133 Rev Biomed 1997; 8:133-138.

Original article

A dengue fever outbreak caused by dengue 4 in the state of Puebla, Mexico.

José L. Imbert-Palafox1, Consuelo Parra1, Jorge M. Villaseca2, Herlinda García2, Celso Ramos2.

1

Centro de Investigaciones Microbiológicas, Benemérita Universidad Autónoma de Puebla, Puebla, México. 2Centro de Investigaciones Sobre Enfermedades Infecciosas, Instituto Nacional de Salud Pública, Cuernavaca, México.

RESUMEN. Introducción. En 1991 ocurrió en el estado de Puebla un brote de fiebre por dengue. El presente trabajo reporta los estudios epidemiológicos y virológicos realizados durante este brote en Ajalpan, Estado de Puebla, México. Material y métodos. La detección de anticuerpos se realizó mediante la técnica de inhibición de la hemaglutinación (IH). El aislamiento se efectuó en células de mosquito (TRA-284) y la identificación del serotipo se realizo por inmunofluorescencia indirecta usando anticuerpos monoclonales. Resultados. El brote de dengue se presentó en mayo de 1991 en la población de Ajalpan, Estado de Puebla. El cuadro clínico se caracterizó por fiebre, mialgias, artralgias, dolor retroocular y cefalea; además se registraron 11 casos con alguna forma leve de hemorragia.

Fueron 133 muestras de suero (fase aguda) y en 57 (42.8%) se aisló el virus y en todos los casos el serotipo 4 fue el identificado; en 47 muestras de fase convalesciente, se presentó un incremento de cuatro veces en el título de anticuerpos IH en 24 (51%). La mayoría de los sueros con aislamiento viral (19/22) tuvieron títulos altos de anticuerpos IH. La frecuencia de infección fue: 68% para el grupo de 15 a 44 años y 8% para menores de 15 años. Discusión. En este estudio se obtuvo una proporcion elevada de virus aislados en células TRA-284, lo cual recomienda su uso como herramienta de diagnóstico. Además se aislaron virus en sueros con altos titulos de anticuerpos IH. Este es el primer reporte del aislamiento del virus dengue serotipo 4 en el Estado de Puebla, donde recientemente se ha incrementado la transmisión del virus.

Correspondence: José Luis Imbert Palafox, Laboratorio Bioquímica - Genética, 14 sur y Av. San Claudio, Colonia San Manuel, Puebla, México. C.P. 72570. Telephone (22) 33-20-10, FAX (22) 44-45-18. Received October 10th, 1996; Accepted May 20th, 1997.

Vol. 8/No. 3/Julio-Septiembre, 1997

134 JL Imbert-Palafox, C Parra, JM Villaseca, H García, C Ramos. Palabras clave: Dengue clásico, virus dengue, epidemiología.

SUMMARY. Introduction. The aim of the present work was to carry out a serologic and virologic study of a dengue fever outbreak which occurred in 1991, in Ajalpan, State of Puebla, Mexico. Materials and methods. Antibodies against dengue virus were detected in serum samples by the Hemagglutination Inhibition Assay (HI). Virus Isolation was carried out on acute sera using the TRA-284 mosquito cell line, and the serotype was identified by indirect immunofluorescence using specific monoclonal antibodies. Results. The main signs and symptoms were characterized by fever, myalgia, arthralgia, retroocular pain and headache; in 11 cases a mild form of hemorrhage was documented. From 133 acute serum samples and dengue virus was isolated from 57 (43%), and in all cases serotype 4 was identified. 47 serum samples were obtained in the convalescent phase; the 51% of convalescent sera (24/47) showed a four-fold increase in the HIA titer. Most viral isolates (19/22, 86%) were obtained from serum samples with high HI antibody titers. The frequency of infection by age group was 68% for 15 to 44 years, and 8% for younger than 15 years. Discussion. In the present study, a high rate of dengue virus isolation was obtained using the TRA284 mosquito cell line. Therefore, its use in virologic diagnosis and surveilance is highly recommended. Additionally, a high proportion of dengue virus isolation was carried out in serum samples having high titers of HI. This is the first report of dengue-4 isolation from dengue fever cases in the state of Puebla, where recently an increase in the transmission of dengue virus has been reported. Keywords: Dengue fever, dengue virus, epidemiology. Revista Biomédica

INTRODUCTION. In the State of Puebla (Mexico), cases of dengue fever have been reported in different regions where approximately a million people lived in 1990(1). In 1981, 888 cases of dengue fever were detected in 11 localities located in the southwest and northern region of Puebla. During the period of 1982-1990, the majority of cases of dengue fever reported were caused by serotypes 1 and 2 (2). The aim of the present work was to carry out a serologic and virologic study of a dengue fever outbreak which occurred in 1991, in Ajalpan, State of Puebla, Mexico.

MATERIALS AND METHODS. Sera. Blood from individuals with signs and symptoms of dengue fever were obtained. Sera were maintained at -70oC until use. Additionally, a questionnaire containing clinical and epidemiological questions was applied by physicians and nurses. Acute (n=133) and convalescent (n=47) serum samples were tested for virus isolation and antibody detection. Hemagglutination inhibition test (HI). Sera were assayed by the HI technique, according to the proceedure of the San Juan Laboratories, Puerto Rico (3). Sera were absorbed with kaolin and goose erythrocytes. Eight hemagglutinating units of dengue-3 antigen were used. Double serial dilutions of sera were incubated with the antigen for 18 h at 4oC and afterwards, a 25% suspension of goose erythrocytes diluted 1 : 24 was added. Titers of HI were detected after one hour of incubation at room temperature. In the present work we only used the dengue-3 viral antigen, because of its availability. Our preliminary analysis using dengue-1 as antigen, showed similar results. Viral antigen. Dengue-3 and dengue-1 antigens were kindly donated by Dr. Duane J Gubler (CDC, Fort Collins, Co.). Their titers were 1:320.

135 Dengue fever in Puebla, Mexico. Cells. The mosquito TRA-284 cell line, was a gift from Dr. Goro Kuno (CDC, San Juan Laboratories, Puerto Rico). Cells were grown in glass tubes at 28oC in Leibowitz medium supplemented with tryptose phosphate broth (vol/vol) and 1% fetal bovine serum (3). Isolation and typification of dengue virus. Monolayers of TRA-284 cells were inoculated with 0.1 mL of serum sterilized by millipore filtration. The four dengue virus prototypes and mock infected cultures were included as positive and negative controls. Cultures were incubated at 28oC for 10 to 12 days. Cells were detached and washed three times with phosphate buffer saline (PBS). A drop of cell suspension was placed in a 12-well slide, Cells were suspended and washed three times in PBS. A drop of cell suspension was placed in a 12-well slide, cells were allowed to dry at room temperature and then fixed with cold acetone for 10 minutes. Cells were stained with a human polyclonal antibody against dengue labeled with fluorescein isothiocyanate (FITC). All the positive cultures were used for dengue serotype identification by indirect immunofluorescence using the specific monoclonal antibodies according

to the method of the Reed Army Institute, Washington, D.C : dengue-1 (15F3-1-15); dengue-2 (3H5-1-21); dengue-3 (5D4-11-24); and dengue-4 (1H10-6-7) (4,5).

RESULTS. From the 133 acute serum samples tested for HI antibodies, 20 (15%) had a HI titer < 1:10, and most of the samples (76%) had titers > 1:100. Dengue virus was isolated from 57 (43%), and in all cases serotype 4 was identified. Most viral isolates (86%) were obtained from serum samples containing HI antibodies titer > 1:100 (table 1) . When 47 paired serum samples were analyzed for HI antibodies, 57% (27/47) showed a fourfold or greater rise in HI titer in the convalescent sample. Sixteen of these serum samples were positive for virus isolation in the acute phase (table 2). The percentage of dengue virus isolation in the different age groups was as follows: 8%, 5-14 years; 68.6%, 15-44 years and 17.5%, greater than 45 years. The percentage of virus isolation was higher in male (51%) than female (39%) (table 3). Table 4 shows the main signs and

Table1 Antibodies titer against dengue antigen in acute serum samples and virus isolation.*

HI titer range † 1:10 1:20 – 1:80 1:160 – 1:320 1:640 – 1:1,280 1:2560 – 1:20,480

Number of samples(%) 20 12 22 25 54

(15%) (9%) (16%) (19%) (41%)

Number of viral Isolates(%) 3 (5%) 5 (8%) 12 (21%) 13 (23%) 24 (42%)

HI = Hemagglutination inhibition test. * : The viral isolation and identification of serotype 4 was carried out in mosquito TRA-284 cell cultures. † : The HI test was done using Dengue-3 antigen.

Vol. 8/No. 3/Julio-Septiembre, 1997

136 JL Imbert-Palafox, C Parra, JM Villaseca, H García, C Ramos. Table2 Comparison of HI titers and virus isolation in paired serum samples. HI Titersinserum Acute

Convalescent

1:10 1:80 1:160 1:160 1:640 1:640 1:5,120 1:10,240 1:20,480 1:10 1:10 1:40 1:80 1:160 1:320 1:640 1:640 1:640 1:640 1:1,280 1:1,280 1:2,560 1:5,120

1:10 1:640 1:160 1:640 1:640 1:2,560 1:10,240 1:20,480 1:20,480 1:640 1:1,280 1:10,240 1:10,240 1:20,480 1:5,120 1:1,280 1:2,560 1:5,120 1:10,240 1:5,120 1:10,240 1:20,480 1:20,480

No. of paired Serum samples

4 2 2 1 1 1 1 1 10 1 1 1 1 2 3 1 2 2 1 2 1 5 1

No. of virus Isolation

1 1 1 0 0 1 0 0 3 1 0 0 0 1 3 1 1 1 1 1 0 4 1

HI = Hemagglutination inhibition test.

symptoms recorded from 118 clinical cases. Prostration, anorexia, chills and other symptoms were not frequent. The stains in face, trunk, extremities and epistaxis were present in 10% of cases.

DISCUSSION. In the State of Puebla, dengue virus serotype 1 was isolated in most cases of dengue fever in the period of 1980 to 1990 ; in 1986 serotypes 1 and 2 was recovered from dengue fever cases (6). In the present work, the high percentage of sera with HI titers >1 :100 suggests a frequent contact of the population with the virus. This is the first report of isolation of dengue-4 in cases of dengue fever in the State of Puebla. Additionally, during this outbreak an important intrafamiliar transmission of dengue virus was recorded, since there was more than one case in the same house in 63% of the studied cases. Our finding of the high number of affected women could be explained to their staying in the house and exposed to the bite of mosquitoes. In 1991 and previously to the begining of the outbreak, an entomological survey showed the following data: Recipient Index = 16.2, Larva Index = 37, and Breteau Index = 63. The number of captured Aedes per house was 6 and the biting average/man/hour was 10.5, these assessments indicates a medium transmission risk.(7). A probable previous dengue fever disease Table3. Relationship of age and sex with virus isolation. Age

No. of virus isolation / No. of analyzed samples

GROUP

FEMALE

MALE

5 - 14

4/6

4/5

15 - 44

20 / 66

14/28

45 - 64

9/13

4/8

³ 65

1/2

0/1

Unknown

1/3

0/1

Total 2

35/90 (39%)

* X = 1.79, p = 0.18 Revista Biomédica

22/43 (51%)*

137 Dengue fever in Puebla, Mexico. Table4 Main signs and symptoms in dengue fever (n=118 patients).

Fever

97 %

Arthralgia

97 %

Retroocular pain

92 %

Headache

91 %

Myalgia

87 %

was communicated by 25% of all patients, (data not shown). Ten percent of cases (n=11) showed at least one mild bleeding sign and dengue-4 was isolated from 5 cases. Nevertheless, no cases of dengue haemorrhagic fever have been reported in the State of Puebla. Additionally, Ramos et al. (8) reported the isolation of dengue-1 from an outbreak of dengue fever occurred in Tepexi de Rodríguez, State of Puebla during August, 1991. Tepexi is a village 60 Km from Ajalpan, and with similar environmental conditions which has showed a cocirculation of both serotypes that have not been reported in Puebla State, where the transmission of dengue virus is increasing. Dengue virus transmission occurs in México during the rainy season with a reach peak in august- october, however, the present outbreak in Ajalpan, Puebla occurred in a region with an altitude of 1,200 meters, and during May which is characterized as being dry. Therefore, the transmission of dengue virus may be explained by the presence of high density of mosquitoes in the house water containers and breeding sites. These statistics demonstrate the need for a closer watch on dengue (9), since classical studies have determined a higher rate of dengue infection under conditions of high temperatures and humidity at low altitude, but not at high altitude (10). An

exception is the first outbreak of classical dengue fever at 1,700 meters reported in Guerrero, state, México (11). The reasons for the worldwide increase in incidence and distribution of dengue are unknow. Perhaps they are closely linked to a better adaptability of the mosquito to higher altitudes (11), lower temperatures (12) and to changes in human ecology and behavior (13).

ACKNOWLEDGMENTS. This work was partially supported by the Epidemiology Council Advisor of Mexico (grant 14-09) and by the National Institute of Public Health, Cuernavaca, Mor., Mexico. We thank to Drs. Blanca L Barron-Romero and Eduardo Garcia-Zepeda for their comments and criticisms of this manuscript. We are also grateful to Dr. Carlos Escamilla for providing experimental animals.

REFERENCES. 1.- Ortega ME. "Programas de Control de Aedes aegypti y dengue durante 1990". Dirección General de Epidemiología. Servicios Coordinados de la Secretaría de Salud, Estado de Puebla, México. 1990; pp 1 - 35. 2.- Gómez DH., Gubler DJ., Zarate AML. Arbovirus, su importancia en América. Publicación Técnica del Instituto Nacional de Diagnóstico y Referencia Epidemiológica, Secretaría de Salud de México, 1992; pp 66. 3.- U.S. Department of Health and Human Service. Public Health Service, Center of Disease Control, Center of Infectious Diseases. Pan American Health Organization, Pan American Sanitary Bureau. Regional Office of the World Health Organization. Dengue Diagnostic Laboratory Procedures for the Americas: A Manual. San Juan-Puerto Rico: San Juan Laboratories, 1991; p.p 1-150. 4.- Gubler DJ, Kuno G, Sather GE, Velez M, Oliver A. Mosquito cell cultures and specific monoclonal antibodies in surveillance for dengue virus. Am J Trop Med Hyg 1984; 33:158-165. 5.- Henchal EG. Rapid identification of dengue virus isolates by using monoclonal antibodies in an indirect immunofluorescence assay. Am J Trop Med Hyg 1983 ; 32:264-268.

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138 JL Imbert-Palafox, C Parra, JM Villaseca, H García, C Ramos. 6.- Gómez DH, Gubler DJ, Zárate AML. El dengue en México, (1979-1991). Publicación Técnica del Instituto Nacional de Diagnóstico y Referencia Epidemiológica No.18, Secretaría de Salud de México, D.F. 1992; pp 65. 7.- Ortega ME. "Programas de Control de Aedes aegypti y dengue durante 1991 y 1992". Dirección General de Epidemiología. Servicios Coordinados de la Secretaría de Salud, Estado de Puebla, México. 1992; pp 1 - 5. 8.- Ramos C, Villaseca JM, Garcia H, Hernandez DG, Ramos-Castaneda J,. Imbert JL. Detection of dengue virus from mosquito cell cultures inoculated with human serum in the presence of actinomycin D. Trans Roy Soc Trop Med Hyg 1995; 89:189-190. 9.- Gomez DH, Tapia CR. Surveillance of Dengue: The identification of a public health problem. DENGUE, a worldwide problem, a common strategy. Proceedings of the International Conference on Dengue and Aedes aegypti community-based control. México, D.F., 1992. 10.- Koopman JS, Prevots DR, Marin VMA, Dantes HG, Zarate AML, Longini Jr IM, Love JS. Determinants and predictors of dengue infection in Mexico. Am J Epidemiol 1991; 133:1168-1178. 11.- Herrera BE, Prevots DR, Zarate ML, Silva JL, Sepulveda AJ. First reported outbreak of classical dengue fever at 1,700 above sea level in Guerrero state, Mexico, June 1988. Am J Trop Med Hyg 1992; 46:649-653. 12.- Watts DM, Burke DS, Harrison BA, Whitmire RE, Nisalak A. Effect of temperature on the vector efficiency of Aedes Aegypti for dengue 2 virus. Am J Trop Med Hyg 1987; 36:143-152. 13.- Monath TP. Dengue: The risk to developed and developing countries. Proc Natl Acad Sci, USA. 1994; 91:2395-2400.

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