VECTOR-BORNE AND ZOONOTIC DISEASES Volume 15, Number 4, 2015 ª Mary Ann Liebert, Inc. DOI: 10.1089/vbz.2014.1724
SHORT COMMUNICATION
Chikungunya from the Caribbean: The Importance of Appropriate Laboratory Tests to Confirm the Diagnosis Fabio Magurano,1,4 Lorenzo Zammarchi,2,4 Melissa Baggieri,1 Claudia Fortuna,1 Alberto Farese,3 Eleonora Benedetti,1 Cristiano Fiorentini,1 Giovanni Rezza,1 Loredana Nicoletti,1 and Alessandro Bartoloni 2,3
Abstract
Chikungunya virus (CHIKV) appeared for the first time in the Western Hemisphere—the French West Indies— in December of 2013. From there, the virus has spread to other Caribbean islands. Following the diagnosis of first autochthonous CHIKV cases in the Caribbean island of Saint Martin, a large outbreak is ongoing in the Americas. As of September 12, 2014, a total of 706,093 suspected and 9803 confirmed CHIKV cases have been reported in the Americas. This case study highlights the possibility of false-negative immunochromatographic CHIKV immunoglobulin M (IgM) tests and the need of confirmatory tests for suspected cases. Moreover, a greater spread of virus together with the presence of a mosquito vector (Aedes albopictus) enhances the risk of autochthonous transmission in Europe. Key Words:
Chikungunya—Dominican Republic—Italy—Rapid diagnostic test—Immunochromatographic IgM
test.
Introduction
C
hikungunya virus (CHIKV) is a member of the Togaviridae family, belonging to genus Alphavirus and classified among the arbovirus group (Burt et al. 2012). CHIKV causes an acute febrile illness, which may be accompanied by skin rash, headache, and polyarthralgia with or without signs of arthritis such as joint swelling. Polyarthralgia is common and may be particularly disabling, persisting for several months, especially in older subjects and in those having previous rheumatologic diseases (Rezza et al. 2007). Rarely, the infection can lead to life-threatening manifestations, including neurological complications, cardiovascular disorders, pneumonia, prerenal failure, and respiratory failure. The incubation period ranges from 1 to 12 days (usually 2–4 days). Phylogenetic analysis showed that CHIKV clustered into three major distinct lineages—Asian, East/Central/South African (ECSA), and West African (WA). The virus is principally transmitted by Aedes (Ae.) mosquitoes, such as Ae. aegypti and Ae. albopictus. Until December, 2013, the geographical distribution of CHIKV was limited to the Old World, namely Africa,
Indian Ocean Islands, the Indian Subcontinent, and South East Asia. Two limited introduced outbreaks were reported from temperate countries, such as Italy (2007) and France (2010) (Rezza et al. 2007, Grandadam et al. 2011). Since December, 2013, following the diagnosis of the first autochthonous CHIKV case in the Caribbean island of Saint Martin, a large outbreak is now underway for the first time in the Americas (LeparcGoffart et al. 2014). As of September 12, 2014, a total of 706,093 suspected and 9803 confirmed CHIKV cases have been reported in the cases from the Americas, including some autochthonous cases acquired in Florida (World Health Organization 2014a, b). We described an imported case of CHIKV from the Caribbean to Italy with laboratory confirmation through PCR. To date, relatively few imported cases from the Caribbean to Europe have been reported. Case Study
On June 10 (day 1), 2014, a previously healthy 50-year-old Dominican female developed severe joint pain 5 days after returning to Italy from a 1-month trip in her country of origin.
1
Department of Infectious, Parasitic and Immune-Mediate Diseases, Istituto Superiore di Sanita`, Rome, Italy. Clinica Malattie Infettive, Dipartimento di Medicina Sperimentale e Clinica, Universita` degli Studi di Firenze, Florence, Italy. SOD Malattie Infettive e Tropicali, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy. 4 These authors contributed equally to this article. 2 3
258
CHIKUNGUNYA FROM THE CARIBBEAN
On June 12, 2014 (day 3), she consulted at the Infectious and Tropical Diseases Unit, Careggi University Hospital, Florence, Italy. Her clinical examination was normal except for altered tympanic body temperature (37.7C). There were no signs of arthritis. Blood tests (day 3) revealed slight normocytic–normochromic anemia (hemoglobin [Hb] 10.5 grams/ dL, reference values 12–16 grams/dL), leukopoenia (2990 white blood cells/lL, reference values 4000–10,000, and white blood cells/lL) with relative monocytosis (14.7%, reference values 3–12%); platelets and transaminases were within the normal ranges. Thin and thick blood slides for malaria and an immunochromatographic test for Plasmodium antigens were all negative. Tests for dengue IgM (Capture ELISA, Panbio Diagnostics) and dengue NS1 antigen (Platelia Dengue NS1 Ag test, Bio-Rad Laboratories) were negative, whereas the test for dengue immunoglobulin G (IgG; Capture ELISA, Panbio Diagnostics) was positive, suggesting a previous dengue infection (all performed on day 3). The immunochromatographic test for chikungunya IgM (OnSite Chikungunya IgM Combo Rapid Test-Cassette, CTK Biotech) was negative, as the test was repeated on day 7. Acute and follow-up serum samples were sent to the Istituto Superiore di Sanita` (Rome, Italy) for CHIKV infection confirmation. An enzyme-linked immunosorbent assay (ELISA) IgM (NovaTec Immunodiagnostica GmbH) test resulted in a borderline result at day 3 and a negative result at day 7 after symptoms onset in a sample taken during a followup visit. A plaque reduction neutralization test by 80% (PRNT80) was negative in the first sample and positive (50%) in the second sample. Real-time PCR performed on the first sample was positive, leading to laboratory confirmation of the suspected CHIKV case. A 512-nucleotide fragment within the envelope protein E1 gene was amplified and sequenced (Edwards et al. 2007). Phylogenetic analysis was performed with MEGA6 software (Tamura et al. 2013). A phylogenetic tree was constructed by using the maximum likelihood method based on the Kimura 2-parameter model
259
(Kimura 1980). The reliability of the tree was estimated with a bootstrap analysis by 1000 replicates. The virus clustered with other Asian strains (Fig. 1), and, as expected, it did not show the single amino acid substitution in the envelope protein gene (E1-A226V) present in the strain responsible for the Indian Ocean islands and Italian outbreaks (Tsetsarkin et al. 2007). The same samples were both negative by an immunochromatographic test for chikungunya IgM (OnSite Chikungunya IgM Combo Rapid Test-Cassette, CTK Biotech). Conclusion
To date, relatively few imported cases transported from the Caribbean to Europe have been reported (Vazeille et al. 2007, Fischer et al. 2013). Italy is a European country heavily infested by Ae. albopictus, a competent vector for different arboviruses, including CHIKV, and a large introduced autochthonous outbreak had already occurred in 2007. Even though the Caribbean isolates do not show the E1-A226V mutation, previous studies have indicated that the CHIKV strains without this mutation are capable of producing high enough viremia in humans to infect Ae. albopictus mosquitoes efficiently (Tsetsarkin et al. 2007). For these reasons, early laboratory diagnosis and notification of suspected CHIKV cases are particularly important in our country, especially during the period of activity of Ae. albopictus (conventionally between June 15 and November 30), so that local transmission can be avoided by implementing adequate vector control measures. However, the diagnosis of imported CHIKV may be challenging for a few reasons. First, given the relatively unspecific signs and symptoms and the new epidemiological scenario of CHIKV in the Americas, clinicians may overlook this condition when managing febrile patients returning from the Caribbean and not requesting the correct diagnostic tests for laboratory confirmation. Second, as highlighted by this report, a false-negative immunochromatographic chikungunya IgM
FIG. 1. The Bayesian Information Criterion (BIC) was used to determine the model of nucleotide substitution that best fit the data using the selection tool available in MEGA6. Evolutionary analyses were conducted using the maximum likelihood method based on the Kimura 2-parameter model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Significant bootstrap values ( > 98%) are shown. ECSA, East/Central/South Africa; WA, West Africa.
260
test is possible, suggesting the need for a confirmatory test for suspected cases with negative rapid test results. The possibility of false-negative rapid tests has already been reported. Kosasish reported a sensitivity of 28.6% for the OnSite Chikungunya IgM rapid diagnostic test in samples tested between 6 and 10 days after onset of symptoms (Kosasih et al. 2012). These results were also confirmed by other authors (Prat et al. 2014). A previous study suggested that the differences between these values could be due to genetic differences in the viruses circulating in the regions where the samples were collected (Yap et al. 2010). Moreover, a remarkable difference in sensitivity of the OnSite Chikungunya IgM rapid diagnostic test according with two different viral strains of the EACS phylogroup has been reported (Vazeille et al. 2007). Among 10 CHIKV-confirmed patients infected with the viral variant V226, none had detectable IgM within 7 days after onset of fever, whereas among patients infected with the viral variant A226 all had positive tests 7 days after onset of symptoms. The most likely explanation for this discrepancy is that the rapid test kit used a recombinant antigen covering the 226 residue of the E1 gene derived from CHIKV-A226. The sequence of the S317 Italy 2014 strain lacking the E1-A226V mutation strongly supports this hypothesis. The occurrence of an outbreak of CHIKV infection in Italy showed that the predicted globalization of humans and vectors has become a reality in countries with a temperate climate. The spread of CHIKV in the Western Hemisphere emphasizes the risk of introduction of this and other arboviral diseases. Given the relatively new epidemiological scenario of CHIKV in the Americas, clinicians should include CHIKV in the differential diagnosis of febrile travelers returning from this region. Early notification and laboratory diagnosis of suspected CHIKV cases are particularly important in the warm season in European countries infested with Ae. albopictus so that adequate vector control measures aimed at avoiding local transmission can be implemented. Acknowledgements
We are grateful to Dr. Maria Elena Remoli, Dr. Antonella Marchi, and Mrs. Paola Bucci for technical support. Author Disclosure Statement
No competing financial interests exist. References
Burt FJ, Rolph MS, Rulli NE, Mahalingam S, et al. Chikungunya: A re-emerging virus. Lancet 2012; 379:662–671. Edwards CJ, Welch SR, Chamberlain J, Hewson R, et al. Molecular diagnosis and analysis of Chikungunya virus. J Clin Virol 2007; 39:271–275. Fischer D, Thomas SM, Suk JE, Sudre B, et al. Climate change effects on chikungunya transmission in Europe: Geospatial
MAGURANO ET AL.
analysis of vector’s climatic suitability and virus’ temperature requirements. Int J Health Geogr. 2013;12:51. Grandadam M, Caro V, Plumet S, Thiberge J-M, et al. Chikungunya virus, southeastern France. Emerg Infect Dis 2011; 17:910–913. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16:111–120. Kosasih H, Widjaja S, Surya E, Hadiwijaya SH, et al. Evaluation of two IgM rapid immunochromatographic tests during circulation of Asian lineage Chikungunya virus. Southeast Asian J Trop Med Public Health 2012; 43:55–61. Leparc-Goffart I, Nougairede A, Cassadou S, Prat C, et al. Chikungunya in the Americas. Lancet 2014; 383:514. Prat CM, Flusin O, Panella A, Tenabray B, et al. Evaluation of commercially available serologic diagnostic tests for chikungunya virus. Emerg Infect Dis 2014; 20:2129–2132. Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, Infection with chikungunya virus in Italy: An outbreak in a temperate region. Lancet. 2007; 370:1840– 1846. Tamura K, Stecher G, Peterson D, Filipski A, et al. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 2013; 30:2725–2729. Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog 2007; 3:e201. Vazeille M, Moutailler S, Coudrier D, Rousseaux C, et al. Two chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus. PLoS One 2007; 2:e1168. World Health Organization, Pan American Health Organization. Number of Reported Cases of Chikungunya Fever in the Americas, by Country or Territory 2013–2014. EW 37, updated September 12, 2014a. Available at www.paho.org/ hq/index.php?option=com_docman&task=doc_download& ltemid=&gid=27274&lang=en World Health Organization, Pan American Health Organization. Countries and territories with autochthonous transmission or imported cases of Chikungunya in the Americas. EW 34, 2014b. Available at www.paho.org/hq/images/stories/AD/ HSD/IR/Viral_Diseases/Chikungunya/CHIKV-Data-Caribe2014-EW-34.jpg?ua = 1 Yap G, Pok KY, Lai YL, Hapuarachchi HC, et al. Evaluation of chikungunya diagnostic assays: differences in sensitivity of serology assays in two independent outbreaks. PLos Negl Trop Dis 2010; 20:e753.
Address correspondence to: Fabio Magurano Department of Infectious, Parasitic and Immune-Mediate Diseases Istituto Superiore di Sanita` Viale Regina Elena 299 Rome 00161, Italy E-mail: [email protected]
SHORT COMMUNICATION
Chikungunya from the Caribbean: The Importance of Appropriate Laboratory Tests to Confirm the Diagnosis Fabio Magurano,1,4 Lorenzo Zammarchi,2,4 Melissa Baggieri,1 Claudia Fortuna,1 Alberto Farese,3 Eleonora Benedetti,1 Cristiano Fiorentini,1 Giovanni Rezza,1 Loredana Nicoletti,1 and Alessandro Bartoloni 2,3
Abstract
Chikungunya virus (CHIKV) appeared for the first time in the Western Hemisphere—the French West Indies— in December of 2013. From there, the virus has spread to other Caribbean islands. Following the diagnosis of first autochthonous CHIKV cases in the Caribbean island of Saint Martin, a large outbreak is ongoing in the Americas. As of September 12, 2014, a total of 706,093 suspected and 9803 confirmed CHIKV cases have been reported in the Americas. This case study highlights the possibility of false-negative immunochromatographic CHIKV immunoglobulin M (IgM) tests and the need of confirmatory tests for suspected cases. Moreover, a greater spread of virus together with the presence of a mosquito vector (Aedes albopictus) enhances the risk of autochthonous transmission in Europe. Key Words:
Chikungunya—Dominican Republic—Italy—Rapid diagnostic test—Immunochromatographic IgM
test.
Introduction
C
hikungunya virus (CHIKV) is a member of the Togaviridae family, belonging to genus Alphavirus and classified among the arbovirus group (Burt et al. 2012). CHIKV causes an acute febrile illness, which may be accompanied by skin rash, headache, and polyarthralgia with or without signs of arthritis such as joint swelling. Polyarthralgia is common and may be particularly disabling, persisting for several months, especially in older subjects and in those having previous rheumatologic diseases (Rezza et al. 2007). Rarely, the infection can lead to life-threatening manifestations, including neurological complications, cardiovascular disorders, pneumonia, prerenal failure, and respiratory failure. The incubation period ranges from 1 to 12 days (usually 2–4 days). Phylogenetic analysis showed that CHIKV clustered into three major distinct lineages—Asian, East/Central/South African (ECSA), and West African (WA). The virus is principally transmitted by Aedes (Ae.) mosquitoes, such as Ae. aegypti and Ae. albopictus. Until December, 2013, the geographical distribution of CHIKV was limited to the Old World, namely Africa,
Indian Ocean Islands, the Indian Subcontinent, and South East Asia. Two limited introduced outbreaks were reported from temperate countries, such as Italy (2007) and France (2010) (Rezza et al. 2007, Grandadam et al. 2011). Since December, 2013, following the diagnosis of the first autochthonous CHIKV case in the Caribbean island of Saint Martin, a large outbreak is now underway for the first time in the Americas (LeparcGoffart et al. 2014). As of September 12, 2014, a total of 706,093 suspected and 9803 confirmed CHIKV cases have been reported in the cases from the Americas, including some autochthonous cases acquired in Florida (World Health Organization 2014a, b). We described an imported case of CHIKV from the Caribbean to Italy with laboratory confirmation through PCR. To date, relatively few imported cases from the Caribbean to Europe have been reported. Case Study
On June 10 (day 1), 2014, a previously healthy 50-year-old Dominican female developed severe joint pain 5 days after returning to Italy from a 1-month trip in her country of origin.
1
Department of Infectious, Parasitic and Immune-Mediate Diseases, Istituto Superiore di Sanita`, Rome, Italy. Clinica Malattie Infettive, Dipartimento di Medicina Sperimentale e Clinica, Universita` degli Studi di Firenze, Florence, Italy. SOD Malattie Infettive e Tropicali, Azienda Ospedaliero-Universitaria Careggi, Florence, Italy. 4 These authors contributed equally to this article. 2 3
258
CHIKUNGUNYA FROM THE CARIBBEAN
On June 12, 2014 (day 3), she consulted at the Infectious and Tropical Diseases Unit, Careggi University Hospital, Florence, Italy. Her clinical examination was normal except for altered tympanic body temperature (37.7C). There were no signs of arthritis. Blood tests (day 3) revealed slight normocytic–normochromic anemia (hemoglobin [Hb] 10.5 grams/ dL, reference values 12–16 grams/dL), leukopoenia (2990 white blood cells/lL, reference values 4000–10,000, and white blood cells/lL) with relative monocytosis (14.7%, reference values 3–12%); platelets and transaminases were within the normal ranges. Thin and thick blood slides for malaria and an immunochromatographic test for Plasmodium antigens were all negative. Tests for dengue IgM (Capture ELISA, Panbio Diagnostics) and dengue NS1 antigen (Platelia Dengue NS1 Ag test, Bio-Rad Laboratories) were negative, whereas the test for dengue immunoglobulin G (IgG; Capture ELISA, Panbio Diagnostics) was positive, suggesting a previous dengue infection (all performed on day 3). The immunochromatographic test for chikungunya IgM (OnSite Chikungunya IgM Combo Rapid Test-Cassette, CTK Biotech) was negative, as the test was repeated on day 7. Acute and follow-up serum samples were sent to the Istituto Superiore di Sanita` (Rome, Italy) for CHIKV infection confirmation. An enzyme-linked immunosorbent assay (ELISA) IgM (NovaTec Immunodiagnostica GmbH) test resulted in a borderline result at day 3 and a negative result at day 7 after symptoms onset in a sample taken during a followup visit. A plaque reduction neutralization test by 80% (PRNT80) was negative in the first sample and positive (50%) in the second sample. Real-time PCR performed on the first sample was positive, leading to laboratory confirmation of the suspected CHIKV case. A 512-nucleotide fragment within the envelope protein E1 gene was amplified and sequenced (Edwards et al. 2007). Phylogenetic analysis was performed with MEGA6 software (Tamura et al. 2013). A phylogenetic tree was constructed by using the maximum likelihood method based on the Kimura 2-parameter model
259
(Kimura 1980). The reliability of the tree was estimated with a bootstrap analysis by 1000 replicates. The virus clustered with other Asian strains (Fig. 1), and, as expected, it did not show the single amino acid substitution in the envelope protein gene (E1-A226V) present in the strain responsible for the Indian Ocean islands and Italian outbreaks (Tsetsarkin et al. 2007). The same samples were both negative by an immunochromatographic test for chikungunya IgM (OnSite Chikungunya IgM Combo Rapid Test-Cassette, CTK Biotech). Conclusion
To date, relatively few imported cases transported from the Caribbean to Europe have been reported (Vazeille et al. 2007, Fischer et al. 2013). Italy is a European country heavily infested by Ae. albopictus, a competent vector for different arboviruses, including CHIKV, and a large introduced autochthonous outbreak had already occurred in 2007. Even though the Caribbean isolates do not show the E1-A226V mutation, previous studies have indicated that the CHIKV strains without this mutation are capable of producing high enough viremia in humans to infect Ae. albopictus mosquitoes efficiently (Tsetsarkin et al. 2007). For these reasons, early laboratory diagnosis and notification of suspected CHIKV cases are particularly important in our country, especially during the period of activity of Ae. albopictus (conventionally between June 15 and November 30), so that local transmission can be avoided by implementing adequate vector control measures. However, the diagnosis of imported CHIKV may be challenging for a few reasons. First, given the relatively unspecific signs and symptoms and the new epidemiological scenario of CHIKV in the Americas, clinicians may overlook this condition when managing febrile patients returning from the Caribbean and not requesting the correct diagnostic tests for laboratory confirmation. Second, as highlighted by this report, a false-negative immunochromatographic chikungunya IgM
FIG. 1. The Bayesian Information Criterion (BIC) was used to determine the model of nucleotide substitution that best fit the data using the selection tool available in MEGA6. Evolutionary analyses were conducted using the maximum likelihood method based on the Kimura 2-parameter model. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Significant bootstrap values ( > 98%) are shown. ECSA, East/Central/South Africa; WA, West Africa.
260
test is possible, suggesting the need for a confirmatory test for suspected cases with negative rapid test results. The possibility of false-negative rapid tests has already been reported. Kosasish reported a sensitivity of 28.6% for the OnSite Chikungunya IgM rapid diagnostic test in samples tested between 6 and 10 days after onset of symptoms (Kosasih et al. 2012). These results were also confirmed by other authors (Prat et al. 2014). A previous study suggested that the differences between these values could be due to genetic differences in the viruses circulating in the regions where the samples were collected (Yap et al. 2010). Moreover, a remarkable difference in sensitivity of the OnSite Chikungunya IgM rapid diagnostic test according with two different viral strains of the EACS phylogroup has been reported (Vazeille et al. 2007). Among 10 CHIKV-confirmed patients infected with the viral variant V226, none had detectable IgM within 7 days after onset of fever, whereas among patients infected with the viral variant A226 all had positive tests 7 days after onset of symptoms. The most likely explanation for this discrepancy is that the rapid test kit used a recombinant antigen covering the 226 residue of the E1 gene derived from CHIKV-A226. The sequence of the S317 Italy 2014 strain lacking the E1-A226V mutation strongly supports this hypothesis. The occurrence of an outbreak of CHIKV infection in Italy showed that the predicted globalization of humans and vectors has become a reality in countries with a temperate climate. The spread of CHIKV in the Western Hemisphere emphasizes the risk of introduction of this and other arboviral diseases. Given the relatively new epidemiological scenario of CHIKV in the Americas, clinicians should include CHIKV in the differential diagnosis of febrile travelers returning from this region. Early notification and laboratory diagnosis of suspected CHIKV cases are particularly important in the warm season in European countries infested with Ae. albopictus so that adequate vector control measures aimed at avoiding local transmission can be implemented. Acknowledgements
We are grateful to Dr. Maria Elena Remoli, Dr. Antonella Marchi, and Mrs. Paola Bucci for technical support. Author Disclosure Statement
No competing financial interests exist. References
Burt FJ, Rolph MS, Rulli NE, Mahalingam S, et al. Chikungunya: A re-emerging virus. Lancet 2012; 379:662–671. Edwards CJ, Welch SR, Chamberlain J, Hewson R, et al. Molecular diagnosis and analysis of Chikungunya virus. J Clin Virol 2007; 39:271–275. Fischer D, Thomas SM, Suk JE, Sudre B, et al. Climate change effects on chikungunya transmission in Europe: Geospatial
MAGURANO ET AL.
analysis of vector’s climatic suitability and virus’ temperature requirements. Int J Health Geogr. 2013;12:51. Grandadam M, Caro V, Plumet S, Thiberge J-M, et al. Chikungunya virus, southeastern France. Emerg Infect Dis 2011; 17:910–913. Kimura M. A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 1980; 16:111–120. Kosasih H, Widjaja S, Surya E, Hadiwijaya SH, et al. Evaluation of two IgM rapid immunochromatographic tests during circulation of Asian lineage Chikungunya virus. Southeast Asian J Trop Med Public Health 2012; 43:55–61. Leparc-Goffart I, Nougairede A, Cassadou S, Prat C, et al. Chikungunya in the Americas. Lancet 2014; 383:514. Prat CM, Flusin O, Panella A, Tenabray B, et al. Evaluation of commercially available serologic diagnostic tests for chikungunya virus. Emerg Infect Dis 2014; 20:2129–2132. Rezza G, Nicoletti L, Angelini R, Romi R, Finarelli AC, Panning M, Infection with chikungunya virus in Italy: An outbreak in a temperate region. Lancet. 2007; 370:1840– 1846. Tamura K, Stecher G, Peterson D, Filipski A, et al. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol 2013; 30:2725–2729. Tsetsarkin KA, Vanlandingham DL, McGee CE, Higgs S. A single mutation in chikungunya virus affects vector specificity and epidemic potential. PLoS Pathog 2007; 3:e201. Vazeille M, Moutailler S, Coudrier D, Rousseaux C, et al. Two chikungunya isolates from the outbreak of La Reunion (Indian Ocean) exhibit different patterns of infection in the mosquito, Aedes albopictus. PLoS One 2007; 2:e1168. World Health Organization, Pan American Health Organization. Number of Reported Cases of Chikungunya Fever in the Americas, by Country or Territory 2013–2014. EW 37, updated September 12, 2014a. Available at www.paho.org/ hq/index.php?option=com_docman&task=doc_download& ltemid=&gid=27274&lang=en World Health Organization, Pan American Health Organization. Countries and territories with autochthonous transmission or imported cases of Chikungunya in the Americas. EW 34, 2014b. Available at www.paho.org/hq/images/stories/AD/ HSD/IR/Viral_Diseases/Chikungunya/CHIKV-Data-Caribe2014-EW-34.jpg?ua = 1 Yap G, Pok KY, Lai YL, Hapuarachchi HC, et al. Evaluation of chikungunya diagnostic assays: differences in sensitivity of serology assays in two independent outbreaks. PLos Negl Trop Dis 2010; 20:e753.
Address correspondence to: Fabio Magurano Department of Infectious, Parasitic and Immune-Mediate Diseases Istituto Superiore di Sanita` Viale Regina Elena 299 Rome 00161, Italy E-mail: [email protected]
