Arch Virol DOI 10.1007/s00705-014-2071-x
BRIEF REPORT
Coxsackievirus A6 and enterovirus 71 causing hand, foot and mouth disease in Cuba, 2011–2013 Magile´ C. Fonseca • Luis Sarmiento • Sonia Resik • Yenisleidys Martı´nez Lai Heng Hung • Luis Morier • Alexander Pin˜o´n • Odalys Valde´z • Vivian Kourı´ • Guelsys Gonza´lez
•
Received: 6 February 2014 / Accepted: 24 March 2014 Ó Springer-Verlag Wien 2014
Abstract Hand, foot and mouth disease (HFMD) is usually caused by coxsackievirus A16 or enterovirus 71 (EV71). Between 2011 and 2013, HFMD cases were reported from different Cuban provinces. A total of 42 clinical specimens were obtained from 23 patients. Detection, identification and phylogenetic analysis of enterovirus-associated HFMD were carried out by virus isolation, specific enterovirus PCR and partial VP1 sequences. HEV was detected in 11 HFMD cases. Emerging genetic variants of coxsackievirus A6 and EV71 were identified as the causative agents of the Cuban HFMD cases.
Outbreaks of hand, foot and mouth disease are usually caused by coxsackievirus A16 (CVA16) or enterovirus 71 (EV71) [1]. A new genetic variant of coxsackievirus A6 (CVA6) has been associated with sporadic HFMD cases and outbreaks in Europe, Southeast Asia, and the USA since 2008 [4]. In Cuba, HFMD is not a notifiable disease, and only sporadic cases have been reported in 2002, 2009, and 2010 associated with CAV16 (data not published). The aim of this work was to identify the etiological agent of HFMD cases reported in different Cuban provinces from 2011 to 2013.
M. C. Fonseca (&) L. Sarmiento S. Resik Y. Martı´nez L. H. Hung L. Morier A. Pin˜o´n O. Valde´z V. Kourı´ G. Gonza´lez ‘‘Pedro Kourı´’’ Tropical Medicine Institute, Avenida Novia del Mediodı´a KM 6 1/2, La Lisa, Marianao 13, P.O. Box 601, CP.11400 Havana, Cuba e-mail: [email protected]; [email protected]
35 cases with clinical symptoms of HFMD were recorded in Cuba from 2011 to 2013, thirty four of which were children and one was an adult. Cienfuegos province reported five suspected HFMD cases from February to April of 2011. Sancti Spı´ritus province reported 24 suspected cases between September and October 2012, most of them from a daycare center. In addition, five cases from Havana and one from Artemisa province (an adult case) were reported from February to July 2013. The age distribution of the 34 cases in children ranged from 9 months to 8 years (mean 1.8 years), and the adult case was 39 years old. 25 (71.4 %) patients showed common clinical signs and symptoms of HFDM, with fever for 1–2 days followed by the appearance of vesicles, mostly on hands, feet and oral mucosa. Six cases (17.1 %) exhibited vesicular rash on the buttocks and perianal area. Four patients, including the adult case (11.4 %), showed severe skin manifestations all over the body, and one required intensive care due to the severity of the manifestations. Interestingly, two out of these four patients experienced pneumonia 1–2 weeks before the beginning of HFMD clinical symptoms. Nail shedding occurred in two cases (5.7 %) within 1–2 months after the onset of disease, but this observation was not obtained in the majority of the cases.There were no reported cases of encephalitis or fatalities. To identify the etiological agent, 23 out of 35 cases with clinical symptoms of HFMD were studied. In total, 42 samples (18 feces, 5 vesicle fluids and 17 nasopharyngeal swabs, 2 rectal swabs) were tested at the National Enterovirus Laboratory (Table 1). First, vesicle fluids and nasopharyngeal swabs were screened for the 50 non-coding region (50 NCR) of the enterovirus genome as described previously [14]. Second, all of the specimens were inoculated into monolayers of
123
M. C. Fonseca et al. Table 1 HFMD cases reported in Cuba, 2011–2013, and laboratory results
City/year
Cienfuegos/ 2011 Santic Spiritus/ 2012 Havana/ 2013
No. of cases reported
Total samples/ specimens
Positive EV PCR specimens
Positive cell culture specimens
Total cases identified/EV serotype
5
5/1 vesicle fluid, 4 feces
–
2 feces
2 /2 CVA6
24
22/13 nasopharyngeal swabs, 9 feces
7 nasopharyngeal swabs
4 nasopharyngeal swabs, 2 feces
6/4CVA6, 2 EV71
11/3 vesicles fluids
2 vesicles fluids, 2 rectal swabs
1 nasopharyngeal swab
2/2 CVA6
5
3 nasopharyngeal swabs,
1 feces 1 rectal swab
4 feces, 2 rectal swabs Artemisa/ 2013
1
3/1 vesicles fluids
–
1 vesicle fluid
1/CA6
7 nasopharyngeal swabs, 2 vesicles fluids, 2 rectal swabs
5 nasopharyngeal swabs
11/9 CVA6 2 EV71
1 nasopharyngeal swabs 1 feces
Total
35
45/5 vesicles fluids 17 nasopharyngeal swabs
1 vesicle fluid 5 feces, 1 rectal swab
18 feces, 2 rectal swabs
rhabdomyosarcoma (RD), fibroblastic diploid embryonic human (PHuE-1) and African green monkey kidney (Vero) cells for virus isolation. Cells were grown in Eagle’s minimal essential medium supplemented with 2 % fetal calf serum at 37 °C. Cytopathic effect (CPE) was monitored daily for up to three passages. Nucleic acids from specimens or isolates were automatically extracted using a QIAampÒ Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and QIAcubeTM extractor (QIAGEN) following the manufacturer’s instructions. cDNA was synthesized using the Invitrogen system with random hexamers as primers. For typing, a partial sequence of the viral protein VP1 was amplified by using the AN88 and AN89 primer pair [8]. Sequencing was performed using a Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Fullerton CA) and an automated sequencer (CEQ TM 8800 Sequence Analysis System, Beckman Coulter) according to the manufacturer’s recommendations. Sequences were subjected to BLAST analysis (http:// blast.ncbi.nlm.nih.gov/Blast.cgi), and phylogenetic trees based on the VP1 region were constructed using MEGA version 5.2.2 [12]. Notably, 11 out of 24 (45.8 %) samples from vesicles fluids, nasopharyngeal and rectal swabs yielded amplification products consistent with enterovirus, and virus isolation was possible in seven of them. In addition, 27.8 % of fecal specimens (5 out of 18) induced extensive enterovirus
123
CPE. All the isolates were obtained on RD cell culture in the second passage, mostly 3–5 days after inoculation. Nine of the twelve isolates were identified as CVA6, and two as EV71 (Table 1). It is important to note that a CVA6 strain was also isolated from a nasopharyngeal swab from a 22-month-old child suffering from pneumonia in late March, 2013. The case was reported from Pinar del Rio province in the framework of the Cuban National surveillance and control of acute respiratory infection. Phylogenetic analysis of eight Cuban CVA6 isolates (GenBank Accession Number: KF196256-KF196263) revealed two genetic groups (Fig. 1). Cuban 2011 CVA6 isolates were related to CVA6 strains detected in Europe and Asia between 2007 and 2011. Nevertheless, Cuban isolates obtained between 2012 and 2013 grouped together with emerging CVA6 genetic variants strains isolated in recent international outbreaks. It is noteworthy that most of those strains displayed close relationships to viruses recovered during outbreaks in 2011 in Spain and in 2008 Finland, but one CVA6 strain (12/2013) corresponding to the adult case, formed a separate distinct branch with strains isolated in China (2010–2012) and Thailand (2012). These results suggest the circulation of two genetically distinct CVA6 strains that could have been independently introduced into Cuba from Europe and Southeast Asia, respectively.
Coxsackievirus A6 and enterovirus 71 in Cuba Fig. 1 Phylogenetic analysis of the partial VP1 CA6 sequences from Cuba. The nucleotide sequences were analyzed using the MEGA 5.2.2 package. The phylogenetic tree was constructed by the neighborjoining method with the bootstrap option. Bootstrap values (%) for 1000 replicated trees are indicated at the nodes; only values [70 % are shown. The isolates are indicated by GenBank Accession Numberstrain name-country-year of isolation. The country abbreviations are as follows: CHN China, ESP Spain, FIN Finland, FRA France, JPN Japan, THA Thailand
123
M. C. Fonseca et al. Fig. 2 Phylogenetic analysis of the partial VP1 sequences of enterovirus 71 strains from Cuba. The nucleotide sequences were analyzed using the MEGA 5.2.2 package. The phylogenetic tree was constructed by the neighbor-joining method with the bootstrap option. Bootstrap values (%) for 1000 replicated trees are indicated at the nodes; only values [70 % are shown. The isolates are indicated by GenBank Accession Numberstrain name-country-year of isolation. The country abbreviations are as follows: AUS Australia, CHN China, FIN Finland, FRA France, GER Germany, JPN Japan, NETH Netherlands
Since 2008, several HFMD outbreaks caused by CVA6 have been reported from Finland, Taiwan, Japan, France, Spain, and, recently, the USA and Thailand, 2012. Most attention has been devoted to the atypical features described in recent CVA6 outbreaks. These include unusual age ranges of patients, including adults; severity of illness, characterized by more-severe and extensive rash, eruptions around the perioral area, buttocks, trunk and/or neck; infrequent seasonality of disease; and the association with
123
cases of onychomadesis from 1 to 2 months after the onset of HFMD [1, 4–7, 10, 13]. The identification of CVA6 in patients with acute encephalitis [1, 5], upper respiratory tract symptoms [4] and acute respiratory infection (in this study) confirm that careful surveillance of this agent it will be necessary to recognize emerging outbreaks in order to improve clinical management within the health care system. Taking into account that HFMD is uncommon and not a notifiable disease in Cuba, it is probable that the number
Coxsackievirus A6 and enterovirus 71 in Cuba
of HFMD cases among children and adults in Cuba that have not yet been studied is much higher. It is noteworthy that this is the first identification of EV71 in Cuba, and we therefore conducted further confirmation by nucleotide sequence analysis of the VP2–VP4 region, specific PCR with forward primer MAS01S and reverse primer MAS02A, and a neutralization test using and specific antiserum [2, 3, 9]. Based on the VP1 region, phylogenetic analysis of Cuban EV71 (GenBank: KF801577–F801578) strains showed that they belonged to genotype C2 and were closely related to viruses of various geographical origins (Fig. 2). Based on the known epidemiological behavior of EV71 in the Americas and the AsiaPacific region, the virus probably exists in most countries, causing sporadic cases or small outbreaks [11]. Nevertheless, the establishment of an international network is an urgent necessity in order to gain an overview of this disease and the serotypes involved. The emergence in Cuba of these two significant human pathogens, EV71 and CVA6, is of global interest because of the understanding it may provide for the increase in emerging and re-emerging viral diseases worldwide. In the future, EV71 and CVA6 activity in our country must be monitored by strengthening and maintaining the virological sentinel surveillance system to trace the propagation of the virus across the country and provide a public-health approach focused on early warning of virus transmission and differential diagnostics in the community. Acknowledgments We thank the patients, parents and physicians for participation in this study. We acknowledge the assistance from Cuban Provincial Centers for Epidemiology, Hygiene and Microbiology from Cienfuegos and Sancti Spiritus provinces for collecting the samples. We also thank Dr. Lı´dice Bernardo for helpful comments, and Dr. Ondrej Mach for reviewing the manuscript. This study was supported by the Cuban Ministry of Health as part of an enterovirus surveillance system maintained by the National Enterovirus Laboratory at Tropical Medicine Institute ‘‘Pedro Kourı´’’.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13. Conflict of interest of interest.
The authors declare that they have no conflict 14.
References
coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland. J Clin Virol 48:49–54 Cardosa MJ, Perera D, Brown BA, Cheon D, Chan HM, Chan KP et al (2003) Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis 9:461–468 Coiras MT, Aguilar JC, Garcia ML, Casas I, Perez-Brena P (2004) Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nestedPCR assays. J Med Virol 72:484–495 Flett K, Youngster I, Huang J, McAdam A, Sandora TJ, Rennick M et al (2012) Hand, foot, and mouth disease caused by coxsackievirus A6. Emerg Infect Dis 18:1702–1704 Fujimoto T, Iizuka S, Enomoto M, Abe K, Yamashita K, Hanaoka N et al (2012) Hand, foot, and mouth disease caused by coxsackievirus A6, Japan, 2011. Emerg Infect Dis 18:337–339 Mirand A, Henquell C, Archimbaud C, Ughetto S, Antona D, Bailly JL et al (2012) Outbreak of hand, foot and mouth disease/ herpangina associated with coxsackievirus A6 and A10 infections in 2010, France: a large citywide, prospective observational study. Clin Microbiol Infect 18:E110–E118 Montes M, Artieda J, Pin˜eiro LD, Gastesi M, Diez-Nieves I, Cilla G (2013) Hand, foot, and mouth disease outbreak and coxsackievirus A6, Northern Spain, 2011. Emerg Infect Dis 19:676–677 Nix WA, Oberste MS, Pallansch MA (2006) Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J Clin Microbiol 44:2698–2704 Perera D, Podin Y, Akin W, Tan WS, Cardosa MJ (2004) Incorrect identification of recent Asian strains of coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT-PCR. BMC Infect Dis 4:11 Puenpa J, Chieochansin T, Linsuwanon P, Korkong S, Thongkomplew S, Vichaiwattana P et al (2013) Hand, foot, and mouth disease caused by coxsackievirus A6, Thailand, 2012. Emerg Infect Dis 19:641–643 Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH (2010) Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 10:778–790 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Wei SH, Huang YP, Liu MC, Tsou TP, Lin HC, Lin TL et al (2011) An outbreak of coxsackievirus A6 hand, foot, and mouth disease associated with onychomadesis in Taiwan, 2010. BMC Infect Dis 11:346 Yang CF, De L, Yang SJ, Go´mez JR, Cruz JR, Halloway MA, Pallansch MA, Kew OM (1992) Genotype-specific in vitro amplification of sequences of the wild type 3 polioviruses from Me´xico and Guatemala. Virus Res 24:277–296
1. Blomqvist S, Klemola P, Kaijalainen S, Pananen A, Simonen ML, Vuorinen T, Roivainen M (2010) Co-circulation of
123
BRIEF REPORT
Coxsackievirus A6 and enterovirus 71 causing hand, foot and mouth disease in Cuba, 2011–2013 Magile´ C. Fonseca • Luis Sarmiento • Sonia Resik • Yenisleidys Martı´nez Lai Heng Hung • Luis Morier • Alexander Pin˜o´n • Odalys Valde´z • Vivian Kourı´ • Guelsys Gonza´lez
•
Received: 6 February 2014 / Accepted: 24 March 2014 Ó Springer-Verlag Wien 2014
Abstract Hand, foot and mouth disease (HFMD) is usually caused by coxsackievirus A16 or enterovirus 71 (EV71). Between 2011 and 2013, HFMD cases were reported from different Cuban provinces. A total of 42 clinical specimens were obtained from 23 patients. Detection, identification and phylogenetic analysis of enterovirus-associated HFMD were carried out by virus isolation, specific enterovirus PCR and partial VP1 sequences. HEV was detected in 11 HFMD cases. Emerging genetic variants of coxsackievirus A6 and EV71 were identified as the causative agents of the Cuban HFMD cases.
Outbreaks of hand, foot and mouth disease are usually caused by coxsackievirus A16 (CVA16) or enterovirus 71 (EV71) [1]. A new genetic variant of coxsackievirus A6 (CVA6) has been associated with sporadic HFMD cases and outbreaks in Europe, Southeast Asia, and the USA since 2008 [4]. In Cuba, HFMD is not a notifiable disease, and only sporadic cases have been reported in 2002, 2009, and 2010 associated with CAV16 (data not published). The aim of this work was to identify the etiological agent of HFMD cases reported in different Cuban provinces from 2011 to 2013.
M. C. Fonseca (&) L. Sarmiento S. Resik Y. Martı´nez L. H. Hung L. Morier A. Pin˜o´n O. Valde´z V. Kourı´ G. Gonza´lez ‘‘Pedro Kourı´’’ Tropical Medicine Institute, Avenida Novia del Mediodı´a KM 6 1/2, La Lisa, Marianao 13, P.O. Box 601, CP.11400 Havana, Cuba e-mail: [email protected]; [email protected]
35 cases with clinical symptoms of HFMD were recorded in Cuba from 2011 to 2013, thirty four of which were children and one was an adult. Cienfuegos province reported five suspected HFMD cases from February to April of 2011. Sancti Spı´ritus province reported 24 suspected cases between September and October 2012, most of them from a daycare center. In addition, five cases from Havana and one from Artemisa province (an adult case) were reported from February to July 2013. The age distribution of the 34 cases in children ranged from 9 months to 8 years (mean 1.8 years), and the adult case was 39 years old. 25 (71.4 %) patients showed common clinical signs and symptoms of HFDM, with fever for 1–2 days followed by the appearance of vesicles, mostly on hands, feet and oral mucosa. Six cases (17.1 %) exhibited vesicular rash on the buttocks and perianal area. Four patients, including the adult case (11.4 %), showed severe skin manifestations all over the body, and one required intensive care due to the severity of the manifestations. Interestingly, two out of these four patients experienced pneumonia 1–2 weeks before the beginning of HFMD clinical symptoms. Nail shedding occurred in two cases (5.7 %) within 1–2 months after the onset of disease, but this observation was not obtained in the majority of the cases.There were no reported cases of encephalitis or fatalities. To identify the etiological agent, 23 out of 35 cases with clinical symptoms of HFMD were studied. In total, 42 samples (18 feces, 5 vesicle fluids and 17 nasopharyngeal swabs, 2 rectal swabs) were tested at the National Enterovirus Laboratory (Table 1). First, vesicle fluids and nasopharyngeal swabs were screened for the 50 non-coding region (50 NCR) of the enterovirus genome as described previously [14]. Second, all of the specimens were inoculated into monolayers of
123
M. C. Fonseca et al. Table 1 HFMD cases reported in Cuba, 2011–2013, and laboratory results
City/year
Cienfuegos/ 2011 Santic Spiritus/ 2012 Havana/ 2013
No. of cases reported
Total samples/ specimens
Positive EV PCR specimens
Positive cell culture specimens
Total cases identified/EV serotype
5
5/1 vesicle fluid, 4 feces
–
2 feces
2 /2 CVA6
24
22/13 nasopharyngeal swabs, 9 feces
7 nasopharyngeal swabs
4 nasopharyngeal swabs, 2 feces
6/4CVA6, 2 EV71
11/3 vesicles fluids
2 vesicles fluids, 2 rectal swabs
1 nasopharyngeal swab
2/2 CVA6
5
3 nasopharyngeal swabs,
1 feces 1 rectal swab
4 feces, 2 rectal swabs Artemisa/ 2013
1
3/1 vesicles fluids
–
1 vesicle fluid
1/CA6
7 nasopharyngeal swabs, 2 vesicles fluids, 2 rectal swabs
5 nasopharyngeal swabs
11/9 CVA6 2 EV71
1 nasopharyngeal swabs 1 feces
Total
35
45/5 vesicles fluids 17 nasopharyngeal swabs
1 vesicle fluid 5 feces, 1 rectal swab
18 feces, 2 rectal swabs
rhabdomyosarcoma (RD), fibroblastic diploid embryonic human (PHuE-1) and African green monkey kidney (Vero) cells for virus isolation. Cells were grown in Eagle’s minimal essential medium supplemented with 2 % fetal calf serum at 37 °C. Cytopathic effect (CPE) was monitored daily for up to three passages. Nucleic acids from specimens or isolates were automatically extracted using a QIAampÒ Viral RNA Mini Kit (QIAGEN, Hilden, Germany) and QIAcubeTM extractor (QIAGEN) following the manufacturer’s instructions. cDNA was synthesized using the Invitrogen system with random hexamers as primers. For typing, a partial sequence of the viral protein VP1 was amplified by using the AN88 and AN89 primer pair [8]. Sequencing was performed using a Dye Terminator Cycle Sequencing Quick Start Kit (Beckman Coulter, Fullerton CA) and an automated sequencer (CEQ TM 8800 Sequence Analysis System, Beckman Coulter) according to the manufacturer’s recommendations. Sequences were subjected to BLAST analysis (http:// blast.ncbi.nlm.nih.gov/Blast.cgi), and phylogenetic trees based on the VP1 region were constructed using MEGA version 5.2.2 [12]. Notably, 11 out of 24 (45.8 %) samples from vesicles fluids, nasopharyngeal and rectal swabs yielded amplification products consistent with enterovirus, and virus isolation was possible in seven of them. In addition, 27.8 % of fecal specimens (5 out of 18) induced extensive enterovirus
123
CPE. All the isolates were obtained on RD cell culture in the second passage, mostly 3–5 days after inoculation. Nine of the twelve isolates were identified as CVA6, and two as EV71 (Table 1). It is important to note that a CVA6 strain was also isolated from a nasopharyngeal swab from a 22-month-old child suffering from pneumonia in late March, 2013. The case was reported from Pinar del Rio province in the framework of the Cuban National surveillance and control of acute respiratory infection. Phylogenetic analysis of eight Cuban CVA6 isolates (GenBank Accession Number: KF196256-KF196263) revealed two genetic groups (Fig. 1). Cuban 2011 CVA6 isolates were related to CVA6 strains detected in Europe and Asia between 2007 and 2011. Nevertheless, Cuban isolates obtained between 2012 and 2013 grouped together with emerging CVA6 genetic variants strains isolated in recent international outbreaks. It is noteworthy that most of those strains displayed close relationships to viruses recovered during outbreaks in 2011 in Spain and in 2008 Finland, but one CVA6 strain (12/2013) corresponding to the adult case, formed a separate distinct branch with strains isolated in China (2010–2012) and Thailand (2012). These results suggest the circulation of two genetically distinct CVA6 strains that could have been independently introduced into Cuba from Europe and Southeast Asia, respectively.
Coxsackievirus A6 and enterovirus 71 in Cuba Fig. 1 Phylogenetic analysis of the partial VP1 CA6 sequences from Cuba. The nucleotide sequences were analyzed using the MEGA 5.2.2 package. The phylogenetic tree was constructed by the neighborjoining method with the bootstrap option. Bootstrap values (%) for 1000 replicated trees are indicated at the nodes; only values [70 % are shown. The isolates are indicated by GenBank Accession Numberstrain name-country-year of isolation. The country abbreviations are as follows: CHN China, ESP Spain, FIN Finland, FRA France, JPN Japan, THA Thailand
123
M. C. Fonseca et al. Fig. 2 Phylogenetic analysis of the partial VP1 sequences of enterovirus 71 strains from Cuba. The nucleotide sequences were analyzed using the MEGA 5.2.2 package. The phylogenetic tree was constructed by the neighbor-joining method with the bootstrap option. Bootstrap values (%) for 1000 replicated trees are indicated at the nodes; only values [70 % are shown. The isolates are indicated by GenBank Accession Numberstrain name-country-year of isolation. The country abbreviations are as follows: AUS Australia, CHN China, FIN Finland, FRA France, GER Germany, JPN Japan, NETH Netherlands
Since 2008, several HFMD outbreaks caused by CVA6 have been reported from Finland, Taiwan, Japan, France, Spain, and, recently, the USA and Thailand, 2012. Most attention has been devoted to the atypical features described in recent CVA6 outbreaks. These include unusual age ranges of patients, including adults; severity of illness, characterized by more-severe and extensive rash, eruptions around the perioral area, buttocks, trunk and/or neck; infrequent seasonality of disease; and the association with
123
cases of onychomadesis from 1 to 2 months after the onset of HFMD [1, 4–7, 10, 13]. The identification of CVA6 in patients with acute encephalitis [1, 5], upper respiratory tract symptoms [4] and acute respiratory infection (in this study) confirm that careful surveillance of this agent it will be necessary to recognize emerging outbreaks in order to improve clinical management within the health care system. Taking into account that HFMD is uncommon and not a notifiable disease in Cuba, it is probable that the number
Coxsackievirus A6 and enterovirus 71 in Cuba
of HFMD cases among children and adults in Cuba that have not yet been studied is much higher. It is noteworthy that this is the first identification of EV71 in Cuba, and we therefore conducted further confirmation by nucleotide sequence analysis of the VP2–VP4 region, specific PCR with forward primer MAS01S and reverse primer MAS02A, and a neutralization test using and specific antiserum [2, 3, 9]. Based on the VP1 region, phylogenetic analysis of Cuban EV71 (GenBank: KF801577–F801578) strains showed that they belonged to genotype C2 and were closely related to viruses of various geographical origins (Fig. 2). Based on the known epidemiological behavior of EV71 in the Americas and the AsiaPacific region, the virus probably exists in most countries, causing sporadic cases or small outbreaks [11]. Nevertheless, the establishment of an international network is an urgent necessity in order to gain an overview of this disease and the serotypes involved. The emergence in Cuba of these two significant human pathogens, EV71 and CVA6, is of global interest because of the understanding it may provide for the increase in emerging and re-emerging viral diseases worldwide. In the future, EV71 and CVA6 activity in our country must be monitored by strengthening and maintaining the virological sentinel surveillance system to trace the propagation of the virus across the country and provide a public-health approach focused on early warning of virus transmission and differential diagnostics in the community. Acknowledgments We thank the patients, parents and physicians for participation in this study. We acknowledge the assistance from Cuban Provincial Centers for Epidemiology, Hygiene and Microbiology from Cienfuegos and Sancti Spiritus provinces for collecting the samples. We also thank Dr. Lı´dice Bernardo for helpful comments, and Dr. Ondrej Mach for reviewing the manuscript. This study was supported by the Cuban Ministry of Health as part of an enterovirus surveillance system maintained by the National Enterovirus Laboratory at Tropical Medicine Institute ‘‘Pedro Kourı´’’.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13. Conflict of interest of interest.
The authors declare that they have no conflict 14.
References
coxsackieviruses A6 and A10 in hand, foot and mouth disease outbreak in Finland. J Clin Virol 48:49–54 Cardosa MJ, Perera D, Brown BA, Cheon D, Chan HM, Chan KP et al (2003) Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes. Emerg Infect Dis 9:461–468 Coiras MT, Aguilar JC, Garcia ML, Casas I, Perez-Brena P (2004) Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nestedPCR assays. J Med Virol 72:484–495 Flett K, Youngster I, Huang J, McAdam A, Sandora TJ, Rennick M et al (2012) Hand, foot, and mouth disease caused by coxsackievirus A6. Emerg Infect Dis 18:1702–1704 Fujimoto T, Iizuka S, Enomoto M, Abe K, Yamashita K, Hanaoka N et al (2012) Hand, foot, and mouth disease caused by coxsackievirus A6, Japan, 2011. Emerg Infect Dis 18:337–339 Mirand A, Henquell C, Archimbaud C, Ughetto S, Antona D, Bailly JL et al (2012) Outbreak of hand, foot and mouth disease/ herpangina associated with coxsackievirus A6 and A10 infections in 2010, France: a large citywide, prospective observational study. Clin Microbiol Infect 18:E110–E118 Montes M, Artieda J, Pin˜eiro LD, Gastesi M, Diez-Nieves I, Cilla G (2013) Hand, foot, and mouth disease outbreak and coxsackievirus A6, Northern Spain, 2011. Emerg Infect Dis 19:676–677 Nix WA, Oberste MS, Pallansch MA (2006) Sensitive, seminested PCR amplification of VP1 sequences for direct identification of all enterovirus serotypes from original clinical specimens. J Clin Microbiol 44:2698–2704 Perera D, Podin Y, Akin W, Tan WS, Cardosa MJ (2004) Incorrect identification of recent Asian strains of coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT-PCR. BMC Infect Dis 4:11 Puenpa J, Chieochansin T, Linsuwanon P, Korkong S, Thongkomplew S, Vichaiwattana P et al (2013) Hand, foot, and mouth disease caused by coxsackievirus A6, Thailand, 2012. Emerg Infect Dis 19:641–643 Solomon T, Lewthwaite P, Perera D, Cardosa MJ, McMinn P, Ooi MH (2010) Virology, epidemiology, pathogenesis, and control of enterovirus 71. Lancet Infect Dis 10:778–790 Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739 Wei SH, Huang YP, Liu MC, Tsou TP, Lin HC, Lin TL et al (2011) An outbreak of coxsackievirus A6 hand, foot, and mouth disease associated with onychomadesis in Taiwan, 2010. BMC Infect Dis 11:346 Yang CF, De L, Yang SJ, Go´mez JR, Cruz JR, Halloway MA, Pallansch MA, Kew OM (1992) Genotype-specific in vitro amplification of sequences of the wild type 3 polioviruses from Me´xico and Guatemala. Virus Res 24:277–296
1. Blomqvist S, Klemola P, Kaijalainen S, Pananen A, Simonen ML, Vuorinen T, Roivainen M (2010) Co-circulation of
123
