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ABSTRACT |
 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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Dengue fever and DHF were first observed in the Yucatan State of Mexico in 1979 and 1984, respectively.3,4 Dengue epidemics have been reported in the Yucatan State in 1979–1982 (DEN-1),3,5 1984 (DEN-1 and DEN-4),4 1991 (DEN-2 and DEN-4), 1994 (DEN-1, DEN-2, and DEN-4), and 1995–1997 (all serotypes) (Farfán-Ale JA, unpublished data). Since 1983, the Laboratorio de Arbovirología of the Universidad Autónoma de Yucatán (UADY) has conducted DEN virus surveillance in the Yucatán State.
Studies on the envelope (E) gene sequences of many strains of DEN-2 virus have shown genetic variation worldwide. The maximum-likelihood trees constructed by Twiddy and others6 with 142 isolates of DEN-2 virus showed that these strains can be classified into five clusters termed genotypes. The American genotype consists of recent isolates from Latin America, including Mexico, and older isolates from India, the Caribbean, and the Pacific Islands. The Asian genotype 1 cluster contains isolates from Thailand and Malaysia. The Asian genotype 2 is composed of isolates from China, the Philippines, Sri Lanka, Taiwan, and Vietnam. The American/Asian genotype consists of isolates from China, Thailand, Vietnam, Brazil, Venezuela, and the Caribbean. The Cosmopolitan genotype cluster contains isolates from Australia, the Pacific Islands, Southeast Asia, the Indian Sub-continent, the Middle East, Africa, and Mexico.
The incidence of epidemic dengue and DHF/DSS has increased dramatically in the last decade. The original emergence of DHF and DSS in the Americas was associated the introduction of a virulent genotype of DEN-2 virus from Southeast Asia.7–9 Introduction of a new genotype of DEN-3 virus was also associated with increased incidence of DHF and DSS in many countries in Latin America.10–12 Clearly, epidemics of DHF and DSS can result from the introduction of new viral genotypes or serotypes, which displace other viruses.9,13,14 More information on the risk factors that condition epidemic dengue and DHF/DSS in Latin America and the clinical and epidemiologic consequences of such introductions is warranted and critical.
Here we report the introduction of a new DEN-2 virus of the American/Asian genotype into the Yucatan State. We also describe the clinical, virologic, and epidemiologic correlates of a DEN-2 virus outbreak in 2002.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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A total of 1,804 serum samples were obtained from 1,560 patients who presented with symptoms consistent with a DEN-like illness. All patients were residents of the Yucatan State. The travel history of each study participant was recorded, and any patient who had traveled outside the Yucatan State in the 15 days prior to disease onset was excluded from the study. The majority of the samples were collected during routine diagnosis of DEN virus infection from January 1 to December 31, 2002. Samples were also collected as part of the human investigations protocol "Molecular Determinants of Dengue Epidemic Potential (A Pilot Study to Define Dengue Virus Variants Circulating in the Yucatan)," which has been reviewed and approved by the institutional review boards of the IMSS, UADY, Colorado State University, and the Division of Microbiology and Infectious Diseases of the National Institutes of Health. Upon presentation at the Laboratorio de Arbovirología, patients were invited to participate in a study to identify the infecting agent. Patients were informed of the clinical and epidemiologic aspects of dengue, and of the importance of providing both acute and convalescent blood samples for laboratory diagnosis.
Standard epidemiologic information was obtained from all patients including age, sex, date of illness onset, date of sample collection, clinical information and results of tourniquet and laboratory tests. The DF and DHF cases were diagnosed by clinical, hematologic, serologic, and/or virologic observations. The DHF cases were graded for severity by using the World Health Organization (WHO) criteria.15 Questionnaire data and clinical and laboratory results were entered in the database and statistical package Epi-Info version 6.04.16
Paired serum samples (acute and convalescent) were obtained from 244 patients and a single serum (acute only) was obtained from 1,316 patients. A signed consent form was obtained from all patients or legal guardians who presented at the Laboratorio de Arbovirología.
Serologic and virologic tests. A patient was considered to have a recent DEN infection if he or she met one or more of the following criteria: 1) IgM antibody was detected in either the acute or convalescent phase serum by antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA), 2) the antibody titer in the convalescent phase serum was at least four-fold greater than in the acute phase serum as determined by hemagglutination-inhibition (HI) assay, 3) DEN virus was isolated by cell culture and detected by indirect immunofluorescence assay (IFA), or 4) DEN virus RNA was amplified by a reverse transcription–polymerase chain reaction (RT-PCR).
Antibody-capture enzyme-linked immunosorbent assay.
The MAC-ELISAs were performed as described by Kuno and others.17 Briefly, sheep anti-human IgM (Kirekegaard and Perry Laboratories, Gaithersburg, MD) was used as the capture antibody and 6B6C-1 was used as the detector antibody.18 The MAC-ELISA antigen was prepared from all four DEN virus serotypes and pooled before use.17 A serum sample was considered positive for antibodies to DEN if the optical density value was 
0.2, as recommended by the Centers for Disease Control and Prevention. All patients were tested for antibodies to DEN virus by MAC-ELISA.
Hemaglutination-inhibition assays. The HI assays were performed following the protocol of Clarke and Casals.19 The assays were conducted typically with DEN-2 virus antigen, and sera were treated with kaolin and erythrocytes to eliminate nonspecific inhibitors. Hemagglutination-inhibition is a sensitive test for detecting antibodies to flaviviruses, but is not specific. However, the test does permit differentiation of primary or secondary DEN infections when acute and convalescent sera are separated by at least seven days. A patient was considered to have a recent flavivirus infection if the antibody titer of the convalescent serum was at least four times higher than the antibody titer of the acute serum. If antibodies to DEN virus were detected in the acute phase serum, but a convalescent phase serum was not available for testing, the patient could not be definitively diagnosed. The HI assays were performed on all patients negative for DEN virus infection by the MAC-ELISA. Generally, patients positive by the MAC-ELISA were not tested by the HI assay.
Virus isolation and IFA. For virus isolation, patient specimens were inoculated onto C6/36 mosquito cells and incubated at 28°C for at least 10 days. Cells were harvested, fixed in acetone, and isolates were serotyped by an indirect IFA.20 The following monoclonal antibodies (MAbs) were used in the IFAs to identify the infecting DEN virus serotype: DEN-1 virus-specific MAb D2-1F1-3, DEN-2 virus-specific MAb 3H5-1-21, DEN-3 virus-specific MAb D6-8A1-12, and DEN-4 virus-specific MAb 1H-10-6-7.21 The majority of sera collected within six days of illness onset were examined by virus isolation and IFA.
Isolation of RNA and RT-PCR.
Dengue virus RNA was isolated from serum samples by using the QIAamp viral RNA kit (Qiagen, Valencia, CA). Seminested RT-PCR amplifications were performed following the protocol of Lanciotti and others.22 Primers used were D1, D2, TS1, TS2, TS3 and TS4 (Table 1
). Briefly, DEN virus RNA was RT-PCR amplified using forward D1 and reverse D2 primers that encompassed a region of the capsid (C) and premembrane (prM) genes of all DEN virus serotypes. This was followed by a nested PCR that used the same forward primer (D1) and a pooled suspension of reverse primers, each specific to one DEN virus serotype. The nested PCR reverse primers were as follows: TS1 (DEN-1 virus-specific), TS2 (DEN-2 virus-specific), TS3 (DEN-3 virus-specific), and TS4 (DEN-4 virus-specific). The expected sizes of the PCR products for DEN-1, DEN-2, DEN-3, and DEN-4 viruses were 482, 119, 290, and 392 base-pairs, respectively. The RT-PCRs were performed on a subset of sera collected within six days of illness onset. However, the majority of sera collected at this time were examined only by virus isolation/IFA because this technique is considerably less expensive than the RT-PCR. No serum sample collected within six days of illness onset was examined by both techniques.
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). Total RNA was then extracted from the cells using a silica-based extraction method (RNeasy; Qiagen). The cDNAs were prepared using primer D2-2588 and Rous-associated virus reverse transcriptase (Amersham, Piscataway, NJ). A 2,474-basepair segment that encompassed that the entire prM and E genes was amplified by PCR using the Expand Long Template PCR System (Roche Diagnostics, Penzburg, Germany) and primers D1 and D2-2588 (Table 1
). The PCR program consisted of 40 cycles of amplification (94°C for 10 seconds, 53°C for 30 seconds, and 68°C for 3 minutes). The PCR products were purified using the Qiaquick PCR Purification Kit (Qiagen) and both strands of the entire E gene were sequenced using the Big Dye terminator cycle sequencing system (Applied Biosystems, Foster City, CA) using eight different primers (Table 1). Maximum parsimony phylogenetic analysis was performed using the Phylogenetic Analysis Using Parsimony (PAUP) 4.10 package.23
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RESULTS |
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Patients were tested for evidence of recent DEN virus infection by MAC-ELISA, HI assay, virus isolation/IFA, and/or RT-PCR. Overall, 282 (18.1%) patients were positive by at least one diagnostic technique, 220 (14.1%) were negative, and diagnostic results were not obtained for 1,058 (67.8%) patients. Of the 1,560 patients tested by MAC-ELISA, 165 (10.6%) were DEN positive. All patients negative by MAC-ELISA were further tested by HI assay. Seventy-six (31.1%) of 244 patients from which paired sera were obtained were positive by HI assay for DEN virus. Sera were obtained from 317 patients within six days of illness onset, and thus examined by RT-PCR or virus isolation/IFA. Eleven (14.1%) of 78 patients were DEN positive by RT-PCR, and 32 (13.4%) of 239 were DEN positive by virus isolation/IFA. Of these, 42 patients were positive for DEN-2 virus and one was positive for DEN-1 virus. A subset of patients were tested for secondary infection using the HI test; 43 (76.8%) of 56 were diagnosed as having a secondary DEN infection.
Dengue disease severity.
Of the 282 DEN-positive cases, 120 (42.6%) were diagnosed as classic DF (without hemorrhagic manifestations), and an additional 75 (26.6%) of the 282 patients were diagnosed as DF with hemorrhagic manifestations (Table 3). Using the WHO criteria, DHF was diagnosed in 87 (30.8%) DEN virus-infected patients. Six DHF cases were considered to be severe (grade IV). Two DHF cases were fatal. The most frequent hemorrhagic clinical manifestations among DHF cases were the presence of petechia (64.4%), a positive tourniquet test result (43.7%), and epistaxis (28.7%) (Table 4). Ninety-seven (34.4%) of the 282 patients with DEN virus infection, including 84 with DHF, were hospitalized. Forty-four (45.4%) of the 97 DEN-infected patients admitted to hospitals were female; 53 (54.6%) were male.
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). When we compared the number of DEN patients with and without hemorrhagic manifestations by chi-square analysis, we observed that 10–14-year-old patients were significantly less likely to develop hemorrhagic symptoms than other age groups (
2 = 10.704) (Table 6). In contrast, 20–25-year-old patients had a significantly greater chance of developing hemorrhagic manifestations (2 = 5.04). The P value obtained in the goodness of fit chi-square test was < 0.005.
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The eight-year-old male patient was a resident of Merida. Clinical manifestations began on October 24, 2002 with general malaise, myalgia, fever, intense headache, severe abdominal pain, and vomiting. Because of fever and uncontrollable vomiting, he was hospitalized. At the time of referral, the patient was severely dehydrated, and had generalized pale, cold, and clammy skin. His pulse became weak and rapid, and he received parenteral solutions. He developed abundant bleeding from mouth, conjunctiva, urethra, and rectum. A blood sample was taken on October 29, and the patient died of DSS later that day. The serum was positive for IgM antibodies to DEN virus by MAC-ELISA. He had experienced a secondary infection as shown by the HI test (antibody titer to DEN-1 virus = 10,240). Dengue 2 virus RNA was amplified from the serum sample by an RT-PCR. Virus was not isolated in cell culture. This patient had the concurrent diagnosis of idiopathic thrombocytopenic purpura.
Epidemiologic results. At least one confirmed DEN case was reported in 42 (39.6%) of the 106 municipalities in Yucatan State. The municipality with the highest number of DEN infections was Merida (n = 144). For most of the confirmed DF and DHF cases (59 and 42 patients, respectively), onset of symptoms occurred during August.
Molecular epidemiology.
Nucleotide sequences representative of previously described genotypes of DEN-2 virus6,8 were obtained from the Genbank database and aligned to the eight Yucatan sequences determined in our studies (Table 2 and Figure 1). The phylogenetic tree constructed from the E gene sequences showed that Mexican DEN-2 virus isolates from the 1980s and early 1990s belong to the American genotype and isolates from the mid 1990s cluster either in the Cosmopolitan or the American genotypes (Figure 1). Isolates from both 2001 and 2002 clustered in the American-Asian genotype. The fact that recent DEN-2 virus isolates are in a different clade from any previous Mexican isolates suggests that the latter are not descendent from the former.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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The DEN-2 viruses belonging to the Southeast Asian genotype appear to be better adapted to mosquito transmission, are associated with more severe clinical outcomes, and are apparently displacing the American genotype of DEN-2 virus in some regions of Latin American.8,9,26 For instance, DEN-2 viruses of the American-Asian genotype were responsible for the first epidemic of DHF in the Americas, which occurred in Cuba in 1981.27,28 Shortly afterwards, viruses of the American-Asian genotype were responsible for the first DHF epidemics in Venezuela (in 1989) and Brazil (in 1990), where they potentially displaced the native American genotype.9,29–31 Thus, the detection of this genotype in the Yucatan State is a major concern to public health authorities. We did not isolate DEN-2 viruses belonging to the American or Cosmopolitan genotypes during the 2002 outbreak. However, additional research is required to determine whether these genotypes have been displaced by the newly-introduced American-Asian genotype. Furthermore, enhanced DEN virus surveillance is needed to determine whether the recent introduction of the American-Asian genotype will coincide with an increase in the incidence and severity of dengue cases in this region.
Indeed, the number of hemorrhagic cases in the Yucatan State since 1984 has increased dramatically. During the 1984 outbreak of DEN-4, there were nine hemorrhagic cases reported, but only one met the WHO criteria for DHF.4 In 2002, 282 DF cases were confirmed. Among these confirmed cases, 162 (57.5%) patients had hemorrhagic manifestations, including 87 cases of DHF, all with severe clinical manifestations.
The procurement of acute and convalescent phase specimens contributed greatly to the diagnosis of DEN infections. The low rate of laboratory confirmation of DEN infections in this study was attributable to a number of factors, but the failure of the patients to return to the laboratory to provide the convalescent phase blood specimen was a major problem. When the acute and convalescent phase specimens were available, DEN infection was confirmed in 41.4% of the patients. These findings are in concordance with other DEN virus outbreaks; typically 30–60% of clinically suspect cases are confirmed to have a DEN virus infection.4,32–34 However, when only the acute phase specimen was available, the confirmation rate decreased to 13.8%. The acute phase sample taken upon presentation (typically five or six days post onset) maximizes potential detection of DEN virus by RT-PCR or virus isolation techniques. The convalescent phase specimen enhances the serologic diagnosis of DEN infections by providing additional time for synthesis of specific IgM for assay by ELISA.
We believe that the total number of cases in this outbreak was underestimated. On September 23, 2002, Hurricane Isidore severely damaged the electrical services, water supplies, and general services in the Yucatán State. Movement and communication between communities were slowed due to extensive road damage and downed power lines. Even when patients were able to access their clinics, samples were not forwarded to the Laboratorio de Arbovirología for diagnosis. For several months following Hurricane Isidore, the only blood samples received at the Laboratorio de Arbovirología for DEN diagnosis were from Merida.
The onset of symptoms for most of the confirmed DF and DHF cases was during August. Presumably rains from April to June contributed to mosquito proliferation, resulting in the increase in DEN virus infections during the following months. Indeed, since 1985, the Yucatan State health authorities have organized campaigns to eliminate mosquito breeding sites and to apply larvicides and adulticides in cities and small rural towns. The most commonly used larvicide is temephos (AbateTM; American Cyanamid, Princeton, NJ) and a pyrethroid adulticide (Aqua ResTM; Bayer Environmental Science, Long Lake [Minneapolis], MN). These activities are conducted before the rainy season, but unfortunately the number of DEN virus infections and more severe disease continue to increase.
Clearly, there are needs to develop less expensive and more specific and sensitive diagnostic techniques. The RT-PCR is too expensive to use as a diagnostic test for all of the suspected cases, including those with classic DF. Therefore, the RT-PCR was only used for diagnosis with hospitalized cases, severe symptoms, and/or hemorrhagic manifestations. New tests, such as the use of an ELISA to detect specific IgA and IgM in sera and saliva of dengue cases,35 offer great potential in this regard for both diagnosis and surveillance. Hopefully, new approaches and techniques will also allow the detection of other medically important flaviviruses such as West Nile, Saint Louis encephalitis, and yellow fever viruses, as well as determination of DEN viruses causing secondary infections. Such tests would be a great addition to the armamentarium of public health practitioners trying to cope with the resurgence of flavivirus diseases in many parts of the world.
In summary, we report the introduction of a new DEN-2 virus of the American/Asian genotype into the Yucatan State. Certainly, expanded surveillance for dengue virus is required in this region to better understand the clinical, virologic, and epidemiologic consequences of this introduction.
Received December 28, 2003. Accepted for publication July 7, 2004.
Acknowledgments: We are grateful to the medical personnel of the Instituto Mexicano del Seguro Social, the Secretaría de Salud, the Instituto de Seguridad y Servicios Sociales de los Trabajadores del Estado, the Servicios Médicos Municipales del Ayuntamiento de Mérida, and all private clinics and physicians for sending us serum samples or the patients to conduct the tests. We thank Dr. Duane J. Gubler and Kathy Wolff (Division of Vector-Borne and Infectious Diseases, Centers for Disease Control and Prevention, Fort Collins, CO) for providing dengue antigens and monoclonal antibodies for the diagnosis of dengue viruses.
Financial support: This study was supported by the National Institutes of Health (grant no. AI-45430) and Sisierra-Conacyt (grant no. 980201).
Authors’ addresses: María A. Loroño-Pino, José A. Farfán Ale, Alicia L. Zapata-Peraza, Elsy P. Rosado-Paredes, Luis F. Flores-Flores and Julián E. García-Rejón, Laboratorio de Arbovirología, Centro de Investigaciones Regionales Dr. Hideyo Noguchi, Universidad Autónoma de Yucatan, Av. Itzáes No. 490 x 59, Centro, Merida, Yucatan, México 97000, Telephone 999-924-6412, Fax: 999-923-1804. Francisco J. Díaz, Bradley J. Blitvich, Carol D. Blair, Ken E. Olson, William Black IV, and Barry J. Beaty, Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, Telephone: 970-491-4383, Fax: 970-491-8323, E-mail: bbeaty{at}colostate.edu. Manuel Andrade-Narváez, Departamento de Medicina Preventiva, Hospital General Regional No. 12, Benito Juárez, Instituto Mexicano del Seguro Social, Av. Colon x Av. Itzáes, Merida, Yucatan, Mexico, Telephone: 999-925-0866, Fax: 999-925-0831. Eusebio Jiménez-Ríos, Dirección de Prevención y Control de Enfermedades, Secretaría de Salud de Yucatán, calle 72 No. 463 x 53 y 55, Merida, Yucatan, México, 97000, Telephone: 999-930-3050.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONREFERENCES |
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