• View in gallery

    Map of Bandung, Indonesia showing the geographic distribution of dengue cases. Each dot represents one case. The locations of the study hospital and textile factories are as indicated. The shaded area represents the city of Bandung.

  • View in gallery

    Distribution of symptomatic dengue (DEN) cases by month, year, and DEN serotype. Each bar represents the total number of cases corresponding to the indicated year and month. The numbers inside the color-coded areas of the bar correspond to the number of identifications for the given dengue serotype. N = no identification made.

  • 1

    Chan VF, 1988. Tropical diseases of public health importance in the Philippines. Southeast Asian J Trop Med Public Health 19 :361–367.

  • 2

    Gubler DJ, Suharyono W, Lubis I, Eram S, Sulianti Saroso J, 1979. Epidemic dengue hemorrhagic fever in rural Indonesia. I. Virological and epidemiological studies. Am J Trop Med Hyg 28 :701–710.

    • Search Google Scholar
    • Export Citation
  • 3

    Eram S, Setyabudi Y, Sadono TI, Sutrisno DS, Gubler DJ, Sulianti Saroso J, 1979. Epidemic dengue hemorrhagic fever in rural Indonesia. II. Clinical studies. Am J Trop Med Hyg 28 :711–716.

    • Search Google Scholar
    • Export Citation
  • 4

    Sumarmo, 1987. Dengue haemorrhagic fever in Indonesia. Southeast Asian J Trop Med Public Health 18 :269–274.

  • 5

    Corwin AL, Larasati RP, Bangs MJ, Wuryadi S, Arjoso S, Sukri N, Listyaningsih E, Hartati S, Namursa R, Anwar Z, Chandra S, Loho B, Ahmad H, Campbell JR, Porter KR, 2001. Epidemic dengue transmission in southern Sumatra, Indonesia. Trans R Soc Trop Med Hyg 95 :257–265.

    • Search Google Scholar
    • Export Citation
  • 6

    Guzman MG, Kouri G, Bravo J, Valdes L, Vazquez S, Halstead SB, 2002. Effect of age on outcome of secondary dengue 2 infections. Int J Infect Dis 6 :118–124.

    • Search Google Scholar
    • Export Citation
  • 7

    Guzman MG, Kouri G, Valdes L, Bravo J, Vazquez S, Halstead SB, 2002. Enhanced severity of secondary dengue-2 infections: death rates in 1981 and 1997 Cuban outbreaks. Rev Panam Salud Publica 11 :223–227.

    • Search Google Scholar
    • Export Citation
  • 8

    Kocijancic I, Vidmar K, Ivanovi-Herceg Z, 2003. Chest sonography versus lateral decubitus radiography in the diagnosis of small pleural effusions. J Clin Ultrasound 31 :69–74.

    • Search Google Scholar
    • Export Citation
  • 9

    1997. Dengue Hemorrhagic Fever: Diagnosis, Treatment, Prevention and Control. Second edition. Geneva: World Health Organization.

  • 10

    Porter KR, Widjaja S, Lohita HD, Hadiwijaya SH, Maroef CN, Suharyono W, Tan R, 1999. Evaluation of a commercially available immunoglobulin M capture enzyme-linked immunosorbent assay kit for diagnosing acute dengue infections. Clin Diagn Lab Immunol 6 :741–744.

    • Search Google Scholar
    • Export Citation
  • 11

    Morens DM, Halstead SB, Repik PM, Putvatana R, Raybourne N, 1985. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microbiol 22 :250–254.

    • Search Google Scholar
    • Export Citation
  • 12

    Clarke DH, Casals J, 1958. Techniques for hemagglutination and hemgglutination-inhibition with arthropod-borne viruses. Am J Trop Med Hyg 7 :561–573.

    • Search Google Scholar
    • Export Citation
  • 13

    Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV, 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30 :545–551.

    • Search Google Scholar
    • Export Citation
  • 14

    Endy TP, Chunsuttiwat S, Nisalak A, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA, 2002. Epidemiology of inapparent and symptomatic acute dengue virus infection: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 156 :40–51.

    • Search Google Scholar
    • Export Citation
  • 15

    Endy TP, Nisalak A, Chunsuttiwat S, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA, 2002. Spatial and temporal circulation of dengue virus serotypes: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 156 :52–59.

    • Search Google Scholar
    • Export Citation
  • 16

    Graham RR, Juffrie M, Tan R, Hayes CG, Laksono I, Ma’roef C, Erlin, Sutaryo, Porter KR, Halstead SB, 1999. A prospective seroepidemiologic study on dengue in children four to nine years of age in Yogyakarta, Indonesia I. studies in 1995–1996. Am J Trop Med Hyg 61 :412–419.

    • Search Google Scholar
    • Export Citation
  • 17

    Sangkawibha N, Rojanasuphot S, Ahandrik S, Viriyapongse S, Jatanasen S, Salitul V, Phanthumachinda B, Halstead SB, 1984. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol 120 :653–669.

    • Search Google Scholar
    • Export Citation
  • 18

    Burke DS, Nisalak A, Johnson DE, Scott RM, 1988. A prospective study of dengue infections in Bangkok. Am J Trop Med Hyg 38 :172–180.

  • 19

    Thein S, Aung MM, Shwe TN, Aye M, Zaw A, Aye K, Aye KM, Aaskov J, 1997. Risk factors in dengue shock syndrome. Am J Trop Med Hyg 56 :566–572.

    • Search Google Scholar
    • Export Citation
  • 20

    Chen WJ, Chen SL, Chien LJ, Chen CC, King CC, Harn MR, Hwang KP, Fang JH, 1996. Silent transmission of the dengue virus in southern Taiwan. Am J Trop Med Hyg 55 :12–16.

    • Search Google Scholar
    • Export Citation
  • 21

    Gubler DJ, Suharyono W, Lubis I, Eram S, Gunarso S, 1981. Epidemic dengue 3 in central Java, associated with low viremia in man. Am J Trop Med Hyg 30 :1094–1099.

    • Search Google Scholar
    • Export Citation
 
 
 

 

 
 
 

 

 

 

 

 

 

EPIDEMIOLOGY OF DENGUE AND DENGUE HEMORRHAGIC FEVER IN A COHORT OF ADULTS LIVING IN BANDUNG, WEST JAVA, INDONESIA

View More View Less
  • 1 Viral Diseases Program, Naval Medical Research Unit No. 2, Jakarta, Indonesia; Medical Faculty, Padjadjaran University Hasan Sadikin Hospital, Bandung, Indonesia; National Institutes of Health Research and Development, Ministry of Health, Jakarta, Indonesia

A prospective study of dengue fever (DF) and dengue hemorrhagic fever (DHF) was conducted in a cohort of adult volunteers from two textile factories located in West Java, Indonesia. Volunteers in the cohort were bled every three months and were actively followed for the occurrence of dengue (DEN) disease. The first two years of the study showed an incidence of symptomatic DEN disease of 18 cases per 1,000 person-years and an estimated asymptomatic/ mild infection rate of 56 cases per 1,000 person-years in areas of high disease transmission. In areas where no symptomatic cases were detected, the incidence of asymptomatic or mild infection was 8 cases per 1,000 person-years. Dengue-2 virus was the predominant serotype identified, but all four serotypes were detected among the cohort. Four cases of DHF and one case of dengue shock syndrome (DSS) were identified. Three of the four DHF cases were due to DEN-3 virus. The one DSS case occurred in the setting of a prior DEN-2 virus infection, followed by a secondary infection with DEN-1 virus. To our knowledge, this is the first report of a longitudinal cohort study of naturally acquired DF and DHF in adults.

INTRODUCTION

Dengue fever (DF) and dengue hemorrhagic fever (DHF) are diseases that have long been endemic in tropical and subtropical regions of the world. Epidemic DHF emerged in southeast Asia following World War II. The first recorded DHF epidemic in this area occurred in Manila, the Philippines, in 1953–1954,1 followed by another in 1956. Fourteen years after the 1953–1954 Philippines epidemic, DHF epidemics were reported in the Indonesian cities of Surabaya and Jakarta. Since then, outbreaks of the disease have spread to involve most of the major urban areas in Indonesia, as well as some of the rural areas of the country.2–4

In 1998, a record number of dengue (DEN) cases occurred in Indonesia, giving rise to thousands of clinically diagnosed DHF cases. An outbreak investigation in Palembang in southern Sumatra from January to April 1998 showed a monthly mean number of outbreak-related DEN cases of 833.5 A review of hospital data from two Palembang hospitals showed that a sizeable number of severe DEN cases occurred in individuals ≥15 years old.

Adult cases of DEN, some severe, were associated with epidemics in Cuba. During the 1981 DEN-2 outbreak in Cuba that followed the 1977 DEN-1 epidemic, many adult secondary infections were documented, but the occurrence of DHF or dengue shock syndrome (DSS) was limited mostly to children.6 Sixteen years later, a second DEN-2 epidemic resulted in a death rate that was significantly higher in adults compared with the earlier DEN-2 epidemic.7 This suggested that the longer interval between primary DEN-1 and secondary DEN-2 virus infections correlated with more severe disease.

The above epidemiologic findings, together with the fact that candidate DEN vaccines must ultimately be evaluated in adult populations in DEN-endemic regions, emphasizes the need to generate more information on DEN virus infections in adults. To further examine the epidemiology of DF and DHF in an adult population, a prospective cohort study was initiated in adult textile factory workers living in Bandung, West Java, Indonesia.

West Java was chosen for the following reasons. Based on the number of cases reported as of September 1998, the provinces recording the most adult ( ≥18 years old) DHF cases were Jakarta (13,813), West Java (10,730), East Java (8,546), Central Java (6,879), and Yogyakarta (3,257). Compared with Jakarta Province, West Java has a larger number of factories from which study volunteers can be recruited. Of the five largest municipalities in West Java, Cirebon, Bogor, and Bandung had the largest number of cases per 100,000 population, with averages over a four-year period (1995 to 1998) of 44, 54.7, and 31.7, respectively. Although Bandung had the lowest case rate of the three, this city (or Kotamadya) was chosen as the desired site because of the presence of highly capable health care personnel and academic research facilities. Located in Bandung, Padjadjaran University is the largest university and medical school in West Java. Hasan Sadikin Hospital, the largest hospital in West Java, is also located in Bandung and serves as a teaching hospital for the medical school. Bandung, the capital city of West Java, is the fourth largest city in Indonesia and has an estimated population of two million. It is located at an altitude of approximately 750 meters above sea level and has a cool climate (18–28°C) throughout the year. The subdistrict within Bandung that consistently reports the largest number of hospitalized adult DHF cases is Cibeunying Kidul. In 1998, 42 DHF cases/ 100,000 population were reported there. In Ujung Berung, another subdistrict 1.5 km east of Cibeunying Kidul, the reported DHF incidence was 40/100,000 population. The results of the first two years of this study (August 2000 through July 2002) are the subject of this report.

MATERIALS AND METHODS

Study site.

The PT Naintex textile factory in Cibeunying Kidul was enlisted to participate in the study. In Acramanik (neighboring Ujung Berung), employees at the PT Grandtex factory were recruited to participate. PT Naintex employs 1,500 people and PT Grandtex employs 4,000 people. The absentee rate due to illness averages seven per day at Naintex and four per day at Grandtex. The ages of the employees ranged from 18 to 66 years old, with the majority (estimated at 90%) being 18–45 years of age. The employees work six days a week in three rotating shifts of eight hours. The shifts are rotated every two weeks. The working hours for each shift are 6:00 am to 2:00 pm, 2:00 pm to 10:00 pm, and 10:00 pm to 6:00 am. These working hours result in all study participants having DEN vector exposures both at work and at home. Fogging takes place at both companies on a regular basis to reduce mosquito populations.

Enrollment and study design.

Prior to enrollment, a series of seminars was conducted to introduce the study to factory personnel and to answer any questions. Consent forms were distributed for advanced reading prior to the start of enrollment. Prior to entering into the study, the consent forms were completed and signed by each volunteer. The cohort study protocol was reviewed and approved by the Institutional Review Boards at the Naval Medical Research Unit No. 2 and the National Institutes of Health Research and Development, Ministry of Health, Indonesia.

All volunteers completed medical history questionnaires and demographic data forms. Physical examinations were conducted on each volunteer and blood samples were obtained for baseline routine laboratory tests that included a complete cell blood count (CBC) with differential, electrolytes, liver enzymes, urinalysis, PT, PTT, protein, and albumin. Baseline tourniquet tests were also performed on each volunteer. Peripheral blood mononuclear cells (PBMCs) and plasma were obtained and stored for later evaluation of baseline anti-DEN immune status. Volunteers were excluded from participation if they were afflicted by, or had a history of, anemia (hemoglobin level < 8 g/dL), infection with human immunodeficiency virus, non-DEN-related bleeding disorder, or any immunologic disorder.

Active surveillance of the volunteer cohort occurred whereby the factory personnel office would notify the study coordinator in the event an employee volunteer failed to show up for work. Evaluation by the factory physician was required for an employee to be officially excused from work. Upon receiving notification of the absenteeism, the study coordinator would immediately contact the volunteer by telephone to determine the reason for the absence. Within 24 hours of making phone contact, a medical evaluation was conducted and blood samples were obtained. For volunteers not possessing a telephone, the study nurse or physician visited his or her residence no later than 24 hours of absenteeism notification. The detection of a febrile illness warranted a medical evaluation. Most of the volunteers who had febrile illnesses were identified through this system.

Follow-up and evaluation of febrile illness.

If absenteeism was due to a febrile illness, a medical evaluation was performed and blood samples were obtained for CBC with differential and platelet count, DEN virus isolation, a DEN reverse transcriptase-polymerase chain reaction (RT-PCR), and serology. Automated CBC and differential counts were performed in the Hasan Sadikin Hospital Hematology Laboratory. Volunteers with thrombocytopenia (platelet count < 100,000/mm3) or those who were severely ill as determined by the attending study physician were hospitalized for observation and/or further evaluation. During their hospital stay, automated hematocrit and platelet determinations were made daily, and sonograms were performed almost daily to look for plasma leakage as shown by hemoconcentration or by the development of ascites and/or pleural effusion. Ultrasound has a higher degree of accuracy with a positive predictive value of 92% compared with a lateral decubitus chest radiographs.8 Because ultrasound does not involve exposure to radiation, this was the technique chosen for use in the study. The hematocrit and platelet counts were performed in the hospital hematology laboratory and the sonograms in the hospital radiology department. Serum protein and albumin measurements were also obtained and used as another tool to monitor volunteers for plasma leakage. These additional tests were performed in the Hasan Sadikin Hospital chemistry laboratory. Febrile volunteers who were not severely ill or who had platelet counts of 100,000–150,000/mm3 were followed as outpatients. Sonograms, serial hematocrits, and platelet determinations were not performed. Seven to 14 days post-illness, convalescent blood samples were obtained from all febrile patients.

Detection of asymptomatic or mild DEN virus infection.

To evaluate the cohort for the occurrence of asymptomatic and mild DEN illness, blood samples were obtained from the entire cohort every three months. Although samples were collected from all volunteers, the hemagglutination-inhibition (HI) test was used to screen serum samples from 250 randomly selected volunteers not experiencing a DEN-related febrile illness to detect DEN seroconversion. Randomization was performed using SPSS software (SPSS Inc., Chicago, IL). A volunteer was said to seroconvert if there was a ≥four-fold increase HI titer between successive serosurveys. The plaque reduction neutralization test (PRNT) was used to confirm HI seroconversions. On alternating blood draws, PBMCs were collected and stored in liquid nitrogen for later evaluation of pre-infection anti-DEN cellular immune status. During the cohort serosurveys, brief questionnaires were administered to the volunteers to inquire about the occurrence of any DEN-like symptoms or illnesses since the preceding serosurvey.

Diagnosis of symptomatic DEN virus infection.

A volunteer was considered to have a DEN virus infection if he or she had a compatible clinical illness and laboratory evidence of DEN. Laboratory evidence of DEN virus infection included either identification of the infecting virus by a nested RT-PCR and/or virus isolation in tissue culture, the presence of IgM antibodies to DEN virus, or a four-fold increase in anti-DEN HI antibody titer. For diagnosing DHF and DSS in hospitalized volunteers, the World Health Organization (WHO) criteria were used.9 Hemoconcentration (> 20%), pleural effusion, and/or ascites were used as indicators of plasma leakage. For assessing hemoconcentration, baseline hematocrits determined at enrollment were used. In some cases, comparisons of admission and post-hydration hematocrit values were used in the assessment. As indicated above, ultrasonography was used to look for ascites and pleural effusions. Blood pressure, pulse rate, and respiration rate were monitored every eight hours for signs of hemodynamic instability, a sign needed to fulfill criteria for DSS.

Virus isolation.

Serum samples obtained during the acute febrile illness were used for virus isolation in tissue culture. To accomplish this, aliquots of serum were diluted 1:10 in sterile tissue culture media and applied to a confluent monolayer of C6/36 cells. Following a one-hour incubation at 25°C, the diluted serum was removed and fresh tissue culture media was added. The plates were then placed at 25°C and observed daily for cytopathic effects (CPEs) for 14 days. Upon development of CPEs or at day 14, cells were scraped from the plates and stained for the presence of virus by standard immunofluorescence using the flavivirus genus-reactive mono-clonal antibody 4G2. Samples reactive against 4G2 were then subtyped by staining with DEN virus serotype-specific mono-clonal antibodies.

Serologic assays for antibodies to DEN virus.

Commercially available enzyme-linked immunosorbent assay kits (Focus Technologies, Cyprus, CA) were used to detect IgM antibodies to DEN virus. The assay was performed according to the manufacturer’s instructions as previously described.10

Anti-DEN neutralizing antibodies were detected by PRNT. Three 10-fold serum dilutions were made in culture media, starting at 1:10 and ending at 1:1,000. Diluted samples were assayed as described previously.11 The dilution that produced a 50% reduction in plaque count compared with the negative control sample (PRNT50) was determined by probit analysis using SPSS software (SPSS Inc). The virus strains used in the assay were isolated from DEN patients in Thailand and included 16682 (DEN-2), 16001 (DEN-1), 16562 (DEN-3), and 1036 (DEN-4).

The HI was performed as previously described.12 To estimate the rate of asymptomatic or mild DEN illness, paired samples from randomly selected volunteers were tested by HI. SPSS Inc. software was used for randomization of volunteers. Samples were tested at serial two-fold dilutions, starting at 1:10 and ending at 1:5,120. A ≥ four-fold increase in HI titer was indicative of a DEN virus infection.

RT-PCR for detecting DEN virus RNA.

Acute serum samples obtained during the febrile illness were assayed for DEN virus RNA by a nested RT-PCR. The method of Lanciotti and others was used to perform this test.13

Data analysis.

Incidence of DEN virus infection was expressed as the number of infections occurring among the cohort per 1,000 person years of follow-up. Each DEN virus infection episode was counted separately, regardless of whether it was the same volunteer or not. Volunteers that dropped out of the study were accounted for in the denominator (total person-years) by including only the length of time they were available for follow-up. Epi-Info Software (Centers for Disease Control and Prevention, Atlanta, GA) was used to compare symptoms and signs between cohort volunteers experiencing either a non-DEN illness or an illness due to DEN.

RESULTS

Study population.

A total of 1,825 volunteers from Grandtex and 711 volunteers from Naintex were recruited to participate in the study. There were 1,198 men and 627 women from Grandtex, and 303 men and 408 women from Naintex. The mean age of volunteers was 37.7 years old (range = 18–64 years) at Grandtex and 33.1 years old (range = 18–66 years) at Naintex. The majority of volunteers were either Javanese (1,144) or Sundanese (1,288). A total of 162 participants dropped out of the study after enrollment, 78 from Naintex and 84 from Grandtex. One hundred forty-five volunteers resigned from their jobs, nine died of study-unrelated reasons, and eight declined further participation for undisclosed reasons. There were no deaths due to DEN viral infection. Of the volunteers who dropped out of the study, only four had experienced a symptomatic DEN virus infection prior to leaving the study. Taking into consideration the date on which the volunteers dropped out and including only the actual time that each volunteer was enrolled, a total of 4,912.8 person-years of cohort follow-up was performed.

Dengue fever and dengue hemorrhagic fever.

There were 565 febrile episodes among the cohort, of which 475 were non-DEN cases (84%), 51 were outpatient DEN cases (9%), and 39 were hospitalized DEN cases (6.9%). No volunteer experienced more than one DEN virus infection. This produced an overall incidence of symptomatic DEN of 18 cases per 1,000 person-years of follow-up. The mean age of the DEN cases was 37 years old in Grandtex and 33 years old in Naintex. For the non-DEN cases, the mean ages were 38 and 33 years old in Grandtex and Naintex, respectively. Symptomatic DEN cases were equally distributed among the various age groups, comprising 3–4% of the volunteers in each group (Table 1). None of the DEN cases were thought to be due to other flavivirus infections such as those with Japanese encephalitis virus, since DEN is the only flavivirus known to circulate widely in West Java.

Of the 39 hospitalized DEN cases, one was classified as DHF grade I, three as DHF grade II, and one as DSS based on WHO criteria. The ages of the DHF/DSS cases were 31, 34, 40, 43, and 45 years old; two were women and three were men. Three DHF cases demonstrated thrombocytopenia and hemoconcentration, and one had thrombocytopenia, hemo-concentration, and pleural effusion. The one DSS case demonstrated hemodynamic instability on day four of hospitalization as shown by restlessness, hypotension, and tachycardia with a weak pulse.

Figure 1 shows the geographic distribution of cases per district. Of the seven districts with DEN cases and more than 50 volunteers, Arcamanik, Rancaekek, Kiarancondong, and Ujung Berung had the highest incidences of DEN virus infection, with 18–30 cases per 1,000 person-years of follow-up (Table 2).

The DEN virus serotypes detected among the cohort and categorized by district where the infected volunteers live are also shown in Table 2. The predominant serotype identified was DEN-2 (24), with fewer cases of DEN-1 (11), DEN-3 (8), and DEN-4 (10). There was one dual infection (DEN 1 plus 2) as indicated by RT-PCR detection of viral RNA. Figure 2 shows the distribution of cases by year, month, and serotype from all districts combined.

For the DHF cases, three were caused by DEN-3 virus and the virus causing the fourth case was not identified (Table 3). The non-shock DHF cases all had pre-existing in vitro neutralizing antibodies against all four serotypes prior to illness. The low titers seen against some of the serotypes (including the infecting serotype) probably represent cross-neutralization due to high titers against one or more of the other serotypes. The one DSS case was due to a DEN-1 virus secondary infection, following a primary infection with DEN-2 virus.

The clinical characteristics of volunteers with symptomatic DEN virus infections are shown in Table 4. Nausea, spontaneous hemorrhage, leukopenia, thrombocytopenia, and elevated levels of liver enzymes were significantly more common in DEN cases than in non-DEN cases. The tourniquet test result was also positive in significantly more volunteers with DEN compared with non-DEN cases. Although retro-orbital eye pain is reported to be a common characteristic of DF, there was no significant difference in this symptom between the two groups. Rash occurred in 4.7% of the DEN cases compared with 0.6% in the non-DEN cases.

Asymptomatic DEN virus infection.

To estimate the level of asymptomatic or mild DEN virus infection among volunteers in the cohort, sera collected during the cohort serosurveys from randomly selected individuals were screened by HI. Although serosurveys were conducted every three months, only the samples collected at every other serosurvey were tested to reduce the number of assays performed. For any HI seroconversions identified, the serum sample collected from the same volunteer three months prior to the collection of the sample showing seroconversion was tested by HI. This was done in an attempt to better pinpoint the time of seroconversion. A total of 250 volunteers were randomly selected for analysis. One hundred twenty-five volunteers were selected from the five districts with the highest number of symptomatic cases. The combined volunteer pool from these five districts totaled 1,604 after excluding the symptomatic DEN cases.

Another 125 volunteers were selected from districts where no DEN cases were identified. These volunteers were selected from a total of 38 districts. Additional districts were used to obtain these 125 volunteers because the total number from each district without detectable symptomatic DEN cases was 25 or less. The total number of study participants in the pool from which the 125 volunteers were randomly selected was 214.

Sera from those volunteers showing HI seroconversion were tested subsequently for increases in neutralizing antibody titers against all four DEN virus serotypes to confirm DEN virus infection. Of the 125 volunteers from areas where symptomatic DEN cases were detected, a ≥ four-fold increase in HI titers was detected in 16 individuals, of which 14 were confirmed DEN virus infections by PRNT50. Review of the symptom questionnaires from these 14 volunteers covering the months preceding the time of seroconversion did not reveal any illness or symptoms indicative of DEN virus infection. Based on these data, the incidence of asymptomatic or mild DEN in this group was calculated to be 56 cases/1,000 person-years. Among the 125 volunteers from areas without DEN cases, three seroconverted by HI and two were confirmed by PRNT50, resulting in a calculated incidence of 8 asymptomatic or mild cases/1,000 person-years. The infecting serotype for these cases could not be determined by the PRNT50 patterns.

DISCUSSION

A prospective cohort study of DEN was initiated in a DEN-endemic region of West Java, Indonesia to define the epidemiology of this disease in an adult population. To date, all other cohort studies of DF and DHF were conducted in pediatric populations in Thailand, Myanmar, and Indonesia.14–19 In preparation for vaccine trials, it is important to understand the epidemiology of DEN in an adult population where the majority of individuals have been exposed to one or more DEN virus infections. To our knowledge, this is the first report of a longitudinal cohort study of naturally acquired DF and DHF in adults. The incidence of symptomatic DEN virus infection among the cohort was 18 cases/1,000 person-years of follow-up, whereas the estimated incidence of asymptomatic or mild infection was more than three times that of symptomatic infection at 56 cases/1,000 person-years. Given that only a fraction of the study population was used to determine the rate of asymptomatic or mild DEN virus infection, the calculated rate may not be representative of the study population at large.

In contrast to the study by Endy and others, where the incidence of symptomatic DEN underestimated the overall incidence of DEN virus infection by 50%,14 our study showed a 76% underestimation in an area of increased DEN transmission. Thus, we estimate that the majority of DEN virus infections in the cohort were asymptomatic or mild, consistent with the findings in other outbreak settings.20,21 The 76% underestimation was calculated using our observed asymptomatic/mild infection rate. Because our estimated asymptomatic/mild infection rate may not be representative of the study population as stated earlier, definitive conclusions regarding the underestimation of DEN virus infection in our cohort cannot be made at this time.

Dengue-2 virus was identified most frequently among the symptomatic cases and it is assumed that this was the predominant serotype circulating during the two years of the study. However, DEN-3 virus was identified in three of the four DHF cases. Although the numbers are too small to make any conclusions, this suggests that DEN-3 virus may be associated with more severe disease. It is also interesting that the infection sequence in the one DSS case was DEN-2 virus followed by DEN-1 virus, the predominant infection sequence among the severe cases in the pediatric cohort study by Graham and others.16

Our data indicate that there was year-round circulation of all four DEN virus serotypes among the cohort. The peak number of cases appeared to trend toward the May-July time frame. This is usually the beginning of the dry season in Indonesia, and the overall higher number of cases may be related to the increased storage of water by volunteers in the cohort. This would in turn result in increased mosquito breeding, with subsequent increases in vector mosquito populations.

Given that Bandung is a DEN-endemic area, it is possible that some of the volunteers acquired their DEN virus infections at work and not at home. To more conclusively determine where the volunteers were infected, sequence comparisons between genomes from viruses isolated from volunteers and viruses isolated from mosquito vectors at the factory would be necessary. Unfortunately, mosquito collections at the factories were not included as part of the approved study protocol. The fact that regular insecticide fogging is performed at both factories to reduce mosquito populations increases the likelihood that the majority of DEN virus infections that occurred in our study cohort were acquired in or around the home.

The clinical symptoms and signs exhibited by symptomatic DEN virus-infected volunteers reflect mostly what has been described in the literature. Retro-orbital eye pain, a symptom frequently associated with DF, did not occur with significantly greater frequency among the DEN cases in our cohort compared with non-DEN cases. The reason for this is not clear. Because resources did not permit the determination of the etiology of the non-DEN febrile illnesses, other infectious agents that produce similar symptoms may have been involved. Infants and young children often experience a maculopapular rash. Among the symptomatic cases identified in our cohort, a rash was seen infrequently (4.7%), but occurred significantly more often than in the non-DEN cases (0.6%). Complaints such as abdominal pain, nausea, and vomiting may be associated with DEN illness in children. Abdominal pain and vomiting were not prominent among our adult cohort and only nausea occurred significantly more often among those with DEN illness.

Our study demonstrates the ability to define the incidence of DEN disease and infection in an adult cohort living in an area where all four virus serotypes circulate. This area is therefore a prime location to conduct Phase 2 and 3 efficacy trials of promising DEN virus vaccine candidates. The study also resulted in the collection of valuable reagents that can be used to study the immunopathogenesis of DEN and to further define correlates of protection against DEN disease and infection. Studies are currently underway to characterize patterns of anti-DEN humoral and cellular immunity that may correlate with protection against DEN disease.

Table 1

Age distribution of dengue cases

Age, years (%)
Category15–2526–3536–45>46
Dengue cases5 (3)40 (4)37 (3)8 (3)
No. of volunteers1741,0391,059264
Table 2

Distribution of symptomatic dengue (DEN) cases among the districts along with the serotype identification of the infecting DEN viruses*

DistrictVirus types†CasesVolunteersIncidence†
* Dashes (–) indicate that the calculation was not performed or no viruses were identified.
†Different DEN serotypes identified from cases in the corresponding district.
†Incidence expressed as number of cases per 1,000 person-years.
ArcamanikDEN-1,2,31322030
Astana Anyar12
Bandung KidulDEN-113
Batununggal234
Cibeunying KalerDEN-1,2223
Cibeunying KidulDEN-2,3934313
CibiruDEN-21668
CicadasDEN-2,3,41653715
CilengkrangDEN-2,3215
CileunyiDEN-2326
Cimeunyan442
CoblongDEN-2115
Dayeuhkolot12
KadungoraDEN-223
KiaracondongDEN-1,2,3,41127520
LembangDEN-211
LengkongDEN-4313
MargaasihDEN-212
MargacintaDEN-113
RancaekekDEN-136025
RancasariDEN-212
RegolDEN-211
Sumedang UtaraDEN-111
Sumendang Selatan11
Ujung BerungDEN-1,3,4822918
Total901,919
Table 3

50% plaque reduction neutralization titers (PRNT50) of pre-illness sera obtained from the dengue hemorrhagic fever/dengue shock syndrome (DSS) cases along with the infecting dengue (DEN) serotype

PRNT50 titer
ID numberDEN-1DEN-2DEN-3DEN-4Infecting serotype
* DSS case.
349*< 10114< 10< 10DEN-1
5335974526444DEN-3
4203071,0126635DEN-3
106612995977Unknown
19291223441627DEN-3
Table 4

Signs and symptoms from dengue and non-dengue cases

Symptom/signDengue (%)Nondengue (%)P
Headache89.989.30.966
Retro-orbital pain36.440.70.531
Sore throat19.141.1< 0.001
Myalgia93.388.60.264
Abdominal pain29.226.10.626
Nausea71.954.3< 0.001
Vomiting21.413.80.096
Spontaneous hemorrhage14.60.4< 0.001
Rash4.70.60.015
Positive tourniquet test result52.334.4< 0.01
Leukopenia (≤4,000 cells/mm3)43.45.7< 0.001
Thrombocytopenia (< 100,000/mm3)37.23.6< 0.001
Elevated liver enzyme levels57.528.3< 0.001
Figure 1.
Figure 1.

Map of Bandung, Indonesia showing the geographic distribution of dengue cases. Each dot represents one case. The locations of the study hospital and textile factories are as indicated. The shaded area represents the city of Bandung.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.60

Figure 2.
Figure 2.

Distribution of symptomatic dengue (DEN) cases by month, year, and DEN serotype. Each bar represents the total number of cases corresponding to the indicated year and month. The numbers inside the color-coded areas of the bar correspond to the number of identifications for the given dengue serotype. N = no identification made.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 72, 1; 10.4269/ajtmh.2005.72.60

Authors’ addresses: Kevin R. Porter, Viral Diseases Department, Naval Medical Research Center, Building 503, Room 3A14A, 503 Robert Grant Avenue, Silver Spring, MD 20910, Telephone: 301-319-7450, Fax: 301-319-7451, E-mail: porterk@nmrc.navy.mil. Charmagne G. Beckett, Herman Kosasih, Ratna Irsiana Tan, Susana Widjaja, Erlin Listiyaningsih, Chairin Nisa Ma’roef, and James L. McArdle, American Embassy Jakarta, U. S. Navy Medical Research Unit No. 2, Unit 8132, FPO, APO 96520-8132. Bachti Alisjahbana, Pandji Irani Fianza Rudiman, Ida Parwati, Primal Sudjana, Hadi Jusuf, and Djoko Yuwono, Internal Medicine, Medical Faculty, Padjadjaran University Hasan Sadikin Hospital, Bandung, Indonesia and Jalan Pasirkaliki No. 35 Bandung, 40161, Jawa Barat, Indonesia. Suharyono Wuryadi, National Institutes of Health Research and Development, Ministry of Health, Jakarta, Indonesia.

Acknowledgments: We thank Sri Hadiwijaya, Dasep Purwaganda, Ungke Antonjaya, Gustiani, Yurike Tobing, Akterono Dwi Budiyati, Antonius Arditya Pradana, Agus Rahmat, and Haditya Leo Mukri for the technical assistance with the study. We also thank Professor Anna Alisjahbana and the World Health Organization Maternal Child Health Collaborating Center for their assistance in data storage and analysis. We are deeply grateful to Drs. Scott Halstead and Curtis Hayes for data reviews and suggestions for the study, and to Terrisita Porter for her assistance in the preparation of this manuscript.

Financial support: This research was supported by the Military Infectious Diseases Research Program and the Naval Medical Research Center for Work unit 62787A 870S.

Disclaimer: The opinions and assertions contained herein are not to be construed as official or as reflecting the views of the Navy services at large.

REFERENCES

  • 1

    Chan VF, 1988. Tropical diseases of public health importance in the Philippines. Southeast Asian J Trop Med Public Health 19 :361–367.

  • 2

    Gubler DJ, Suharyono W, Lubis I, Eram S, Sulianti Saroso J, 1979. Epidemic dengue hemorrhagic fever in rural Indonesia. I. Virological and epidemiological studies. Am J Trop Med Hyg 28 :701–710.

    • Search Google Scholar
    • Export Citation
  • 3

    Eram S, Setyabudi Y, Sadono TI, Sutrisno DS, Gubler DJ, Sulianti Saroso J, 1979. Epidemic dengue hemorrhagic fever in rural Indonesia. II. Clinical studies. Am J Trop Med Hyg 28 :711–716.

    • Search Google Scholar
    • Export Citation
  • 4

    Sumarmo, 1987. Dengue haemorrhagic fever in Indonesia. Southeast Asian J Trop Med Public Health 18 :269–274.

  • 5

    Corwin AL, Larasati RP, Bangs MJ, Wuryadi S, Arjoso S, Sukri N, Listyaningsih E, Hartati S, Namursa R, Anwar Z, Chandra S, Loho B, Ahmad H, Campbell JR, Porter KR, 2001. Epidemic dengue transmission in southern Sumatra, Indonesia. Trans R Soc Trop Med Hyg 95 :257–265.

    • Search Google Scholar
    • Export Citation
  • 6

    Guzman MG, Kouri G, Bravo J, Valdes L, Vazquez S, Halstead SB, 2002. Effect of age on outcome of secondary dengue 2 infections. Int J Infect Dis 6 :118–124.

    • Search Google Scholar
    • Export Citation
  • 7

    Guzman MG, Kouri G, Valdes L, Bravo J, Vazquez S, Halstead SB, 2002. Enhanced severity of secondary dengue-2 infections: death rates in 1981 and 1997 Cuban outbreaks. Rev Panam Salud Publica 11 :223–227.

    • Search Google Scholar
    • Export Citation
  • 8

    Kocijancic I, Vidmar K, Ivanovi-Herceg Z, 2003. Chest sonography versus lateral decubitus radiography in the diagnosis of small pleural effusions. J Clin Ultrasound 31 :69–74.

    • Search Google Scholar
    • Export Citation
  • 9

    1997. Dengue Hemorrhagic Fever: Diagnosis, Treatment, Prevention and Control. Second edition. Geneva: World Health Organization.

  • 10

    Porter KR, Widjaja S, Lohita HD, Hadiwijaya SH, Maroef CN, Suharyono W, Tan R, 1999. Evaluation of a commercially available immunoglobulin M capture enzyme-linked immunosorbent assay kit for diagnosing acute dengue infections. Clin Diagn Lab Immunol 6 :741–744.

    • Search Google Scholar
    • Export Citation
  • 11

    Morens DM, Halstead SB, Repik PM, Putvatana R, Raybourne N, 1985. Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization. J Clin Microbiol 22 :250–254.

    • Search Google Scholar
    • Export Citation
  • 12

    Clarke DH, Casals J, 1958. Techniques for hemagglutination and hemgglutination-inhibition with arthropod-borne viruses. Am J Trop Med Hyg 7 :561–573.

    • Search Google Scholar
    • Export Citation
  • 13

    Lanciotti RS, Calisher CH, Gubler DJ, Chang GJ, Vorndam AV, 1992. Rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction. J Clin Microbiol 30 :545–551.

    • Search Google Scholar
    • Export Citation
  • 14

    Endy TP, Chunsuttiwat S, Nisalak A, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA, 2002. Epidemiology of inapparent and symptomatic acute dengue virus infection: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 156 :40–51.

    • Search Google Scholar
    • Export Citation
  • 15

    Endy TP, Nisalak A, Chunsuttiwat S, Libraty DH, Green S, Rothman AL, Vaughn DW, Ennis FA, 2002. Spatial and temporal circulation of dengue virus serotypes: a prospective study of primary school children in Kamphaeng Phet, Thailand. Am J Epidemiol 156 :52–59.

    • Search Google Scholar
    • Export Citation
  • 16

    Graham RR, Juffrie M, Tan R, Hayes CG, Laksono I, Ma’roef C, Erlin, Sutaryo, Porter KR, Halstead SB, 1999. A prospective seroepidemiologic study on dengue in children four to nine years of age in Yogyakarta, Indonesia I. studies in 1995–1996. Am J Trop Med Hyg 61 :412–419.

    • Search Google Scholar
    • Export Citation
  • 17

    Sangkawibha N, Rojanasuphot S, Ahandrik S, Viriyapongse S, Jatanasen S, Salitul V, Phanthumachinda B, Halstead SB, 1984. Risk factors in dengue shock syndrome: a prospective epidemiologic study in Rayong, Thailand. I. The 1980 outbreak. Am J Epidemiol 120 :653–669.

    • Search Google Scholar
    • Export Citation
  • 18

    Burke DS, Nisalak A, Johnson DE, Scott RM, 1988. A prospective study of dengue infections in Bangkok. Am J Trop Med Hyg 38 :172–180.

  • 19

    Thein S, Aung MM, Shwe TN, Aye M, Zaw A, Aye K, Aye KM, Aaskov J, 1997. Risk factors in dengue shock syndrome. Am J Trop Med Hyg 56 :566–572.

    • Search Google Scholar
    • Export Citation
  • 20

    Chen WJ, Chen SL, Chien LJ, Chen CC, King CC, Harn MR, Hwang KP, Fang JH, 1996. Silent transmission of the dengue virus in southern Taiwan. Am J Trop Med Hyg 55 :12–16.

    • Search Google Scholar
    • Export Citation
  • 21

    Gubler DJ, Suharyono W, Lubis I, Eram S, Gunarso S, 1981. Epidemic dengue 3 in central Java, associated with low viremia in man. Am J Trop Med Hyg 30 :1094–1099.

    • Search Google Scholar
    • Export Citation
Save