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ABSTRACT |
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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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.
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MATERIALS AND METHODS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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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.
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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).
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. 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.
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). 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.
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. 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.
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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.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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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.
Received March 14, 2004. Accepted for publication September 14, 2004.
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.
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{at}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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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