1906–12. http://dx.doi.org/10. many provinces during 1976–1995 Oxford, Oxford, UK (S. Dittrich, S.D. Black- 1056/NEJM199912163412507 (4). All of the CHIKV strains found sell, A. Dubot-Pérès, J. Castonguay-Vanier, 8. Greenwood BM, Bradley-Moore AM, in Thailand at that time were of the P.N. Newton, D.H. Paris); Faculty of Tropi- Bryceson AD, Palit A. Immunosuppres- Asian genotype. The virus has since sion in children with malaria. Lancet. cal Medicine, , , 1972;1:169–72. http://dx.doi.org/10.1016/ reemerged during 2008–2009 and Thailand (T. Anantatat, S.D. Blacksell, D.H. S0140-6736(72)90569-7 caused large outbreaks in southern Paris); Service Fraternel d’Entraide–Laos 9. Socolovschi C, Pages F, Ndiath MO, Thailand, affecting >50,000 persons Medical Projects, Luang Namtha, Laos (G. Ratmanov P, Raoult D. Rickettsia spe- (5). These outbreaks were attributed cies in African Anopheles mosquitoes. Slesak); Tropenklinik Paul-Lechler-Kran- PLoS ONE. 2012;7:e48254. http://dx.doi. to the ECSA genotype. We report an kenhaus, Tübingen, Germany (G. Slesak); org/10.1371/journal.pone.0048254 outbreak of CHIKV infection in the Aix Marseille University, Medical University, 10. Choi YJ, Lee EM, Park JM, Lee KM, northeastern province of Marseille, France (A. Dubot-Pérès); and Han SH, Kim JK, et al. Molecular de- in 2013. tection of various rickettsiae in mites The Geelong Hospital, Geelong, Victoria, (acari: trombiculidae) in southern is located Australia (J. Stenos) Jeolla Province, Korea. Microbiol Im- on the River on the foothills munol. 2007;51:307–12. http://dx.doi. DOI: http://dx.doi.org/10.3201/eid2008.131308 of the mountainous region of Laos to org/10.1111/j.1348-0421.2007.tb03912.x the north. An outbreak of suspected dengue cases was reported during the References Address for correspondence: Daniel H. Paris, rainy season during April–September Mahidol-Oxford Tropical Medicine Research 1. Phongmany S, Rolain JM, Phetsou- 2013(6). Beginning in September, Unit, Faculty of Tropical Medicine, Mahidol vanh R, Blacksell SD, Soukkhaseum V, however, hospital physicians noticed Rasachack B, et al. Rickettsial infec- University, 3rd Floor, 60th Anniversary that patients were reporting fever with tions and fever, Vientiane, Laos. Emerg Chalermprakiat Building, 420/6 Ratchawithi Infect Dis. 2006;12:256–62. http://dx.doi. moderate to severe joint pain result- Rd., Ratchathewi District, 10400-Bangkok, org/10.3201/eid1202.050900 ing in limitation of movement that Thailand; email: [email protected] 2. Mayxay M, Castonguay-Vanier J, Chansa- lasted for weeks. Serum samples were mouth V, Dubot- Pérés A, Paris DH, Phet- souvanh R, et al. Causes of non-malarial collected from 109 persons (hospital- fever in Laos: a prospective study. Lancet ized and outpatient) in October. Clini- Glob Health. 2013;1:e46–54. http://dx.doi. cal data showed that 38 (34.9%) had org/10.1016/S2214-109X(13)70008-1 moderate to severe joint pain; median 3. Nilsson K, Wallmenius K, Hartwig S, Norlander T, Pahlson C. Bell’s palsy duration of illness was 4 days (range and sudden deafness associated with 1–7). Median timing of sample col- Rickettsia spp. infection in Sweden. A lection from the onset of illness was 8 retrospective and prospective serologi- days (range 1–21). cal survey including PCR findings. Eur Chikungunya J Neurol. 2014;21:206–14. http://dx.doi. Outbreak in Bueng Samples were sent to Chulalong- org/10.1111/ene.12218 korn University Hospital in Bangkok 4. Jiang J, Maina AN, Knobel DL, Kan Province, to screen for mosquitoborne viruses. Cleaveland S, Laudisoit A, Wamburu K, The study protocol was approved by et al. Molecular detection of Rickettsia Thailand, 2013 felis and Candidatus Rickettsia asem- the Institutional Review Board of Ch- boensis in fleas from human habitats, To the Editor: Chikungunya fe- ulalongkorn University and consents Asembo, Kenya. Vector Borne Zoo- ver is a dengue-like syndrome charac- were waived because all samples were notic Dis. 2013;13:550–8. http://dx.doi. terized by acute fever, arthralgia, and stored as anonymous. Viral genomic org/10.1089/vbz.2012.1123 5. Maina AN, Knobel DL, Jiang J, Hal- maculopapular rash. The causative RNA was assayed by using semin- liday J, Feikin DR, Cleaveland S, et al. agent is chikungunya virus (CHIKV), ested reverse transcription PCR (RT- Rickettsia felis infection in febrile pa- which is transmitted by Aedes aegypti PCR) for CHIKV nucleic acid (7). tients, western Kenya, 2007–2010. Emerg and Aedes albopictus mosquitoes (1). Serum samples were tested for IgM Infect Dis. 2012;18:328–31. http://dx.doi. org/10.3201/eid1802.111372 Based on the genome and the viral en- against CHIKV by using SD BIO- 6. Kernif T, Socolovschi C, Wells K, velope E1 sequences, CHIKV is clas- LINE Chikungunya IgM Test (Stan- Lakim MB, Inthalad S, Slesak G, et al. sified into 3 genetic lineages: Asian, dard Diagnostics Inc., Kyonggi-do,

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South Korea) (8). In our study, the criteria for diagnosis of CHIKV infec- tion included the detection of CHIKV nucleic acid by RT-PCR or IgM anti- bodies against CHIKV. Of the 109 samples tested, 51 (46.8%) had evidence of CHIKV in- fection, as 25 (22.9%) were positive for CHIKV RNA by RT-PCR, and 32 (29.3%) were positive for IgM an- tibodies. Both CHIKV nucleic acid and IgM were found in 6 samples. To further characterize the phylogenetic relationships between the CHIKV strains in this outbreak with strains previously found in Thailand and neighboring regions, we performed full-length viral genomic sequencing from srains from 4 samples by primer walking (4). We subjected sequences to BLAST analysis (BLAST, http:// blast.ncbi.nlm.nih.gov), aligned using the BioEdit program v7.1.9 (http:// www.mbio.ncsu.edu/bioedit/bioedit. html), and performed sequence as- sembly using the DNASTAR v6.0 (DNA Star, Madison, WI, USA). We performed maximum likelihood phy- logenetic analysis of a set of 11,710 nt, including the sequences identified in this study (THA/Bueng Kan/BK46/ 2013, THA/Bueng Kan/BK57/2013, Figure. Phylogenetic analysis of whole genome nucleotide sequences of chikungunya virus THA/Bueng Kan/BK63/2013, THA/ (CHIKV) isolated during the 2013 outbreak in Bueng Kan Province, Thailand. The trees were generated by maximum-likelihood method, and the numbers along the branches indicate Bueng Kan/BK68/2013; accession nos. bootstrap values. Scale bar denotes nucleotide substitutions per site. Branch support and KJ579184–7) by using the MEGA pro- nodal confidence was assessed by using a general time reversible +I−4 nt substitution gram v6.0 (http://www.megasoftware. model with 1,000 bootstrap resampling. All sequences are labeled with GenBank accession net). Taking into consideration pos- number, country (3 letter code) and city of origin, strain name, and year of sampling. Bold text sible co-circulation of the Asian and indicates CHIKV isolates identified in this study. Black and white circles on the tree indicate E1-A226V mutant and nonmutant strains, respectively. ESCA, East/Central/South African. ECSA genotypes in Thailand, we also examined intergenotypic recom- bination using the Recombination was confirmed by analysis of the non- previous CHIKV outbreaks in south- Detection Program, v4.22 (http:// structural, structural, and E1 encoding ern Thailand (7), and those in the re- en.bio-soft.net/tree/RDP.html). regions (data not shown). The THA/ cent outbreak in Cambodia (10). This Phylogenetic analysis, compar- Bueng Kan strains all possessed an suggests the presence and continued ing the 4 strains of CHIKV identi- alanine to valine change at residue circulation of the E1-A226V virus in fied from Bueng Kan to 52 additional 226 (A226V) in the E1 gene, which Thailand since 2008. However, an- whole genomic sequences from Gen- was noted as one of the crucial sub- other mutation specific to the Indian Bank, revealed that the 4 strains are stitutions for increased transmissibil- Ocean isolates, D284E in the E1 gene, closely related and share >99.8% pair- ity by Aedes albopictus mosquitoes was not found in THA/Bueng Kan wise nucleotide identity (Figure). The reported for the Réunion Island iso- strains. No evidence of recombination Bueng Kan isolates grouped to the lates (9). This substitution was shared has been found in the THA/Bueng ECSA genotype within the recent In- among strains isolated in Thailand in Kan strains and other strains identified dian Ocean clade (9). This relationship 2008, which were responsible for the in Thailand thus far.

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Viral infections by mosquitoes References Address for correspondence: Yong Poovorawan, continue to challenge public health in Center of Excellence in Clinical Virology, 1 Weaver SC, Osorio JE, Livengood JA, Department of Pediatrics, Faculty of Medicine, Thailand. In evidence of this, we dem- Chen R, Stinchcomb DT. Chikungunya onstrated that CHIKV has become virus and prospects for a vaccine. Expert University, Bangkok 10330, established in northeastern Thailand. Rev Vaccines. 2012;11:1087–101. http:// Thailand; email: [email protected] Surveillance and clinical recognition dx.doi.org/10.1586/erv.12.84 2. Volk SM, Chen R, Tsetsarkin KA, will not prevent future outbreaks, but Adams AP, Garcia TI, Sall AA, et al. rather will assist in organizing an early Genome-scale phylogenetic analyses of response to outbreaks and thus mini- chikungunya virus reveal independent mize unnecessary illness and death. emergences of recent epidemics and vari- ous evolutionary rates. J Virol. 2010;84: 6497–504 http://dx.doi.org/10.1128/JVI. Acknowledgments 01603-09. We thank Padet Siriyasatien for pro- 3. Hammon WM, Rudnick A, Sather GE. Decline in Japanese viding us with laboratory test kits and pro- Viruses associated with epidemic hemor- rhagic fevers of the Philippines and Thai- Encephalitis, tocols. We thank the staff of Bueng Kan land. Science. 1960;131:1102–3. http:// Kushinagar District, Provincial Hospital for their contribution dx.doi.org/10.1126/science.131.3407.1102 in sample collection and the staff at the 4. Pongsiri P, Auksornkitti V, Theamboon- Uttar Pradesh, Center of Excellence in Clinical Virology, lers A, Luplertlop N, Rianthavorn P, Poovorawan Y. Entire genome charac- India Faculty of Medicine, Chulalongkorn Uni- terization of Chikungunya virus from the versity, for their hard work in performing 2008–2009 outbreaks in Thailand. Trop To the Editor: Kakkar et al. re- laboratory tests. Biomed. 2010;27:167–76. cently concluded that the low-quality 5. Suangto P, Uppapong T. Chikungunya surveillance data on acute encepha- This research is supported by Na- fever. Annual epidemiological surveil- litis syndrome (AES)/Japanese en- tional Research University Project; Office lance report 2009, Bureau of Epidemiol- ogy, Department of Disease Control, Min- cephalitis (JE) in Kushinagar Dis- of Higher Education Commission (WCU- istry of Public Health. [cited 2014 Mar trict, India, provide little evidence to 007-HR-57); Integrated Innovation Aca- 18]. http://www.boe.moph.go.th/Annual/ support development of prevention demic Center, Chulalongkorn University; Annual%202552/Main.html and control measures and to estimate Centenary Academic Development Project 6. Epidemiological Information Center, Communicable Disease Epidemiological the effect of interventions (1). Analy- (CU56-HR01); the Ratchadaphiseksom- Section, Bureau of Epidemiology. Re- sis of the surveillance data, however, phot Endowment Fund of Chulalongkorn ported Cases of Diseases under Surveil- does provide evidence supporting University (RES560530093); the Outstand- lance 506, 53rd week [cited 2014 Mar the effect of an ongoing intervention ing Professor of the Thailand Research 18]. http://203.157.15.4/wesr/file/y56/F56 533_1393.pdf (i.e., JE vaccination). Fund (DPG5480002); and King Chulalong- 7. Rianthavorn P, Prianantathavorn K, In accordance with the surveil- korn Memorial Hospital Thai Red Cross Wuttirattanakowit N, Theamboonlers A, lance protocol, cerebrospinal fluid Society; the Ratchadaphiseksomphot En- Poovorawan Y. An outbreak of chikun- and/or serum samples from AES pa- dowment Fund (S. Vongpunsawad and P. gunya in from 2008 to 2009 caused by African strains with tients admitted to the Baba Raghav Linsuwanon); MK Restaurant Company A226V mutation. Int J Infect Dis. Das Medical College (Gorakhpur, In- Limited; and The Siam Cement Pcl. 2010;3:e161–5. http://dx.doi.org/10.1016/ dia) are tested for IgM against JE at the j.ijid.2010.01. 001. field laboratory of National Institute of Nasamon Wanlapakorn, 8. Kosasih H, Widjaja S, Surya E, Hadiwijaya SH, Butarbutar DP, Jaya UA, Virology (NIV) at Gorakhpur (2). The Thanunrat Thongmee, et al. Evaluation of two IgM rapid im- samples are tested by using the ELISA Piyada Linsuwanon, munochromatographic tests during cir- developed by NIV Pune (Pune, India), Paiboon Chattakul, culation of Asian lineage Chikungunya which has a specificity of 85% (range Sompong Vongpunsawad, virus. Southeast Asian J Trop Med Public Health. 2012;43:55–61. 77%–95%) and sensitivity of 71% Sunchai Payungporn, 9. Schuffenecker I, Iteman I, Michault A, (range 71%–75%) (3). Patients with and Yong Poovorawan Murri S, Frangeul L, Vaney MC, et al. samples negative for JE are considered Author affiliations: Chulalongkorn Univer- Genome microevolution of chikungunya to have JE-negative AES. sity, Bangkok, Thailand (N. Wanlapakorn, viruses causing the Indian Ocean outbreak. PLoS Med. 2006;3:e263. http://dx.doi. We obtained the line-list of AES T. Thongmee, P. Linsuwanon, S. Vongp- org/10.1371/journal.pmed.0030263 patients from the NIV laboratory at unsawad, S. Payungporn, Y. Poovorawan); 10. Duong V, Andries AC, Ngan C, Sok T, Gorakhpur for 2008–2012. Analysis and Bueng Kan Provincial Hospital, Bueng Richner B, Asgari-Jirhandeh N, et al. Re- of the surveillance data indicated that Kan, Thailand (P. Chattakul) emergence of Chikungunya virus in Cam- bodia. Emerg Infect Dis. 2012;18:2066–9 251 (8.2%, range 4%–14.7%) of the DOI: http://dx.doi.org/10.3201/eid2008.140481 http://dx.doi.org/10.3201/eid1812.120471. 3,047 AES patients from Kushinagar

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