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MODEL
Travel Medicine and Infectious Disease (2014) xx, 1e5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevierhealth.com/journals/tmid
Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013 nculescu-Ga tej c,1, Sorin Dinu a,b,1, Ioana R. Pa Simin A. Florescu d,e, Corneliu P. Popescu d,e, Anca Sıˆrbu f, descu c, Leticia Franco g, Gabriela Opris‚an a, Daniela Ba Cornelia S. Ceianu c,* a Molecular Epidemiology Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independent‚ei, 050096 Bucharest, Romania b Genetics Department, Faculty of Biology, University of Bucharest, 1-3 Aleea Portocalelor, 060101 Bucharest, Romania c Vector-Borne Infections and Medical Entomology Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independent‚ei, 050096 Bucharest, Romania d Clinical Hospital of Infectious and Tropical Diseases “Dr. Victor Babes‚”, 281 S‚os. Mihai Bravu, 030303 Bucharest, Romania e University of Medicine “Carol Davila”, 37 Dionisie Lupu, 020021 Bucharest, Romania f National Institute of Public HealtheNational Centre for Surveillance and Control of Communicable Diseases, 1-3 Dr. Leonte Anastasievici, 050463, Bucharest, Romania g Arbovirus and Imported Viral Diseases, National Centre of Microbiology, Instituto de Salud Carlos III, Ctra. de Majadahonda-Pozuelo, km. 2,200, 28220 Majadahonda, Madrid, Spain
Received 30 September 2014; received in revised form 3 November 2014; accepted 4 November 2014
KEYWORDS Mosquito-borne infection; Dengue viruses; RT-PCR; DNA sequencing; Phylogenetic analysis
Summary Background: Dengue fever is the commonest arthropod-borne infection worldwide. In recent years, rapid growth in global air travel has resulted in a considerable increase in the incidence of imported cases. In Romania it is now the second most frequent cause for hospitalization (after malaria) in patients arriving from tropical regions. Methods: Serological and molecular diagnostics were applied to samples obtained between 2008 and 2013 from travelers with suspected dengue. Molecular typing was performed by RT-PCR followed by sequencing of the E-NS1 junction.
* Corresponding author. Tel.: þ40 213069243. E-mail address: [email protected] (C.S. Ceianu). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.tmaid.2014.11.001 1477-8939/ª 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
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S. Dinu et al. Results: Twelve of 37 suspected cases were confirmed and three remained probable. The infections were acquired in endemic regions in Asia, Africa and in Europe (Madeira Island). Dengue virus nucleic acid was detected and sequenced in nine cases. Phylogenetic analysis indicated that the viruses were of genotypes I and V of serotype 1, cosmopolitan genotype of serotype 2 and genotypes I and III of serotype 3. Conclusions: Romanian tourists traveling to dengue-endemic countries are at risk of acquiring dengue infection. Appropriate prevention measures prior to travel and upon return should be taken, particularly as the dengue secondary vector Aedes albopictus is now established in Bucharest. ª 2014 Elsevier Ltd. All rights reserved.
1. Introduction Dengue fever is the most widely distributed arthropodborne infection worldwide. According to World Health Organization 2.5 billion humans living in tropical and subtropical regions are at risk of acquiring dengue infection [1], with an estimated 390 million cases per year [2]. The dengue viruses (DENVs) comprise a distinct serocomplex of the family Flaviviridae in the genus Flavivirus [3,4]. There are four divergent serotypes (DENV-1 to DENV-4), based on genetic and antigenic properties. These display 63e68% amino acid sequence homology [5,6]. In addition, a new viral serotype (DENV-5) has been identified in a human outbreak in Malaysia in 2007; there is evidence that this circulates among macaques [7]. Serotypes 1e4 are further classified into genotypes according to nucleotide divergence of up to 6% in the envelope glycoprotein coding region. DENV-1 comprises five genotypes (IeV) with a broad distribution: Southeast Asia, China, East Africa (genotype I); Thailand (genotype II); Malaysia (genotype III); West Pacific islands and Australia (genotype IV); America, West Africa and Asia (genotype V) [8]. Six genotypes have been proposed for DENV-2: Asian I (Southeast Asia and East Asia); Asian II (Southeast Asia, East Asia, Oceania, Caribbean region and the Americas); Asian/American (Southeast Asia, Caribbean region and Americas); a cosmopolitan genotype (India, South-Central Asia, East Asia, East Africa, Middle East, Southeast Asia and Oceania); an American genotype (India, Caribbean region, South America, Central America, Oceania and Trinidad) and sylvatic genotype (West Africa and Malaysia) [9]. DENV-3 isolates are assigned to four genotypes: genotype I (Indonesia, Malaysia, Philippines and South Pacific islands), genotype II (Thailand, Vietnam and Bangladesh), genotype III (Sri Lanka, India, Africa and Samoa), and genotype IV (Puerto Rico, Latin and Central America and Tahiti) [10]. Phylogenetic analysis indicates the existence of four genotypes within the DENV-4 serotype: genotype I (Thailand, the Philippines, Sri Lanka and Japan), genotype II (Indonesia, Malaysia, Tahiti, the Caribbean and the Americas), genotype III (Thailand) and genotype IV (sylvatic strains from Malaysia) [10]. The virus is transmitted by mosquitoes of the genus Aedes, primarily Aedes aegypti and Ae. albopictus, and is maintained in urban cycles in humans and sylvatic cycles in non-human primates [1,11]. Spillover from the sylvatic cycle to the urban cycle has been documented [12,13]. The
infection can be asymptomatic or range from an undifferentiated acute febrile illness to “classic” dengue fever (DF) which, in some cases, can progress to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS) [14]. At present there are no specific treatments or commercially available vaccines [15]. One of the largest epidemics of dengue ever recorded occurred world-wide was in refugee camps in Greece (1927e1928), with at least 650,000 cases and 1,000 deaths. The vector was Ae. aegypti [16]. In recent years there has been a sharp rise in the number of imported DF in Europe, attributable to a rapid rise in international air travel and high rates of transmission in endemic countries [17]. The introduction of Ae. albopictus followed by rapid colonization has given rise to autochthonous cases in mainland France (2010, 2013) and Croatia (2010) [18e20]. In addition, more than 2,000 cases were recorded in Madeira (2012), an autonomous region of Portugal and therefore technically a part of Europe. In this case the vector was Ae. aegypti, a recent arrival in the archipelago [21,23]. The rapid dissemination and establishment of the Ae. albopictus in Southern Europe, and the intense increase in traveling fuel the introduction of dengue in non-endemic areas [24]. This species was detected in 2012 in Bucharest, Romania, and has remained present in subsequent years (Prioteasa et al., personal communication). Dengue is now included in the list of notifiable communicable diseases; laboratory diagnosis by the Romanian National Reference Centre for Vector-Borne Infections was implemented starting 2008. In the present study we present the molecular epidemiology of imported DF cases in Romania between 2008 and 2013.
2. Material and methods 2.1. Patients and laboratory diagnosis Sera from travelers with clinical suspicion of dengue fever after return from dengue-endemic regions were received by the National Reference Centre for Vector-Borne Infections (“Cantacuzino” N.I.R.D.M.I.) from Public Health Departments and from Clinical Hospital of Infectious and Tropical Diseases “Dr. Victor Babes‚” in Bucharest. The samples were tested for DENVs antibodies with IgM and IgG indirect ELISA (Euroimmun, Lu ¨beck, Germany). Paired sera were requested but convalescent serum was not available
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
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Molecular epidemiology of dengue fever cases e Romania for all patients. Sera taken in the acute phase of the disease (up to eight days from fever onset) were subjected to molecular analysis. A case was classified as probable when IgM was detected in serum obtained during the acute phase. Confirmed cases were defined by IgM and IgG, seroconversion for IgG in paired sera, and/or detection of DENVs nucleic acid in sera by RT-PCR and DNA sequencing.
2.2. Molecular typing Viral RNA was extracted from 140 mL of serum using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the instructions provided by the manufacturer. Reverse transcription and PCR were carried out using primers described earlier [25] that amplify a fragment located within the E and NS1 viral genomic regions junction. Serotype assignment was made based on the size of the DNA fragment obtained. The amplicons were gel purified (Wizard SV PCR and Gel Clean-Up System; Promega, Madison, WI) and sequenced with BigDye Terminator v3.1 on a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The DNA sequences were visually inspected and aligned with BioEdit version 7.0.5.3 [26]. Phylogenetic analysis was conducted using Mega 6 software [27], Neighbor-joining algorithm with 1,000 bootstrap replicates. Sequences obtained in this study were submitted to GenBank.
3. Results and discussion From 2008 to 2013, serum samples from 37 suspected patients returning from endemic countries were tested for the presence of IgM and IgG dengue-specific antibodies (Table 1). The patients declared a history of recent travel in Asia, Africa and Europe (Madeira). One patient, a cruise ship worker, took a voyage in Southeast Asia and islands in the Indian Ocean. For six DF suspected patients we did not receive any information regarding their traveling history. Twelve DF cases were confirmed in patients with recent travel to India, Indonesia, Vietnam-Thailand, Angola, Kenya and Portugal (Madeira). Three more cases, returning from
Table 1 Summary of the dengue fever suspected cases investigated by The Romanian National Reference Centre for Vector-Borne Infections between 2008 and 2013. Year
Number of suspected cases tested
2008 2009 2010 2011
2 0 1 7
2012
11
2013
16
Total
37
Number of confirmed cases
Number of probable cases
1 (India) 0 0 4 (Indonesia, VietnameThailand, Angola, Kenya) 3 (VietnameThailand, Madeira Island, India) 4 (Indonesia-3, VietnameThailand) 12
0 0 0 0
1 (Vietname Thailand) 2 (Sri Lanka, India) 3
3 Vietnam-Thailand, India and Sri Lanka, were classified as probable (Table 1). Nine DNA sequences were obtained from patients returning from India, Indonesia, Vietnam-Thailand and Portugal (Madeira). The isolates were of DENV-1 (genotypes I and V), DENV-2 (cosmopolitan genotype) and DENV-3 (genotypes I and III) (Table 2). In half of the confirmed and probable cases, the infected persons arrived during the Ae. albopictus season in Romania, months May to October.
3.1. DENV-1 phylogenetic analysis Five out of the nine analyzed sequences cluster into DENV1. The first sequence (GenBank acc. no. HE565699), belonging to genotype I, was obtained from the serum of a patient working on a cruise ship in 2011 who declared a history of travel in Southeast Asia (Vietnam and Thailand) and islands of Indian Ocean and was repatriated to Romania from Maldives after the illness onset. The phylogenetic analysis indicates 100% identity between our sequence and sequences obtained in recent years in Southeast Asia (Cambodia, Malaysia, Thailand, Vietnam) where genotype I is circulating [8]. A similar strain was involved in a major outbreak in Colombo, Sri Lanka (2012) after its introduction in 2009 [28,29]. Phylogeographic analysis performed by Ocwieja et al. indicates that the origins of this strain might be the virus causing severe dengue in Thailand (2001) and which later spread to neighboring Cambodia and Vietnam [29]. Two other sequences clustering into genotype I of DENV-1 were obtained from patients who traveled to Indonesia in 2013 (GenBank acc. no. HG918038-9). These were remarkably similar to sequences obtained in 2004, 2011 and 2013 in China, as well as Singapore (2003) and Malaysia (2005). Two sequences fell into genotype V of DENV-1. The first was obtained from a patient returning from India in 2008 (GenBank acc. no. FR874940) and was identical to others obtained from viremic subjects in that country in 2010 and 2011. Similar sequences had been obtained from India (1982 and 2009) and Sri Lanka (1997 and 2004). It is noteworthy that the first autochthonous cases of DF in Croatia were due to DENV-1 in the same cluster of
Table 2
Results of molecular typing.
Serotype
Genotype
Year
History of traveling
GenBank accession number
DENV-1
I
2011
Vietname Thailand Indonesia Indonesia India Madeira Island India Indonesia Vietname Thailand Vietname Thailand
HE565699
2013
DENV-2 DENV-3
V
2008 2012
Cosmopolitan I III
2012 2011 2012 2013
HG918038 HG918039 FR874940 HF955512 HF955511 FR822184 HE800554 HG918040
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
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isolates [30]. The second sequence (GenBank acc. no. HF955512) falling into genotype V of DEN-1 was amplified from the serum of a patient traveling to Madeira in 2012 at the time of the outbreak mentioned above [22]. Our sequence is 99.94% identical with a sequence derived from an isolate obtained in 2008 in Colombia, South America. As shown earlier, the isolate from Madeira probably originated in Brazil, Columbia or Venezuela [31,32].
Cornelia S. Ceianu performed the serological diagnosis. Simin A. Florescu and Corneliu P. Popescu performed clinical diagnosis. Anca Sıˆrbu managed dengue fever surveilnculescu-Ga tej wrote the lance. Sorin Dinu and Ioana R. Pa paper. Leticia Franco advised regarding the molecular approach and provided a critical review of the manuscript. Cornelia S. Ceianu coordinated the study.
3.2. DENV-2 phylogenetic analysis
Conflict of interest
The only DENV-2 sequence obtained in this study (GenBank acc. no. HF955511) was from a patient arriving from India in 2012. A similar sequence was found in 2011 in an Irish patient with an unknown travel history. Phylogenetic analysis indicates that this isolate belonged to the cosmopolitan genotype of DENV-2. Similar sequences were previously obtained from patients in India as early as 1996 or in the neighboring country Bangladesh or in Sri Lanka.
None.
3.3. DENV-3 phylogenetic analysis DENV-3 sequences belong to genotypes I and III. One sequence (GenBank acc. no. FR822184) from a patient arriving from Indonesia (2011) clusters with genotype I sequences retrieved from East Timor (2000), Australia (2008), as well as endemic viruses from Indonesia (2004). The Indonesian sequences belong to the Sumatran-Javan clade, a viral lineage that appears to have a superior level of evolutionary fitness and epidemic potential [33]. The sequences belonging to genotype III (GenBank acc. no. HE800554 and HG918040) were obtained from two patients arriving from Vietnam-Thailand in 2012 and 2013, respectively. The two sequences are highly similar to each other and share a high degree of similarity with sequences obtained from Cambodia (2011) and Pakistan (2005e2009). As previously shown, the Pakistanis strains emerged from isolates already circulating in Indian subcontinent before 2005e2006 [34].
4. Conclusions This is the first report on the molecular epidemiology of DF imported into Romania. Given the small number of cases, the widespread geographic origins of these cases is remarkable, a graphic demonstration of the global mobility of this virus. DF is a potentially serious infection. For this reason it should be included in differential diagnosis in patients with fever and recent travel in endemic regions. Molecular investigation of samples from viremic patients is of interest for typing the viruses, characterizing their genome and detecting their origin and evolution in endemic regions. Autochthonous cases are likely wherever competent vectors are present, which now includes continental Europe.
Contributors nculescu-Ga tej and Gabriela Opris‚an Sorin Dinu, Ioana R. Pa descu and performed molecular analysis. Daniela Ba
Acknowledgments This work was partially supported by grant PN09220102 funded by the Ministry of National Education, Romania. This study was partially funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext279 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and don’t necessarily reflect the views of the European Commission. The authors are grateful to Prof. Paul Reiter, Paris Pasteur Institute, for valuable suggestions and revision of the manuscript.
References [1] Dengue guidelines for diagnosis, treatment, prevention and control. A joint publication of the World Health Organization (WHO) and the Special Programme for Research and Training in Tropical Diseases (TDR). 2009. http://www.who.int/tdr/ publications/documents/dengue-diagnosis.pdf. [2] Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature 2013;496:504e7. [3] Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, et al. Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J Gen Virol 1989;70:37e43. [4] http://ictvdb.bio-mirror.cn/Ictv/fs_flavi.htm#Genus1. [5] Russell PK, Nisalak A. Dengue virus identification by the plaque reduction neutralization test. J Immunol 1967;99:291e6. [6] Mason PW, McAda PC, Mason TL, Fournier MJ. Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1. Virology 1987;161:262e7. [7] Normile D. Tropical medicine. Surprising new dengue virus throws a spanner in disease control efforts. Science;342:415. [8] Goncalvez AP, Escalante AA, Pujol FH, Ludert JE, Tovar D, Salas RA, et al. Diversity and evolution of the envelope gene of dengue virus type 1. Virology 2002;303:110e9. [9] Walimbe AM, Lotankar M, Cecilia D, Cherian SS. Global phylogeography of dengue type 1 and 2 viruses reveals the role of India. Infect Genet Evol 2014;22:30e9. [10] Weaver SC, Vasilakis N. Molecular evolution of dengue viruses: contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease. Infect Genet Evol 2009;9:523e40. [11] Holmes EC, Twiddy SS. The origin, emergence and evolutionary genetics of dengue virus. Infect Genet Evol 2003;3: 19e28.
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
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MODEL
Molecular epidemiology of dengue fever cases e Romania [12] Carey DE, Causey OR, Reddy S, Cooke AR. Dengue viruses from febrile patients in Nigeria, 1964e68. Lancet 1971;1:105e6. [13] Vasilakis N, Tesh RB, Weaver SC. Sylvatic dengue virus type 2 activity in humans, Nigeria, 1966. Emerg Infect Dis 2008;14: 502e4. [14] Ba ¨ck AT, Lundkvist A. Dengue viruses e an overview. Infect Ecol Epidemiol 2013;3. [15] da Costa VG, Marques-Silva AC, Floriano VG, Moreli ML. Safety, immunogenicity and efficacy of a recombinant tetravalent dengue vaccine: a meta-analysis of randomized trials. Vaccine 2014;32:4885e92. [16] Halstead SB, Papaevangelou G. Transmission of dengue 1 and 2 viruses in Greece in 1928. Am J Trop Med Hyg 1980;29:635e7. [17] Jelinek T. Trends in the epidemiology of dengue fever and their relevance for importation to Europe. Euro Surveill 2009; 14(25). pii: 19250. [18] La Ruche G, Souare `s Y, Armengaud A, Peloux-Petiot F, Delaunay P, Despre `s P, et al. First two autochthonous dengue virus infections in metropolitan France, September 2010. Euro Surveill 2010;15:19676. [19] Gjenero-Margan I, Aleraj B, Krajcar D, Lesnikar V, Klobucar A, Pem-Novosel I, et al. Autochthonous dengue fever in Croatia, AugusteSeptember 2010. Euro Surveill 2011;16(9). pii: 19805. [20] Marchand E, Prat C, Jeannin C, Lafont E, Bergmann T, Flusin O, et al. Autochthonous case of dengue in France, October 2013. Euro Surveill 2013;18:20661. [21] Margarita Y, Santos Gra ´cio AJ, Lencastre I, Silva AC, Novo T, Sousa C, et al. First record of Aedes (Stegomia) aegypti (Linnaeus, 1762) (Diptera, Culicidae) in Madeira Island e Portugal (Portuguese, English abstract). Acta Parasitolo ´gica Port 2006; 13:59e61. [22] Sousa CA, Clairouin M, Seixas G, Viveiros B, Novo MT, Silva AC, et al. Ongoing outbreak of dengue type 1 in the autonomous region of Madeira, Portugal: preliminary report. Euro Surveill 2012;17(49). pii: 20333. [23] Lourenc ¸o J, Recker M. The 2012 Madeira dengue outbreak: epidemiological determinants and future epidemic potential. PLoS Negl Trop Dis 2014;8(8):e3083. [24] Medlock JM, Hansford KM, Schaffner F, Versteirt V, Hendrickx G, Zeller H, et al. A review of the invasive
5
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
mosquitoes in Europe: ecology, public health risks, and control options. Vector Borne Zoonotic Dis 2012;12:435e47. Domingo C, Palacios G, Niedrig M, Cabrerizo M, Jabado O, Reyes N, et al. A new tool for the diagnosis and molecular surveillance of dengue infections in clinical samples. Dengue Bull 2004;28:87e95. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis for Windows 95/98/NT. Nucleic Acids Symp 1999;41:95e8. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725e9. Tissera HA, Ooi EE, Gubler DJ, Tan Y, Logendra B, Wahala WM, et al. New dengue virus type 1 genotype in Colombo, Sri Lanka. Emerg Infect Dis 2011;17:2053e5. Ocwieja KE, Fernando AN, Sherrill-Mix S, Sundararaman SA, Tennekoon RN, Tippalagama R, et al. Phylogeography and molecular epidemiology of an epidemic strain of dengue virus type 1 in Sri Lanka. Am J Trop Med Hyg 2014;91:225e34. Kurolt IC, Betica-Radic L, Dakovic-Rode O, Franco L, Zelena ´ H, Tenorio A, et al. Molecular characterization of dengue virus 1 from autochthonous dengue fever cases in Croatia. Clin Microbiol Infect 2013;19:E163e5. Alves MJ, Fernandes PL, Amaro F, Oso ´rio H, Luz T, Parreira P, et al. Clinical presentation and laboratory findings for the first autochthonous cases of dengue fever in Madeira island, Portugal, October 2012. Euro Surveill 2013;18(6). pii: 20398. Wilder-Smith A, Quam M, Sessions O, Rocklov J, LiuHelmersson J, Franco L, et al. The 2012 dengue outbreak in Madeira: exploring the origins. Euro Surveill 2014;19:20718. Erratum in: Euro Surveill 2014;19:20725. Ong SH, Yip JT, Chen YL, Liu W, Harun S, Lystiyaningsih E, et al. Periodic re-emergence of endemic strains with strong epidemic potential-a proposed explanation for the 2004 Indonesian dengue epidemic. Infect Genet Evol 2008;8: 191e204. Koo C, Nasir A, Hapuarachchi HC, Lee KS, Hasan Z, Ng LC, et al. Evolution and heterogeneity of multiple serotypes of dengue virus in Pakistan, 2006e2011. Virol J 2013;10:275.
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
MODEL
Travel Medicine and Infectious Disease (2014) xx, 1e5
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevierhealth.com/journals/tmid
Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013 nculescu-Ga tej c,1, Sorin Dinu a,b,1, Ioana R. Pa Simin A. Florescu d,e, Corneliu P. Popescu d,e, Anca Sıˆrbu f, descu c, Leticia Franco g, Gabriela Opris‚an a, Daniela Ba Cornelia S. Ceianu c,* a Molecular Epidemiology Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independent‚ei, 050096 Bucharest, Romania b Genetics Department, Faculty of Biology, University of Bucharest, 1-3 Aleea Portocalelor, 060101 Bucharest, Romania c Vector-Borne Infections and Medical Entomology Laboratory, “Cantacuzino” National Institute of Research-Development for Microbiology and Immunology, 103 Splaiul Independent‚ei, 050096 Bucharest, Romania d Clinical Hospital of Infectious and Tropical Diseases “Dr. Victor Babes‚”, 281 S‚os. Mihai Bravu, 030303 Bucharest, Romania e University of Medicine “Carol Davila”, 37 Dionisie Lupu, 020021 Bucharest, Romania f National Institute of Public HealtheNational Centre for Surveillance and Control of Communicable Diseases, 1-3 Dr. Leonte Anastasievici, 050463, Bucharest, Romania g Arbovirus and Imported Viral Diseases, National Centre of Microbiology, Instituto de Salud Carlos III, Ctra. de Majadahonda-Pozuelo, km. 2,200, 28220 Majadahonda, Madrid, Spain
Received 30 September 2014; received in revised form 3 November 2014; accepted 4 November 2014
KEYWORDS Mosquito-borne infection; Dengue viruses; RT-PCR; DNA sequencing; Phylogenetic analysis
Summary Background: Dengue fever is the commonest arthropod-borne infection worldwide. In recent years, rapid growth in global air travel has resulted in a considerable increase in the incidence of imported cases. In Romania it is now the second most frequent cause for hospitalization (after malaria) in patients arriving from tropical regions. Methods: Serological and molecular diagnostics were applied to samples obtained between 2008 and 2013 from travelers with suspected dengue. Molecular typing was performed by RT-PCR followed by sequencing of the E-NS1 junction.
* Corresponding author. Tel.: þ40 213069243. E-mail address: [email protected] (C.S. Ceianu). 1 These two authors contributed equally to this work. http://dx.doi.org/10.1016/j.tmaid.2014.11.001 1477-8939/ª 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
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S. Dinu et al. Results: Twelve of 37 suspected cases were confirmed and three remained probable. The infections were acquired in endemic regions in Asia, Africa and in Europe (Madeira Island). Dengue virus nucleic acid was detected and sequenced in nine cases. Phylogenetic analysis indicated that the viruses were of genotypes I and V of serotype 1, cosmopolitan genotype of serotype 2 and genotypes I and III of serotype 3. Conclusions: Romanian tourists traveling to dengue-endemic countries are at risk of acquiring dengue infection. Appropriate prevention measures prior to travel and upon return should be taken, particularly as the dengue secondary vector Aedes albopictus is now established in Bucharest. ª 2014 Elsevier Ltd. All rights reserved.
1. Introduction Dengue fever is the most widely distributed arthropodborne infection worldwide. According to World Health Organization 2.5 billion humans living in tropical and subtropical regions are at risk of acquiring dengue infection [1], with an estimated 390 million cases per year [2]. The dengue viruses (DENVs) comprise a distinct serocomplex of the family Flaviviridae in the genus Flavivirus [3,4]. There are four divergent serotypes (DENV-1 to DENV-4), based on genetic and antigenic properties. These display 63e68% amino acid sequence homology [5,6]. In addition, a new viral serotype (DENV-5) has been identified in a human outbreak in Malaysia in 2007; there is evidence that this circulates among macaques [7]. Serotypes 1e4 are further classified into genotypes according to nucleotide divergence of up to 6% in the envelope glycoprotein coding region. DENV-1 comprises five genotypes (IeV) with a broad distribution: Southeast Asia, China, East Africa (genotype I); Thailand (genotype II); Malaysia (genotype III); West Pacific islands and Australia (genotype IV); America, West Africa and Asia (genotype V) [8]. Six genotypes have been proposed for DENV-2: Asian I (Southeast Asia and East Asia); Asian II (Southeast Asia, East Asia, Oceania, Caribbean region and the Americas); Asian/American (Southeast Asia, Caribbean region and Americas); a cosmopolitan genotype (India, South-Central Asia, East Asia, East Africa, Middle East, Southeast Asia and Oceania); an American genotype (India, Caribbean region, South America, Central America, Oceania and Trinidad) and sylvatic genotype (West Africa and Malaysia) [9]. DENV-3 isolates are assigned to four genotypes: genotype I (Indonesia, Malaysia, Philippines and South Pacific islands), genotype II (Thailand, Vietnam and Bangladesh), genotype III (Sri Lanka, India, Africa and Samoa), and genotype IV (Puerto Rico, Latin and Central America and Tahiti) [10]. Phylogenetic analysis indicates the existence of four genotypes within the DENV-4 serotype: genotype I (Thailand, the Philippines, Sri Lanka and Japan), genotype II (Indonesia, Malaysia, Tahiti, the Caribbean and the Americas), genotype III (Thailand) and genotype IV (sylvatic strains from Malaysia) [10]. The virus is transmitted by mosquitoes of the genus Aedes, primarily Aedes aegypti and Ae. albopictus, and is maintained in urban cycles in humans and sylvatic cycles in non-human primates [1,11]. Spillover from the sylvatic cycle to the urban cycle has been documented [12,13]. The
infection can be asymptomatic or range from an undifferentiated acute febrile illness to “classic” dengue fever (DF) which, in some cases, can progress to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/ DSS) [14]. At present there are no specific treatments or commercially available vaccines [15]. One of the largest epidemics of dengue ever recorded occurred world-wide was in refugee camps in Greece (1927e1928), with at least 650,000 cases and 1,000 deaths. The vector was Ae. aegypti [16]. In recent years there has been a sharp rise in the number of imported DF in Europe, attributable to a rapid rise in international air travel and high rates of transmission in endemic countries [17]. The introduction of Ae. albopictus followed by rapid colonization has given rise to autochthonous cases in mainland France (2010, 2013) and Croatia (2010) [18e20]. In addition, more than 2,000 cases were recorded in Madeira (2012), an autonomous region of Portugal and therefore technically a part of Europe. In this case the vector was Ae. aegypti, a recent arrival in the archipelago [21,23]. The rapid dissemination and establishment of the Ae. albopictus in Southern Europe, and the intense increase in traveling fuel the introduction of dengue in non-endemic areas [24]. This species was detected in 2012 in Bucharest, Romania, and has remained present in subsequent years (Prioteasa et al., personal communication). Dengue is now included in the list of notifiable communicable diseases; laboratory diagnosis by the Romanian National Reference Centre for Vector-Borne Infections was implemented starting 2008. In the present study we present the molecular epidemiology of imported DF cases in Romania between 2008 and 2013.
2. Material and methods 2.1. Patients and laboratory diagnosis Sera from travelers with clinical suspicion of dengue fever after return from dengue-endemic regions were received by the National Reference Centre for Vector-Borne Infections (“Cantacuzino” N.I.R.D.M.I.) from Public Health Departments and from Clinical Hospital of Infectious and Tropical Diseases “Dr. Victor Babes‚” in Bucharest. The samples were tested for DENVs antibodies with IgM and IgG indirect ELISA (Euroimmun, Lu ¨beck, Germany). Paired sera were requested but convalescent serum was not available
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
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Molecular epidemiology of dengue fever cases e Romania for all patients. Sera taken in the acute phase of the disease (up to eight days from fever onset) were subjected to molecular analysis. A case was classified as probable when IgM was detected in serum obtained during the acute phase. Confirmed cases were defined by IgM and IgG, seroconversion for IgG in paired sera, and/or detection of DENVs nucleic acid in sera by RT-PCR and DNA sequencing.
2.2. Molecular typing Viral RNA was extracted from 140 mL of serum using QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the instructions provided by the manufacturer. Reverse transcription and PCR were carried out using primers described earlier [25] that amplify a fragment located within the E and NS1 viral genomic regions junction. Serotype assignment was made based on the size of the DNA fragment obtained. The amplicons were gel purified (Wizard SV PCR and Gel Clean-Up System; Promega, Madison, WI) and sequenced with BigDye Terminator v3.1 on a 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). The DNA sequences were visually inspected and aligned with BioEdit version 7.0.5.3 [26]. Phylogenetic analysis was conducted using Mega 6 software [27], Neighbor-joining algorithm with 1,000 bootstrap replicates. Sequences obtained in this study were submitted to GenBank.
3. Results and discussion From 2008 to 2013, serum samples from 37 suspected patients returning from endemic countries were tested for the presence of IgM and IgG dengue-specific antibodies (Table 1). The patients declared a history of recent travel in Asia, Africa and Europe (Madeira). One patient, a cruise ship worker, took a voyage in Southeast Asia and islands in the Indian Ocean. For six DF suspected patients we did not receive any information regarding their traveling history. Twelve DF cases were confirmed in patients with recent travel to India, Indonesia, Vietnam-Thailand, Angola, Kenya and Portugal (Madeira). Three more cases, returning from
Table 1 Summary of the dengue fever suspected cases investigated by The Romanian National Reference Centre for Vector-Borne Infections between 2008 and 2013. Year
Number of suspected cases tested
2008 2009 2010 2011
2 0 1 7
2012
11
2013
16
Total
37
Number of confirmed cases
Number of probable cases
1 (India) 0 0 4 (Indonesia, VietnameThailand, Angola, Kenya) 3 (VietnameThailand, Madeira Island, India) 4 (Indonesia-3, VietnameThailand) 12
0 0 0 0
1 (Vietname Thailand) 2 (Sri Lanka, India) 3
3 Vietnam-Thailand, India and Sri Lanka, were classified as probable (Table 1). Nine DNA sequences were obtained from patients returning from India, Indonesia, Vietnam-Thailand and Portugal (Madeira). The isolates were of DENV-1 (genotypes I and V), DENV-2 (cosmopolitan genotype) and DENV-3 (genotypes I and III) (Table 2). In half of the confirmed and probable cases, the infected persons arrived during the Ae. albopictus season in Romania, months May to October.
3.1. DENV-1 phylogenetic analysis Five out of the nine analyzed sequences cluster into DENV1. The first sequence (GenBank acc. no. HE565699), belonging to genotype I, was obtained from the serum of a patient working on a cruise ship in 2011 who declared a history of travel in Southeast Asia (Vietnam and Thailand) and islands of Indian Ocean and was repatriated to Romania from Maldives after the illness onset. The phylogenetic analysis indicates 100% identity between our sequence and sequences obtained in recent years in Southeast Asia (Cambodia, Malaysia, Thailand, Vietnam) where genotype I is circulating [8]. A similar strain was involved in a major outbreak in Colombo, Sri Lanka (2012) after its introduction in 2009 [28,29]. Phylogeographic analysis performed by Ocwieja et al. indicates that the origins of this strain might be the virus causing severe dengue in Thailand (2001) and which later spread to neighboring Cambodia and Vietnam [29]. Two other sequences clustering into genotype I of DENV-1 were obtained from patients who traveled to Indonesia in 2013 (GenBank acc. no. HG918038-9). These were remarkably similar to sequences obtained in 2004, 2011 and 2013 in China, as well as Singapore (2003) and Malaysia (2005). Two sequences fell into genotype V of DENV-1. The first was obtained from a patient returning from India in 2008 (GenBank acc. no. FR874940) and was identical to others obtained from viremic subjects in that country in 2010 and 2011. Similar sequences had been obtained from India (1982 and 2009) and Sri Lanka (1997 and 2004). It is noteworthy that the first autochthonous cases of DF in Croatia were due to DENV-1 in the same cluster of
Table 2
Results of molecular typing.
Serotype
Genotype
Year
History of traveling
GenBank accession number
DENV-1
I
2011
Vietname Thailand Indonesia Indonesia India Madeira Island India Indonesia Vietname Thailand Vietname Thailand
HE565699
2013
DENV-2 DENV-3
V
2008 2012
Cosmopolitan I III
2012 2011 2012 2013
HG918038 HG918039 FR874940 HF955512 HF955511 FR822184 HE800554 HG918040
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isolates [30]. The second sequence (GenBank acc. no. HF955512) falling into genotype V of DEN-1 was amplified from the serum of a patient traveling to Madeira in 2012 at the time of the outbreak mentioned above [22]. Our sequence is 99.94% identical with a sequence derived from an isolate obtained in 2008 in Colombia, South America. As shown earlier, the isolate from Madeira probably originated in Brazil, Columbia or Venezuela [31,32].
Cornelia S. Ceianu performed the serological diagnosis. Simin A. Florescu and Corneliu P. Popescu performed clinical diagnosis. Anca Sıˆrbu managed dengue fever surveilnculescu-Ga tej wrote the lance. Sorin Dinu and Ioana R. Pa paper. Leticia Franco advised regarding the molecular approach and provided a critical review of the manuscript. Cornelia S. Ceianu coordinated the study.
3.2. DENV-2 phylogenetic analysis
Conflict of interest
The only DENV-2 sequence obtained in this study (GenBank acc. no. HF955511) was from a patient arriving from India in 2012. A similar sequence was found in 2011 in an Irish patient with an unknown travel history. Phylogenetic analysis indicates that this isolate belonged to the cosmopolitan genotype of DENV-2. Similar sequences were previously obtained from patients in India as early as 1996 or in the neighboring country Bangladesh or in Sri Lanka.
None.
3.3. DENV-3 phylogenetic analysis DENV-3 sequences belong to genotypes I and III. One sequence (GenBank acc. no. FR822184) from a patient arriving from Indonesia (2011) clusters with genotype I sequences retrieved from East Timor (2000), Australia (2008), as well as endemic viruses from Indonesia (2004). The Indonesian sequences belong to the Sumatran-Javan clade, a viral lineage that appears to have a superior level of evolutionary fitness and epidemic potential [33]. The sequences belonging to genotype III (GenBank acc. no. HE800554 and HG918040) were obtained from two patients arriving from Vietnam-Thailand in 2012 and 2013, respectively. The two sequences are highly similar to each other and share a high degree of similarity with sequences obtained from Cambodia (2011) and Pakistan (2005e2009). As previously shown, the Pakistanis strains emerged from isolates already circulating in Indian subcontinent before 2005e2006 [34].
4. Conclusions This is the first report on the molecular epidemiology of DF imported into Romania. Given the small number of cases, the widespread geographic origins of these cases is remarkable, a graphic demonstration of the global mobility of this virus. DF is a potentially serious infection. For this reason it should be included in differential diagnosis in patients with fever and recent travel in endemic regions. Molecular investigation of samples from viremic patients is of interest for typing the viruses, characterizing their genome and detecting their origin and evolution in endemic regions. Autochthonous cases are likely wherever competent vectors are present, which now includes continental Europe.
Contributors nculescu-Ga tej and Gabriela Opris‚an Sorin Dinu, Ioana R. Pa descu and performed molecular analysis. Daniela Ba
Acknowledgments This work was partially supported by grant PN09220102 funded by the Ministry of National Education, Romania. This study was partially funded by EU grant FP7-261504 EDENext and is catalogued by the EDENext Steering Committee as EDENext279 (http://www.edenext.eu). The contents of this publication are the sole responsibility of the authors and don’t necessarily reflect the views of the European Commission. The authors are grateful to Prof. Paul Reiter, Paris Pasteur Institute, for valuable suggestions and revision of the manuscript.
References [1] Dengue guidelines for diagnosis, treatment, prevention and control. A joint publication of the World Health Organization (WHO) and the Special Programme for Research and Training in Tropical Diseases (TDR). 2009. http://www.who.int/tdr/ publications/documents/dengue-diagnosis.pdf. [2] Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL, et al. The global distribution and burden of dengue. Nature 2013;496:504e7. [3] Calisher CH, Karabatsos N, Dalrymple JM, Shope RE, Porterfield JS, Westaway EG, et al. Antigenic relationships between flaviviruses as determined by cross-neutralization tests with polyclonal antisera. J Gen Virol 1989;70:37e43. [4] http://ictvdb.bio-mirror.cn/Ictv/fs_flavi.htm#Genus1. [5] Russell PK, Nisalak A. Dengue virus identification by the plaque reduction neutralization test. J Immunol 1967;99:291e6. [6] Mason PW, McAda PC, Mason TL, Fournier MJ. Sequence of the dengue-1 virus genome in the region encoding the three structural proteins and the major nonstructural protein NS1. Virology 1987;161:262e7. [7] Normile D. Tropical medicine. Surprising new dengue virus throws a spanner in disease control efforts. Science;342:415. [8] Goncalvez AP, Escalante AA, Pujol FH, Ludert JE, Tovar D, Salas RA, et al. Diversity and evolution of the envelope gene of dengue virus type 1. Virology 2002;303:110e9. [9] Walimbe AM, Lotankar M, Cecilia D, Cherian SS. Global phylogeography of dengue type 1 and 2 viruses reveals the role of India. Infect Genet Evol 2014;22:30e9. [10] Weaver SC, Vasilakis N. Molecular evolution of dengue viruses: contributions of phylogenetics to understanding the history and epidemiology of the preeminent arboviral disease. Infect Genet Evol 2009;9:523e40. [11] Holmes EC, Twiddy SS. The origin, emergence and evolutionary genetics of dengue virus. Infect Genet Evol 2003;3: 19e28.
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
+
MODEL
Molecular epidemiology of dengue fever cases e Romania [12] Carey DE, Causey OR, Reddy S, Cooke AR. Dengue viruses from febrile patients in Nigeria, 1964e68. Lancet 1971;1:105e6. [13] Vasilakis N, Tesh RB, Weaver SC. Sylvatic dengue virus type 2 activity in humans, Nigeria, 1966. Emerg Infect Dis 2008;14: 502e4. [14] Ba ¨ck AT, Lundkvist A. Dengue viruses e an overview. Infect Ecol Epidemiol 2013;3. [15] da Costa VG, Marques-Silva AC, Floriano VG, Moreli ML. Safety, immunogenicity and efficacy of a recombinant tetravalent dengue vaccine: a meta-analysis of randomized trials. Vaccine 2014;32:4885e92. [16] Halstead SB, Papaevangelou G. Transmission of dengue 1 and 2 viruses in Greece in 1928. Am J Trop Med Hyg 1980;29:635e7. [17] Jelinek T. Trends in the epidemiology of dengue fever and their relevance for importation to Europe. Euro Surveill 2009; 14(25). pii: 19250. [18] La Ruche G, Souare `s Y, Armengaud A, Peloux-Petiot F, Delaunay P, Despre `s P, et al. First two autochthonous dengue virus infections in metropolitan France, September 2010. Euro Surveill 2010;15:19676. [19] Gjenero-Margan I, Aleraj B, Krajcar D, Lesnikar V, Klobucar A, Pem-Novosel I, et al. Autochthonous dengue fever in Croatia, AugusteSeptember 2010. Euro Surveill 2011;16(9). pii: 19805. [20] Marchand E, Prat C, Jeannin C, Lafont E, Bergmann T, Flusin O, et al. Autochthonous case of dengue in France, October 2013. Euro Surveill 2013;18:20661. [21] Margarita Y, Santos Gra ´cio AJ, Lencastre I, Silva AC, Novo T, Sousa C, et al. First record of Aedes (Stegomia) aegypti (Linnaeus, 1762) (Diptera, Culicidae) in Madeira Island e Portugal (Portuguese, English abstract). Acta Parasitolo ´gica Port 2006; 13:59e61. [22] Sousa CA, Clairouin M, Seixas G, Viveiros B, Novo MT, Silva AC, et al. Ongoing outbreak of dengue type 1 in the autonomous region of Madeira, Portugal: preliminary report. Euro Surveill 2012;17(49). pii: 20333. [23] Lourenc ¸o J, Recker M. The 2012 Madeira dengue outbreak: epidemiological determinants and future epidemic potential. PLoS Negl Trop Dis 2014;8(8):e3083. [24] Medlock JM, Hansford KM, Schaffner F, Versteirt V, Hendrickx G, Zeller H, et al. A review of the invasive
5
[25]
[26]
[27]
[28]
[29]
[30]
[31]
[32]
[33]
[34]
mosquitoes in Europe: ecology, public health risks, and control options. Vector Borne Zoonotic Dis 2012;12:435e47. Domingo C, Palacios G, Niedrig M, Cabrerizo M, Jabado O, Reyes N, et al. A new tool for the diagnosis and molecular surveillance of dengue infections in clinical samples. Dengue Bull 2004;28:87e95. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis for Windows 95/98/NT. Nucleic Acids Symp 1999;41:95e8. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 2013;30:2725e9. Tissera HA, Ooi EE, Gubler DJ, Tan Y, Logendra B, Wahala WM, et al. New dengue virus type 1 genotype in Colombo, Sri Lanka. Emerg Infect Dis 2011;17:2053e5. Ocwieja KE, Fernando AN, Sherrill-Mix S, Sundararaman SA, Tennekoon RN, Tippalagama R, et al. Phylogeography and molecular epidemiology of an epidemic strain of dengue virus type 1 in Sri Lanka. Am J Trop Med Hyg 2014;91:225e34. Kurolt IC, Betica-Radic L, Dakovic-Rode O, Franco L, Zelena ´ H, Tenorio A, et al. Molecular characterization of dengue virus 1 from autochthonous dengue fever cases in Croatia. Clin Microbiol Infect 2013;19:E163e5. Alves MJ, Fernandes PL, Amaro F, Oso ´rio H, Luz T, Parreira P, et al. Clinical presentation and laboratory findings for the first autochthonous cases of dengue fever in Madeira island, Portugal, October 2012. Euro Surveill 2013;18(6). pii: 20398. Wilder-Smith A, Quam M, Sessions O, Rocklov J, LiuHelmersson J, Franco L, et al. The 2012 dengue outbreak in Madeira: exploring the origins. Euro Surveill 2014;19:20718. Erratum in: Euro Surveill 2014;19:20725. Ong SH, Yip JT, Chen YL, Liu W, Harun S, Lystiyaningsih E, et al. Periodic re-emergence of endemic strains with strong epidemic potential-a proposed explanation for the 2004 Indonesian dengue epidemic. Infect Genet Evol 2008;8: 191e204. Koo C, Nasir A, Hapuarachchi HC, Lee KS, Hasan Z, Ng LC, et al. Evolution and heterogeneity of multiple serotypes of dengue virus in Pakistan, 2006e2011. Virol J 2013;10:275.
Please cite this article in press as: Dinu S, et al., Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013, Travel Medicine and Infectious Disease (2014), http://dx.doi.org/10.1016/j.tmaid.2014.11.001
