Symptomatic Dengue in Children in 10 Asian and Latin American Countries

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1 The new england journal of medicine Original Article Symptomatic Dengue in Children in 10 Asian and Latin American Countries Maïna L Azou, M.Sc., Annick Moureau, M.Sc., Elsa Sarti, Ph.D., Joshua Nealon, M.Sc., Betzana Zambrano, M.D., T. Anh Wartel, M.D., Luis Villar, M.D., Maria R.Z. Capeding, M.D., and R. Leon Ochiai, Ph.D., for the CYD14 and CYD15 Primary Study Groups* ABSTRACT BACKGROUND The control groups in two phase 3 trials of dengue vaccine efficacy included two large regional cohorts that were followed up for dengue infection. These cohorts provided a sample for epidemiologic analyses of symptomatic dengue in children across 10 countries in Southeast Asia and Latin America in which dengue is endemic. METHODS We monitored acute febrile illness and virologically confirmed dengue (VCD) in 3424 healthy children, 2 to 16 years of age, in Asia (,, the,, and ) from June 2011 through December 2013 and in 6939 children, 9 to 18 years of age, in Latin America (,,,, and ) from June 2011 through April Acute febrile episodes were determined to be VCD by means of a nonstructural protein 1 antigen immunoassay and reverse-transcriptase polymerase-chain-reaction assays. Dengue hemorrhagic fever was defined according to 1997 World Health Organization criteria. RESULTS Approximately 10% of the febrile episodes in each cohort were confirmed to be VCD, with 319 VCD episodes (4.6 episodes per 100 person-years) occurring in the Asian cohort and 389 VCD episodes (2.9 episodes per 100 person-years) occurring in the Latin American cohort; no trend according to age group was observed. The incidence of dengue hemorrhagic fever was less than 0.3 episodes per 100 personyears in each cohort. The percentage of VCD episodes requiring hospitalization was 19.1% in the Asian cohort and 11.1% in the Latin American cohort. In comparable age groups (9 to 12 years and 13 to 16 years), the burden of dengue was higher in Asia than in Latin America. From Global Epidemiology, Sanofi Pasteur, Lyon (M.L., R.L.O.), and the Global Clinical Department, Sanofi Pasteur, Marcy l Etoile (A.M.) both in France; Sanofi Pasteur Latin America, Coyoacán, (E.S.); Sanofi Pasteur Asia Pacific Region, Singapore, Singapore (J.N., T.A.W.); Sanofi Pasteur Uruguay, Montevideo (B.Z.); Clinical Epidemiology Unit, School of Medicine, Universidad Industrial de Santander, Bucaramanga, (L.V.); and the Research Institute for Tropical Medicine, Alabang, Muntinlupa City, (M.R.Z.C.). Address reprint requests to Dr. Ochiai at Global Epidemiology, Sanofi Pasteur, 2 Ave. Pont Pasteur, Lyon, France, or at leon.ochiai@sanofipasteur. com. * The complete list of the members of the CYD14 and CYD15 Primary Study Groups is provided in the Supplementary Appendix, available at NEJM.org. N Engl J Med 2016;374: DOI: /NEJMoa Copyright 2016 Massachusetts Medical Society. CONCLUSIONS The burdens of dengue were substantial in the two regions and in all age groups. Burdens varied widely according to country, but the rates were generally higher and the disease more frequently severe in Asian countries than in Latin American countries. (Funded by Sanofi Pasteur; CYD14 and CYD15 ClinicalTrials.gov numbers, NCT and NCT ) n engl j med 374;12 nejm.org March 24,

2 The new england journal of medicine In the past several decades, the burden of dengue has expanded to place almost 3.9 billion people at risk for infection. 1,2 From an evidence-based global map, it was estimated that 390 million infections occur annually, 96 million of which are symptomatic. 1 Furthermore, dengue is essentially endemic throughout the tropics and has expanded into 128 countries as dengue virus serotypes continue to spread into new areas. 2 In areas where dengue is endemic, all populations and age groups are at risk, and the burden of cases that require hospitalization can be substantial, especially during outbreaks, with attendant strains on health care resources and utilization. 3-5 Dengue has no specific treatment, and the vaccine that was most advanced in its clinical development was licensed in, the, and in Precise estimates of the burden of dengue disease have been difficult to determine. 1,2 Although dengue is generally a reportable disease, the totals that are reported by national health authorities are likely to underestimate the true burden of disease because medical care is not sought for many mild-to-moderate cases. 9 Furthermore, national surveillance, reporting, and laboratory-confirmation practices vary considerably, and the clinical definitions recommended by the World Health Organization (WHO) have not been used or applied consistently. 15,16 Prospective longitudinal cohort studies that have used active surveillance allow the incidence of symptomatic dengue in a defined population to be measured with precision and reliability. 1,10-13 Disease burdens in such cohorts vary over time but tend to be higher than nationally reported totals. However, because locations, periods, and methods usually differ among cohort studies, these studies are often difficult to compare. Sanofi Pasteur developed a recombinant live, attenuated, tetravalent dengue vaccine (chimeric yellow fever dengue tetravalent dengue vaccine [CYD-TDV]). 6,7,17 Two phase 3 clinical trials that included more than 30,000 children, 2 to 16 years of age, were conducted in Southeast Asia and Latin America. 6,7 Recruitment of the participants, active surveillance, definition of episodes, clinical assessment, and laboratory confirmation were standardized across all the study sites. Thus, the unvaccinated control groups in the two studies could be considered to be two comparable cohorts for the study of dengue disease across 10 countries in which dengue is endemic. Here, we present data regarding the burdens of symptomatic dengue and seropositivity results in the various age groups in these two cohorts during 25 months of follow-up that occurred in the period from June 2011 through April Methods Study Population and Design The children included in the descriptive analyses presented here were in the control groups of two phase 3, randomized, observer-blind, placebocontrolled clinical trials to assess the safety and efficacy of CYD-TDV in preventing dengue infection and reducing disease severity. 6,7 Details of the study procedures have been reported previously. 6,7 In brief, the studies began in 2011 and included healthy children 2 to 14 years of age in Southeast Asia, with multiple centers in,, the,, and (Fig. S1A in the Supplementary Appendix, available with the full text of this article at NEJM.org), and children 9 to 16 years of age in Latin America, with multiple centers in,,,, and (Fig. S1B in the Supplementary Appendix). These countries and the age ranges of the participants were selected because they had a high incidence of dengue disease, which was confirmed for most countries in two prospective cohort studies that were conducted before the vaccine trials. 18,19 Participants were randomly assigned, in a 2:1 ratio, to receive three doses of CYD-TDV (treatment group) or placebo (0.9% sodium chloride; control group) within 1 year. In addition, 20% of the participants in Asia and 10% of those in Latin America were randomly assigned to a subgroup for the assessment of vaccine reactogenicity, immunogenicity, and baseline seropositivity. Participants were followed for 25 months after the first dose of vaccine or placebo. Each study was conducted in compliance with Good Clinical Practice guidelines, the principles of the Declaration of Helsinki, and the regulations of the relevant country. Each study was approved by the appropriate ethics review committee. Written informed consent was obtained from a parent or guardian for all the partici n engl j med 374;12 nejm.org March 24, 2016

3 Symptomatic Dengue in Children in Asia and Latin America pants in the two trials, with assent obtained depending on the participant s age. With respect to the current analysis, the sponsor (Sanofi Pasteur) participated in the design of the study and the collection, analysis, and interpretation of the data. The sponsor also participated in the writing of the report and in the decision to submit the manuscript for publication. Medical writing services were provided by 4Clinics France, with funding from the sponsor. Study Procedures Children were actively monitored for acute febrile illness (temperature 38 C for 2 consecutive days) by means of weekly contact with parents or guardians in each cohort and by surveillance of school absenteeism in the Asian cohort. Participants who had an acute febrile episode were provided standard care, and routine biologic tests were performed; children were hospitalized if necessary, according to local practices. Consecutive febrile episodes were considered to be independent if they occurred more than 14 days apart. To confirm the presence of dengue virus infection, a blood sample obtained within 5 days after the onset of fever was tested by an enzymelinked immunosorbent assay for nonstructural protein 1 antigen (Platelia, Bio-Rad Laboratories), by a quantitative reverse-transcriptase polymerasechain-reaction (PCR) screening assay for dengue, and by a serotype-specific real-time PCR assay (Simplexa Dengue, Focus Diagnostics). Febrile episodes were considered to be virologically confirmed dengue (VCD) if any of these tests had a positive result. Dengue hemorrhagic fever was defined according to the 1997 WHO criteria, which included fever persisting for 2 to 7 days accompanied by hemorrhage (or a positive tourniquet test), thrombocytopenia ( 100,000 cells per cubic millimeter), and evidence of plasma leakage (hematocrit that is 20% above the normal value for age or that decreased by 20% after fluid-replacement therapy), pleural effusion, ascites, or hypoproteinemia. 20 Dengue hemorrhagic fever was classified in four grades of severity. Grade 1 is defined by fever accompanied by nonspecific constitutional symptoms; the only hemorrhagic manifestation is a positive tourniquet test, easy bruising, or both. Grade 2 is defined by spontaneous bleeding (usually in the forms of skin or other hemorrhages) in addition to the manifestations of grade 1. Grade 3 is defined by circulatory failure manifested by a rapid, weak pulse and narrowing of pulse pressure or hypotension, with the presence of cold, clammy skin and restlessness. Grade 4 is defined by profound shock with undetectable blood pressure or pulse. 20 The seropositivity analysis was performed with baseline data from participants in the immunogenicity subgroup who received at least one injection of vaccine or placebo. The baseline titers of dengue-neutralizing antibodies in the blood samples obtained before vaccination were measured with the use of a plaque-reduction seroneutralization assay, in which the titer was the reciprocal of the serum dilution that reduced the number of plaques in the control by 50%, and seropositivity was defined as a titer of 10 or more. 21 Assays were performed under blinded conditions at the Global Clinical Immunology laboratories of the sponsor, at the Center for Vaccine Development at Mahidol University in Bangkok,, and at Focus Diagnostics. Statistical Analysis Descriptive analyses of the acute febrile episodes, VCD, hospitalization, and dengue hemorrhagic fever according to country and age group included all the participants in the control group of each study who received at least one injection of placebo. All the initial and recurrent episodes of VCD were considered in the incidencedensity calculation for the entire follow-up period. Incidence density was determined according to age group on the basis of the age at fever onset for the episode and the number of person-years followed, which was determined weekly as the cumulative time in years that each participant contributed to each age group during the active surveillance period. Participants were stratified according to age groups that were applicable to each cohort according to the age at the onset of fever (2 to 4 years, 5 to 8 years, 9 to 12 years, 13 to 16 years, and 17 to 18 years); thus, some children were outside the inclusion age range of each cohort, and only the age groups of 9 to 12 years and 13 to 16 years were comparable across the two cohorts. The 95% confidence intervals for incidence density and percentages were computed with the exact n engl j med 374;12 nejm.org March 24,

4 The new england journal of medicine Table 1. Characteristics of the Study Populations at Inclusion in the Asian and Latin American Cohorts.* Characteristic Asia Latin America (N = 6939) (N = 440) (N = 1149) (N = 931) (N = 3245) (N = 1174) (N = 3424) (N = 778) (N = 392) (N = 1166) (N = 465) (N = 623) Percent of participants 8/11 3/14 6/11 3/14 6/11 4/14 6/11 3/14 8/11 3/14 9/11 12/13 10/11 12/13 6/11 12/13 6/11 11/13 6/11 9/13 Surveillance period Male:female ratio Age yr Mean 8.5± ± ± ± ± ± ± ± ± ± ± ±2.1 Range no. (%) 2 4 yr 96 (15.4) 74 (15.9) 273 (23.4) 78 (19.9) 67 (8.6) 588 (17.2) NA NA NA NA NA NA 5 8 yr 269 (43.2) 155 (33.3) 337 (28.9) 129 (32.9) 289 (37.1) 1179 (34.4) NA NA NA NA NA NA 9 12 yr 204 (32.7) 168 (36.1) 361 (31.0) 130 (33.2) 338 (43.4) 1201 (35.1) 721 (61.4) 1949 (60.1) 578 (62.1) 699 (60.8) 221 (50.2) 4168 (60.1) yr 54 (8.7) 68 (14.6) 195 (16.7) 55 (14.0) 84 (10.8) 456 (13.3) 453 (38.6) 1296 (39.9) 353 (37.9) 450 (39.2) 219 (49.8) 2771 (39.9) * Plus minus values are means ±SD. Participants were in the control groups of two phase 3, randomized, placebo-controlled clinical trials of the chimeric yellow fever dengue tetravalent dengue vaccine (CYD-TDV). NA denotes not applicable. binomial distribution for percentages (Clopper Pearson method). 22 Statistical analyses were performed with the use of SAS software, version 9.2 (SAS Institute). Results Demographic Characteristics of the Cohorts The Asian cohort included 3424 participants, 2 to 14 years of age, who were followed for 25 months in the period between June 3, 2011, and December 16, 2013 (Table 1). 6 The country subcohorts in Asia varied in size, with the smallest in and the largest in the. The Latin American cohort included 6939 participants, 9 to 16 years of age, who were followed for 25 months in the period between June 8, 2011, and April 3, The smallest country subcohort in Latin America was in and the largest was in. The mean age of the participants was 8.8 years in the Asian cohort and 12.5 years in the Latin American cohort. The mean ages and the sex ratios of the country subcohorts were similar at inclusion within each geographic region. Acute Febrile Episodes In the Asian cohort, 3109 febrile episodes were observed during 6934 person-years (2.03 years per participant), for an overall incidence of 44.8 episodes per 100 person-years (Table 2, and Table S1 in the Supplementary Appendix). In the Latin American cohort, 3615 febrile episodes were observed during 13,527 person-years (1.95 years per participant), for an overall incidence of 26.7 episodes per 100 person-years. The incidence of febrile episodes in the subcohorts ranged from 20.7 episodes per 100 person-years (in ) to 60.0 episodes per 100 personyears (in the ); the incidence decreased with increasing age of the participants. The overall incidence in each comparable age group (9 to 12 years and 13 to 16 years) was similar in the two cohorts, although the incidence rates varied according to subcohort. Virologically Confirmed Dengue The overall incidence of VCD in the Asian cohort was 4.6 episodes per 100 person-years (range, 2.2 [in ] to 6.6 [in the ]); the incidence in the Latin American cohort was 2.9 episodes per 100 person-years (range, 1.5 [in 1158 n engl j med 374;12 nejm.org March 24, 2016

5 Symptomatic Dengue in Children in Asia and Latin America ] to 4.1 [in ]) (Table 2, and Table S1 in the Supplementary Appendix). The age groups that were most affected varied according to subcohort, with no clearly observable trends. The percentages of VCD episodes among febrile episodes in each cohort were similar (10.3% in Asia and 10.8% in Latin America), with percentages in the subcohorts ranging from 6.3% (in ) to 12.3% (in ) in Asia and from 7.0% (in ) to 14.7% (in ) in Latin America. Overall, and within most countries, this percentage tended to increase with increasing age, but in the two comparable age groups (9 to 12 years and 13 to 16 years), the percentage was slightly higher in the Asian cohort than in the Latin American cohort. Hospitalization and Dengue Hemorrhagic Fever The overall incidence of hospitalization for VCD episodes was 0.9 hospitalizations per 100 person-years (range, 0.2 [in ] to 1.6 [in and ]) in the Asian cohort and 0.3 hospitalizations per 100 person-years (range, 0.1 [in and ] to 0.4 [in and ]) in the Latin American cohort (Table 3, and Table S2 in the Supplementary Appendix). The percentage of VCD episodes requiring hospitalization was 19.1% (range, 5.9 [in ] to 45.5 [in ]) in the Asian cohort and 11.1% (range, 4.9 [in ] to 17.0 [in ]) in the Latin American cohort. There was no age-group trend for either the incidence or the percentage of VCD episodes that required hospitalization. However, both these values in the two comparable age groups tended to be higher in the Asian cohort than in the Latin American cohort. A total of 30 episodes of VCD were diagnosed as dengue hemorrhagic fever, as defined according to the 1997 WHO criteria, with 20 episodes occurring in the Asian cohort and 10 in the Latin American cohort (Table 3, and Table S2 in the Supplementary Appendix). Most episodes of dengue hemorrhagic fever (28 of 30) were classified as grade 1 or 2. The overall incidence of episodes of dengue hemorrhagic fever was 0.3 episodes per 100 person-years (range, 0.1 [in and ] to 0.6 [in ]) in the Asian cohort and 0.1 episodes per 100 personyears (range, 0.0 [in and ] to 0.1 [in,, and ]) in the Latin American cohort, with no episodes of dengue hemorrhagic fever occurring in or. The percentage of VCD episodes that were diagnosed as dengue hemorrhagic fever was 6.3% (range, 3.9 [in ] to 18.2 [in ]) in Asia and 2.6% (range, 0.0 [in and ] to 7.7 [in ]) in Latin America. The percentage in (18.2%) was much higher than that in any other country (all other countries, <7.7%). There were no agegroup trends with respect to the incidence of dengue hemorrhagic fever or the percentage of episodes of VCD that were diagnosed as dengue hemorrhagic fever. No deaths due to dengue occurred in either cohort. Serotype Distribution All four dengue virus serotypes cocirculated in the five Asian countries during the study period, although the distributions and predominant serotypes differed among countries (Fig. 1A). Shifts in serotype predominance occurred in (serotype 2 to serotype 1), the (serotype 1 to serotype 4), and (serotype 2 to serotype 3) (Fig. S2 in the Supplementary Appendix). In the Latin American cohort, serotype distributions also varied according to country (Fig. 1B). Single serotypes predominated in (serotype 4) and (serotype 1), whereas two or more serotypes predominated in,, and (which was the only Latin American subcohort affected by all four serotypes). No clear changes in serotype predominance occurred in the Latin American subcohorts (Fig. S3 in the Supplementary Appendix). Dengue Seropositivity In the Asian immunogenicity subgroup, 1345 of 1999 participants (67.3%) were seropositive at baseline. In the Latin American subgroup, 1585 of 2000 participants (79.2%) were seropositive (Table 4). Overall rates of seropositivity varied according to country and were between 47.0% () and 80.9% () in the Asian cohort and between 52.6% () and 92.3% () in the Latin American cohort. The rates of seropositivity according to age group were similar overall in the Asian cohort and the Latin American cohort: in the group of partici- n engl j med 374;12 nejm.org March 24,

6 The new england journal of medicine Table 2. Incidences of Febrile Episodes and Episodes of Virologically Confirmed Dengue (VCD) in the Asian and Latin American Cohorts.* Variable Asia Latin America (N = 623) (N = 465) (N = 1166) (N = 392) (N = 778) (N = 3424) (N = 1174) (N = 3245) (N = 931) (N = 1149) (N = 440) (N = 6939) Person-yr no ,527 person-yr 2 4 yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr , yr , yr NA NA NA NA NA NA Febrile episodes no ,615 Incidence no./100 person-yr Overall yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr yr yr NA NA NA NA NA NA Tested for dengue no VCD episodes no Incidence no./100 person-yr Overall (95% CI) 3.6 ( ) 2.2 ( ) 6.6 ( ) 5.9 ( ) 3.2 ( ) 4.6 ( ) 2 4 yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr yr yr NA NA NA NA NA NA ( ) 2.6 ( ) 4.1 ( ) 2.5 ( ) 1.5 ( ) ) 1160 n engl j med 374;12 nejm.org March 24, 2016

7 Symptomatic Dengue in Children in Asia and Latin America Variable Asia Latin America (N = 6939) (N = 440) (N = 1149) (N = 931) (N = 3245) (N = 1174) (N = 3424) (N = 778) (N = 392) (N = 1166) (N = 465) (N = 623) Percent of VCD episodes among febrile episodes Overall (95% CI) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 4 yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr yr yr NA NA NA NA NA NA * All age groups were defined according to age at episode onset. CI denotes confidence interval. pants 9 to 12 years of age, the rates were 75.7% in the Asian cohort and 76.4% in the Latin American cohort, and in the group of those 13 to 16 years of age, the rates were 87.4% and 84.0%, respectively. Discussion These two studies, conducted across 10 countries in which dengue is endemic, were similarly designed and used the same methods and case definitions, which enabled the capture of data regarding symptomatic dengue episodes as well as comparisons across countries, regions, and age groups. We found that dengue-specific burdens varied extensively within each geographic region and that all pediatric age groups were affected. The overall incidences of febrile episodes varied according to country and decreased with increasing age. Although the overall incidence appeared to be higher in the Asian cohort than in the Latin American cohort, this disparity was probably due in part to the younger age range of the participants included in this cohort, among whom the incidence of acute febrile disease is expected to be higher. 23,24 The similar overall incidence in the comparable age groups (9 to 12 years of age and 13 to 16 years of age) suggests that the burden of febrile disease among children 9 to 16 years of age was similar in the two cohorts. We found substantial burdens of dengue disease in all 10 countries; however, the incidence of VCD overall and in the two comparable age groups across the subcohorts was generally higher in the Asian cohort than in the Latin American cohort. Although the incidence rates of dengue differ according to location and fluctuate according to year and season, our results (4.6 episodes per 100 person-years in the Asian cohort and 2.9 per 100 person-years in the Latin American cohort) fall within the incidence ranges observed in other dengue cohort studies conducted in some of the same countries. 18,19,25-27 For example, the incidence of VCD among children was reported to be 1.77 to 5.74 episodes per 100 person-years in in the period from 2006 through 2009, to 4.04 episodes per 100 person-years in in the period from 2005 through 2007, 25 and 1.34 episodes per 100 person-years in in the period from n engl j med 374;12 nejm.org March 24,

8 The new england journal of medicine Table 3. Incidences of Hospitalization and Dengue Hemorrhagic Fever (DHF) among Participants with VCD in the Asian and Latin American Cohorts.* Variable Asia Latin America (N = 623) (N = 465) (N = 1166) (N = 392) (N = 778) (N = 3424) (N = 1174) (N = 3245) (N = 931) (N = 1149) (N = 440) (N = 6939) Hospitalization no Incidence no./100 person-yr Overall (95% CI) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 4 yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr yr yr NA NA NA NA NA NA Percent of hospitalizations among VCD episodes Overall (95% CI) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 2 4 yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr yr yr NA NA NA NA NA NA DHF No. of episodes Incidence no./100 person-yr Overall (95% CI) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) 1162 n engl j med 374;12 nejm.org March 24, 2016

9 Symptomatic Dengue in Children in Asia and Latin America Table 3. (Continued.) Variable Asia Latin America (N = 623) (N = 465) (N = 1166) (N = 392) (N = 778) (N = 3424) (N = 1174) (N = 3245) (N = 931) (N = 1149) (N = 440) (N = 6939) 2 4 yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr yr yr NA NA NA NA NA NA Percent of DHF episodes among VCD episodes Overall (95% CI) 18.2 ( ) 4.8 ( ) 4.5 ( ) 4.3 ( ) 3.9 ( ) 6.3 ( ) 0.0 ( ) 4.2 ( ) 2.7 ( ) 0.0 ( ) 7.7 ( ) 2 4 yr NA NA NA NA NA NA 5 8 yr NA NA NA NA NA NA 9 12 yr yr yr NA NA NA NA NA NA ( ) * s were stratified according to age at episode onset. n engl j med 374;12 nejm.org March 24,

10 The new england journal of medicine A Asian Cohort Participants (%) (N=42) (N=20) (N=159) (N=46) (N=48) B Latin American Cohort Serotype 1 Serotype 2 Serotype 3 Serotype Participants (%) (N=81) Serotype 1 Serotype 2 Serotype 3 Serotype 4 (N=167) (N=65) (N=57) (N=13) Figure 1. Serotype Distributions in Episodes of Virologically Confirmed Dengue, According to Country Subcohort in Asia and Latin America. Dengue virus serotypes in blood samples that were obtained during episodes of virologically confirmed dengue were determined by means of reversetranscriptase polymerase-chain-reaction assay with the use of serotypespecific primers. The overall serotype distributions during the surveillance period are shown for each country. Results include five episodes in the Asian cohort (one in and four in the ) and eight in the Latin American cohort (four in, one in, two in, and one in ) that were coinfections with two dengue virus serotypes through VCD accounted for approximately 10% of the febrile episodes across the two cohorts. This percentage tended to increase with age in the two cohorts and within most countries; in some of the oldest age groups, it was more than 20%. A total of 30 episodes of dengue hemorrhagic fever were observed in the studies. The incidence in the Asian cohort (0.3 episodes per 100 person-years) was similar to the incidence of severe dengue that has been found in other Asian cohort studies (0.1 to 0.7%). 27 Owing to the active nature of the surveillance used in our studies, we consider these incidence rates of dengue hemorrhagic fever to be accurate measures of this disease in these otherwise-healthy, pediatric community-based cohorts. Although the indicators of dengue severity (i.e., incidences of hospitalizations for VCD or dengue hemorrhagic fever and the percentages of VCD episodes for which the participant was hospitalized or was found to have dengue hemorrhagic fever) varied according to country, they were generally higher in Asia than in Latin America, both overall and in the comparable age groups. Whether this means that VCD is more severe in Asia than in Latin America continues to be a topic of discussion that involves complex factors, including differences in disease burden and virus genotype. 29,30 Finally, since more than 70% of the hospitalizations for VCD episodes (74 of 104 episodes overall) were not associated with dengue hemorrhagic fever, it appears that this milder presentation exerts a substantial hospitalization burden in some countries. All the age groups in the subcohorts were affected by dengue. Overall, no age group in either regional cohort was more clearly affected than any other, although some age groups had higher incidence rates in some countries. In the Asian subcohorts, the incidence of VCD appeared to be highest in either the group of participants who were 5 to 8 years of age or the group of those who were 9 to 12 years of age (depending on country). In the Latin American cohort, the highest incidence rates occurred in different age groups (the group that was 17 to 18 years of age in,, and and the group that was 9 to 12 years of age in and ). These findings are consistent with recent reviews of reports that were based on national surveillance. 3,10-13,31-33 There were no observable trends according to age group with respect to the percentages of VCD episodes that either required hospitalization or were found to be dengue hemorrhagic fever. Between 60 and 98% of the children who were older than 13 years of age in these countries had been exposed to dengue before the 1164 n engl j med 374;12 nejm.org March 24, 2016

11 Symptomatic Dengue in Children in Asia and Latin America start of the study. However, a link between the various seropositivity rates in the subcohorts and the incidence of VCD was not evident, which further highlights the extensive geographic and temporal variability of dengue epidemiology. All four virus serotypes cocirculated in all five Asian countries, with variations according to country and over time. Serotype distributions in the Latin American countries usually consisted of one or two predominant serotypes, with minor changes over time. The different serotype-distribution profiles in these two geographic regions have been linked to their different histories of dengue, with sustained hyperendemicity in Southeast Asia since World War II versus widespread eradication of the Aedes aegypti vector in Latin America in the 1950s, followed by progressive reintroduction of the mosquito and of dengue viruses. 29 As serotypes continue to spread in Latin America, serotype distributions and the epidemiologic features of dengue may eventually resemble those in Asia. Our analysis has a few limitations. First, since the countries were chosen for their high burdens of dengue, 20,21 generalization of these findings to other countries in the region may be limited. Furthermore, the cohorts included only healthy children whose parents were willing to have their children participate in a vaccine trial; the disease-burden estimates in these children may differ from those in other children in these countries. Second, the ages of the participants were selected on the basis of the highest incidence of dengue in each cohort but did not include the youngest children (<2 years of age) in either geographic region and were not the same across the two regions, which limited some comparisons. Finally, with only 30 episodes of dengue hemorrhagic fever observed in the two cohorts, interpretations of the incidence of this disease should be made with caution. Our analysis of these pediatric cohorts showed substantial and varied burdens of dengue disease among participants 2 to 16 years of age in five Asian countries and among participants 9 to 18 years of age in five Latin American countries. The disease burden was severe enough to cause considerable rates of hospitalization and accounted for up to approximately 15% of the episodes of febrile disease in some countries. Supported by Sanofi Pasteur. Table 4. Dengue Seropositivity at Inclusion in the Asian and Latin American Cohorts. Variable Asia Latin America (N = 6939) (N = 440) (N = 1149) (N = 931) (N = 3245) (N = 1174) (N = 3424) (N = 778) (N = 392) (N = 1166) (N = 465) (N = 623) Blood samples no Seropositive specimens no ( ) 55.9 ( ) 52.6 ( ) 86.3 ( ) 92.3 ( ) 73.0 ( ) 67.3 ( ) 54.2 ( ) 67.7 ( ) 78.1 ( ) 47.0 ( ) % (95% CI) 80.9 ( ) no. (%)* 2 4 yr 50 (57.5) 22 (32.4) 87 (58.0) 33 (48.5) 43 (44.3) 235 (50.0) NA NA NA NA NA NA 5 8 yr 76 (84.4) 28 (34.1) 125 (74.9) 73 (61.9) 70 (49.3) 372 (62.1) NA NA NA NA NA NA 9 12 yr 101 (87.1) 59 (57.8) 139 (88.5) 93 (79.5) 90 (62.1) 482 (75.7) 136 (67.7) 526 (90.9) 154 (82.4) 97 (48.3) 42 (51.2) 955 (76.4) yr 56 (98.2) 32 (66.7) 119 (93.0) 32 (84.2) 17 (77.3) 256 (87.4) 83 (83.8) 324 (94.7) 105 (92.9) 75 (59.5) 43 (61.4) 630 (84.0) * s were stratified according to age at inclusion. The value in parentheses is the seropositivity rate in the age group of the country subcohort. n engl j med 374;12 nejm.org March 24,

12 Symptomatic Dengue in Children in Asia and Latin America Disclosure forms provided by the authors are available with the full text of this article at NEJM.org. We thank all the parents and children who agreed to participate in these trials; the trial staff in the countries; the clinical research organization staff who contributed to the successful completion of the trials; Sandra Besada-Lombana, Paul Commander, Valentine Delore, Laure Durand, Michael Greenberg, Chris Nelson, and Jo-Ann West for critical review of an earlier version of the manuscript; and Kurt Liittschwager, 4Clinics France, for medical writing services. References 1. Bhatt S, Gething PW, Brady OJ, et al. The global distribution and burden of dengue. Nature 2013; 496: Brady OJ, Gething PW, Bhatt S, et al. Refining the global spatial limits of dengue virus transmission by evidence-based consensus. PLoS Negl Trop Dis 2012; 6(8): e L Azou M, Brett J, Marsh G, Sarti E. 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PLoS Negl Trop Dis 2013; 7(4): e Mohd-Zaki AH, Brett J, Ismail E, L Azou M. Epidemiology of dengue disease in ( ): a systematic literature review. PLoS Negl Trop Dis 2014; 8(11): e Villar LA, Rojas DP, Besada-Lombana S, Sarti E. Epidemiological trends of dengue disease in ( ): a systematic review. PLoS Negl Trop Dis 2015; 9(3): e Copyright 2016 Massachusetts Medical Society n engl j med 374;12 nejm.org March 24, 2016