Tens of millions of dengue cases and approximately 500,000 life-threatening complications occur annually. New tools are needed to distinguish dengue from other febrile illnesses. In addition, the natural history of pediatric dengue early in illness in a community-based setting has not been well-defined.
Data from the multi-year, ongoing Pediatric Dengue Cohort Study of approximately 3,800 children aged 2–14 years in Managua, Nicaragua, were used to examine the frequency of clinical signs and symptoms by day of illness and to generate models for the association of signs and symptoms during the early phase of illness and over the entire course of illness with testing dengue-positive. Odds ratios (ORs) and 95% confidence intervals were calculated using generalized estimating equations (GEE) for repeated measures, adjusting for age and gender.
One-fourth of children who tested dengue-positive did not meet the WHO case definition for suspected dengue. The frequency of signs and symptoms varied by day of illness, dengue status, and disease severity. Multivariable GEE models showed increased odds of testing dengue-positive associated with fever, headache, retro-orbital pain, myalgia, arthralgia, rash, petechiae, positive tourniquet test, vomiting, leukopenia, platelets ≤150,000 cells/mL, poor capillary refill, cold extremities and hypotension. Estimated ORs tended to be higher for signs and symptoms over the course of illness compared to the early phase of illness.
Day-by-day analysis of clinical signs and symptoms together with longitudinal statistical analysis showed significant associations with testing dengue-positive and important differences during the early phase of illness compared to the entire course of illness. These findings stress the importance of considering day of illness when developing prediction algorithms for real-time clinical management.
Dengue virus causes an estimated 50 million dengue cases and approximately 500,000 life-threatening complications annually. New tools are needed to distinguish dengue from other febrile illnesses. In addition, the natural history of pediatric dengue early in illness in a community-based setting has not been well-defined. Here, we describe the clinical spectrum of pediatric dengue over the course of illness in a community setting by using five years of data from an ongoing prospective cohort study of children in Managua, Nicaragua. Day-by-day analysis of clinical signs and symptoms together with longitudinal statistical analysis showed significant associations with testing dengue-positive and important differences during the early phase of illness compared to the entire course of illness. These findings are important for clinical practice since outside of the hospital setting, clinicians may see dengue patients toward the beginning of their illness and utilize that information to decide whether their patient has dengue or another febrile illness. The results of these models should be extended for the development of prediction algorithms to aid clinicians in diagnosing suspected dengue.
Citation: Biswas HH, Ortega O, Gordon A, Standish K, Balmaseda A, Kuan G, et al. (2012) Early Clinical Features of Dengue Virus Infection in Nicaraguan Children: A Longitudinal Analysis. PLoS Negl Trop Dis 6(3): e1562. https://doi.org/10.1371/journal.pntd.0001562
Editor: Benedito A. Lopes da Fonseca, Universidade de São Paulo, Brazil
Received: October 20, 2011; Accepted: January 25, 2012; Published: March 6, 2012
Copyright: © 2012 Biswas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant VE-1 from the Pediatric Dengue Vaccine Initiative to EH and the University of California, Berkeley Center for Global Health and Graduate Division Fellowships to HHB. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Dengue virus (DENV) causes the most prevalent mosquito-borne viral disease affecting humans, with 2.5–3 billion people at risk for infection and approximately 50 million cases of dengue each year , . The four DENV serotypes are transmitted to humans by Aedes aegypti and Ae. albopictus mosquitoes, primarily in urban and peri-urban areas in tropical and subtropical countries worldwide. Most cases present as classic dengue fever (DF), a debilitating but self-limited illness that manifests with high fever, retro-orbital pain, severe myalgia/arthralgia, and rash. However, in some cases, mainly children, illness progresses to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS), characterized by vascular leakage leading to hypovolemic shock and a case fatality rate up to 5% , , . Currently, no licensed vaccine or antiviral therapy exists for dengue. Early identification of patients at risk of developing severe dengue is critical to provide timely supportive care, which can reduce the risk of mortality to <1% , . However, distinguishing dengue from other febrile illnesses (OFIs) early in illness is challenging, since symptoms are non-specific and common to other febrile illnesses such as malaria, leptospirosis, rickettsiosis, and typhoid fever – in dengue-endemic countries. In addition, many distinguishing clinical features of DHF/DSS generally emerge only after 4–5 days, at defervescence, when the patient is already critically ill.
Although the World Health Organization (WHO) has recently established new clinical guidelines to classify dengue severity , serological, virological, and molecular biological tests are required to definitively diagnose DENV infection. In many endemic countries, laboratory diagnosis of dengue is often problematic due to lack of reagents, expense, or delay in obtaining results. Patients with suspected dengue are often hospitalized for close monitoring to ensure proper treatment if they begin to develop severe dengue; however, up to 38–52% are later diagnosed with OFIs ,  and thus were hospitalized unnecessarily at great financial cost to their family and society . New tools are therefore needed to distinguish dengue from OFIs to prevent deaths from severe dengue and to mitigate the economic burden of excess hospitalization.
Recent approaches using multivariable logistic or linear regression models have shown that petechiae, thrombocytopenia (platelet count ≤100,000 cells/mm3), positive tourniquet test, rash, and other signs and symptoms can distinguish dengue from OFIs –; however, results were not consistent across studies. Only two studies considered clinical and laboratory features according to day of illness –, but as these were hospital-based studies, the results likely reflect patients with more severe symptoms and not the clinical spectrum of all symptomatic cases in dengue-endemic populations. Furthermore, none of these studies analyzed data using longitudinal statistical methods, which account for correlations between repeated measures on individuals over time. The use of longitudinal statistical methods to analyze cohort data is essential to utilize all of the data available for analysis and appropriately estimate the within-person and between-person variance in measures over time.
In this study, we used five years of data from an ongoing prospective cohort study of approximately 3,800 children aged 2–14 years in Managua, Nicaragua, to examine the frequency of clinical signs and symptoms by day of illness and to generate models for the association of signs and symptoms during the early phase of illness and over the entire course of illness with testing dengue-positive. In order to account for the longitudinal structure of the data, odds ratios (ORs) and 95% confidence intervals were calculated using generalized estimating equations (GEE), adjusting for age and gender.
Study site and participants
In August and September 2004, a community-based pediatric cohort was established in District II of Managua, a low-to-middle income area with a population of approximately 62,500 . Study activity was based in the Health Center Sócrates Flores Vivas (HCSFV), a public facility that is the primary source of health care for District II residents. Briefly, participants aged 2–9 years were recruited through house-to-house visits, and additional two year-olds were enrolled each year to maintain the age structure of the cohort . Children were eligible to remain in the study until age 12 or until they moved from the study area. The parent/legal guardian of each participant signed an informed consent form, and children ≥6 years old provided verbal assent. In 2007, participants ≤11 years old were given the opportunity to continue for an additional 3 years, and a second informed consent was performed.
The study was approved by the Institutional Review Boards of the University of California, Berkeley, the Nicaraguan Ministry of Health, and the International Vaccine Institute in Seoul, Korea. Parents or legal guardians of all subjects in both studies provided written informed consent, and subjects 6 years of age and older provided assent.
Upon enrollment, parents/legal guardians of all participants were encouraged to bring their child(ren) to the HCSFV at first sign of illness or fever. Study physicians and nurses, trained in identification of possible dengue cases, provided medical care for study participants. Febrile illnesses that met the WHO criteria for suspected dengue (Table 1) and those without other apparent origin (undifferentiated febrile illnesses) were treated as possible dengue cases and followed daily while fever or symptoms persisted through visits with study medical personnel (Figure 1). Complete blood counts (CBCs) were completed every 48 hours or more frequently as necessary, as indicated by the physician. Cases were monitored closely for severe manifestations and were transferred by study personnel to the Infectious Disease Ward of the Manuel de Jesús Rivera Children's Hospital, the national pediatric reference hospital, when they presented with any sign of alarm (Table 1). In addition, an annual healthy blood sample was collected to identify all DENV infections during the previous year and for baseline CBC values. Study physicians in both the hospital and HCSFV completed systematic data collection forms that contained approximately 80 variables (Table 1). In the hospital, additional clinical data, including fluid balance and treatment, were collected daily during hospitalization or through ambulatory follow-up visits by a team of study physicians and nurses. Data were also recorded on medical tests ordered and treatments prescribed.
Of the 1,974 episodes of febrile illness in the Pediatric Dengue Cohort Study from August 2004 to June 2009 that met the WHO classification criteria for suspected dengue or were diagnosed with undifferentiated fever, 405 patients presented with febrile illness on 2 occasions, 105 presented on 3 occasions, 21 presented on 4 occasions, and 5 presented on 5 occasions. One patient presented after day 8 of illness and was excluded from analysis. Twenty-nine patients had cause of fever identified later in the course of illness. CBC, complete blood count; WHO, World Health Organization; UTI, urinary tract infection.
A case was considered laboratory-confirmed dengue when acute DENV infection was demonstrated by: detection of DENV RNA by RT-PCR; isolation of DENV; seroconversion of DENV-specific IgM antibodies observed by MAC-ELISA in paired acute- and convalescent-phase samples; and/or a ≥4-fold increase in anti-DENV antibody titer measured using Inhibition ELISA – in paired acute and convalescent samples. DENV serotypes were identified by RT-PCR and/or virus isolation.
Laboratory-confirmed dengue cases were further classified by severity. DHF and DSS were defined according to the traditional WHO criteria (Table 1) . Additional categories of severity were included for those cases presenting with shock without thrombocytopenia and/or hemoconcentration (dengue with signs associated with shock (DSAS))  or dengue fever with compensated shock (DFCS)  (Table 1). Laboratory-confirmed cases were defined as primary DENV infections if acute-phase antibody titer, as measured by Inhibition ELISA, was <1∶10 or if convalescent phase antibody titer was <1∶2560, and as secondary infections if the acute titer was ≥1∶10 or convalescent titer was ≥1∶2560 –.
Data from the first five years of the study (August 30, 2004–June 30, 2009) were used for analysis. The first three days after onset of fever were considered the early febrile phase of illness. Day of illness at presentation was determined by the date of fever onset, which was defined as the first day of illness as reported by the parent/guardian. Variable definitions are described in Table 1. Positive tourniquet test was examined using cut-offs of ≥10 petechiae/in2 and ≥20 petechiae/in2. Platelet count was dichotomized using a cut-off of ≤150,000 cells/mm3 to enable comparisons during days 1–3. Only data from days 1–8 of illness were included for analysis.
The frequency of dengue testing results (laboratory-confirmed dengue-positive versus dengue-negative) and disease severity (DF versus severe dengue) was examined by year, demographics, serotype and immune response. The frequency of the WHO case definition for suspected dengue was examined by dengue testing results and age, and a chi-square test for trend was performed. The frequency of clinical signs and symptoms by day of illness and dengue severity was also examined using chi-square tests.
To examine the association between clinical signs and symptoms and the odds of testing dengue-positive versus dengue-negative, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated using GEE models assuming an exchangeable correlation structure with robust standard errors to account for the correlations between repeated measures on the same patients over time. First, ORs were calculated using bivariable models that included only dengue testing results and each of the signs or symptoms. All signs and symptoms were then examined in multivariable models that adjusted for age and gender. Data from the first three days of illness and from all days of illness only were analyzed separately. Finally, for comparison, we used traditional logistic regression models to analyze the association between signs and symptoms and testing dengue-positive with data collapsed by illness episode to disregard repeated measures on the same patients, using the same model generation process as for the GEE models. All analyses were conducted using STATA 10 (StataCorp LP, College Station, TX).
From August 2004 to June 2009, 22,778 episodes of febrile illness were evaluated, of which 1,974 episodes were suspected dengue or undifferentiated fever (Figure 1). Of the 1,974 possible dengue cases, 1,793 (91%) tested negative and 181 (9%) were laboratory-confirmed as dengue-positive, of which 161 (89%) were classified as DF, 9 (5%) as DHF, 4 (2%) as DSS, 3 (2%) as DSAS and 4 (2%) as DFCS (Table 1). Nearly all (95%) of the severe dengue cases but only 116 (72%) of the DF cases met the WHO case definition for dengue. The proportion of laboratory-confirmed DENV infections that met the WHO case definition significantly increased by age (chi-square test for trend 5.977, p = 0.01), while younger children experienced significantly more undifferentiated febrile illness due to DENV infection (Figure 2). The median age for cases meeting the dengue case definition was 8 years (range 2–13) and that of undifferentiated febrile illness due to DENV infection was 6 years (range 2–10).
Upon presentation to the health center or hospital, children with a febrile illness were classified according to whether or not they met the WHO classification criteria for suspected dengue. One patient had two dengue virus infections over the course of the study and is represented twice. n = 6 for age 2, n = 10 for age 3, n = 18 for age 4, n = 23 for age 5, n = 21 for age 6, n = 16 for age 7, n = 21 for age 8, n = 23 for age 9, n = 24 for age 10, n = 19 for age 11+. Chi-square test for trend 5.977, p = 0.01. WHO, World Health Organization.
The number of confirmed dengue-positive cases varied by year, as expected (Table 2) . Both genders were equally represented, with a slightly higher percentage of females experiencing severe dengue, though this difference was not statistically significant. The majority of DF cases were DENV-2 (58%), followed by DENV-1 (21%) and DENV-3 (9%), while 60% of severe dengue cases were DENV-2, followed by DENV-3 (25%) and DENV-1 (10%). In addition, there were nearly equal proportions of primary and secondary immune responses among DF cases, whereas the majority (70%) of severe dengue cases were secondary DENV infection (Table 2). The median day of illness at presentation was day 2 for all patients, and almost all presented on days 1–3 of illness (90%). The total follow-up time of all children in the cohort was 17,931 person-years with a median follow-up of 3.9 years per child.
As shown in Figure 3, several signs and symptoms appeared to differentiate OFIs from DF cases, and DF cases from severe dengue cases, according to day of illness. In particular, higher proportions of DF and severe dengue cases experienced petechiae, platelets ≤150,000 cells/mm3, leukopenia, and positive tourniquet test compared to patients with OFIs. Higher proportions of severe cases experienced petechiae, platelets ≤150,000 cells/mm3, myalgia/arthralgia and abdominal pain compared to DF cases and patients with OFIs. Abdominal pain differentiated severe dengue cases from DF and OFI only beginning on day 3 of illness (for severe dengue compared to DF: chi-square 0.144, p = 0.70 for days 1–2 versus chi-square 16.910, p<0.0001 for day ≥3).
Over the course of an episode of febrile illness, signs and symptoms were observed by medical personnel or reported by children and/or their parent/guardian. Selected signs and symptoms are shown here. A, Petechiae; OFI versus DF: chi-square test for trend 21.313, p<0.0001; day 1, n = 606; day 2, n = 1,243; day 3, n = 1,066; day 4, n = 876; day 5, n = 675; day 6, n = 481; day 7, n = 291; day 8, n = 175; B, Platelet count ≤150,000 cells/mm3; OFI versus DF: chi-square test for trend 14.928, p = 0.0001; day 1, n = 604; day 2, n = 970; day 3, n = 615; day 4, n = 568; day 5, n = 348; day 6, n = 234; day 7, n = 122; day 8, n = 65; C, Myalgia/arthralgia; OFI versus DF: chi-square test for trend 4.569, p = 0.03; day 1, n = 612; day 2, n = 1,253; day 3, n = 1,075; day 4, n = 877; day 5, n = 671; day 6, n = 477; day 7, n = 289; day 8, n = 181; D, Leukopenia; OFI versus DF: chi-square test for trend 6.449, p = 0.01; day 1, n = 604; day 2, n = 971; day 3, n = 615; day 4, n = 568; day 5, n = 348; day 6, n = 234; day 7, n = 122; day 8, n = 65; E, Positive tourniquet test; OFI versus DF: chi-square test for trend 20.124, p<0.0001; day 1, n = 256; day 2, n = 496; day 3, n = 402; day 4, n = 308; day 5, n = 202; day 6, n = 156; day 7, n = 78; day 8, n = 38; F, Abdominal pain; OFI versus DF: chi-square test for trend 9.149, p = 0.002; DF versus severe dengue: chi-square test for trend 4.127, p = 0.04; day 1, n = 609; day 2, n = 1,245; day 3, n = 1,066; day 4, n = 877; day 5, n = 675; day 6, n = 482; day 7, n = 290; day 8, n = 174; All other chi-square tests for trend comparing DF to severe dengue were non-significant. OFI, other febrile illness; DF, dengue fever; Severe dengue = dengue hemorrhagic fever, dengue shock syndrome, dengue with signs associated with shock, or dengue fever with compensated shock. Leukopenia is defined as WBC ≤5000 cells/mm3 and positive tourniquet test is defined as ≥10 petechiae/in2.
Bivariable and multivariable analyses were performed using GEE models to examine signs and symptoms early in illness and over the course of illness (Table 3). On days 1–3 of illness, dengue-positive cases had up to 2-fold increased odds of fever, headache, retro-orbital pain, myalgia, arthralgia, and vomiting compared to patients with OFIs. They also had from 3-fold to 9-fold increased odds of rash, petechiae, positive tourniquet test with cut-offs of ≥10 and ≥20 petechiae/in2, leukopenia, platelets ≤150,000 cells/mm3, poor capillary refill, cold extremities and hypotension compared to patients with OFIs. In contrast, they had decreased odds of abdominal pain, likely because this feature appears later in the entire course of dengue illness. On all days of illness, dengue-positive cases had increased odds of the same signs and symptoms as on days 1–3 of illness; however, the magnitude of the point estimates tended to be higher. This difference was most pronounced for rash and platelets ≤150,000 cells/mm3, which had ORs approximately double in magnitude. In addition, dengue-positive cases had increased odds of three additional signs and symptoms: poor appetite, absence of cough, and increased hematocrit. When GEE analyses on data with the longitudinal structure preserved were compared to traditional logistic regression analyses on data collapsed on febrile episode, the point estimates for the ORs were similar, although the 95% confidence intervals for the logistic regression models tended to be slightly narrower (data not shown).
In this study, we describe the clinical spectrum of pediatric dengue starting early in illness in a community setting. Longitudinal statistical analysis of day-by-day clinical signs and symptoms revealed significant associations with testing dengue-positive and important differences during the early phase of illness compared to the entire course of illness. These results stress the importance of considering day of illness when developing prediction algorithms for real-time clinical management.
The early identification of dengue cases and particularly those at risk for severe dengue is critical for preventing severe illness and death. We found that 25% of laboratory-confirmed dengue cases did not meet the WHO case definition, suggesting that the WHO criteria are not sufficient to identify dengue at younger ages. Younger children may experience different signs and symptoms from adults or may be unable to communicate their symptoms to their parents, health care providers, or both. Previous studies demonstrated that children may experience significantly more cough, vomiting, abdominal pain, rash, epistaxis, oliguria, thrombocytopenia, hepatomegaly, and shock compared to adults, although the direction of these differences was not consistent across studies , , –. A recent study of dengue in adults showed significant differences in clinical features and outcomes across ten-year age groups, indicating that signs and symptoms associated with DENV infection may continue to evolve past childhood . If these differences are confirmed, the WHO case definition may need to be adjusted to be age-specific to function effectively for younger children and older age groups.
Retro-orbital pain and low platelets were among the clinical features independently associated with DENV infection in this study. These results are supported by a study of dengue patients in Puerto Rico in which data were recorded at the time of initial consult rather than at hospitalization , and by a study of Thai children . Moreover, our results showing increased frequency of abdominal pain in patients beginning at day 3 of illness are consistent with a prospective study of adults admitted to an emergency department in Martinique . A positive tourniquet test using cut-offs of ≥10 and ≥20 petechiae/in2 was also independently associated with DENV infection. Both cut-offs were used because studies have indicated that a cut-off of ≥10 may improve discrimination of DENV infection , ; however, the 1997 WHO classification scheme specified a cut-off of ≥20 . Our results support using a cut-off of ≥10 petechiae/in2, and this cut-off has been specified in the 2011 WHO clinical guidelines .
A major strength of this study is the use of statistical models designed for analysis of longitudinal data. Few other prospective community-based cohort studies have analyzed early clinical features in pediatric dengue compared to OFI , –, and none that we are aware of were analyzed using longitudinal statistical methods that account for correlations between repeated measures on patients. Here, we preserved the longitudinal structure of the dataset by using statistical models that support repeated measurements on subjects over time and account for correlations between signs and symptoms experienced within the same individual on different days of illness and in multiple episodes. Longitudinal data have long been collected in dengue research but have rarely been analyzed using appropriate statistical methods. This may introduce bias into findings, as studies may overestimate the magnitude of association or reduce the statistical power of the study as data are lost when they are collapsed for non-longitudinal analysis.
An additional strength of this study is that it is community-based , enabling day-by-day capture of information on the early course of illness and on the full clinical spectrum of symptomatic dengue. In contrast, nearly all previous studies enrolled patients upon presentation to a hospital , where patients present later; thus, these studies were unable to capture information on the early days of illness or on mild disease. By examining the clinical spectrum of dengue by day of illness, we were able to detect differences in the prevalence of signs and symptoms that could not be revealed by simply analyzing whether they ever occurred over the course of illness. In addition, through multivariable longitudinal models, we were able to identify distinguishing features of dengue during the early phase of illness compared to the entire course of illness. These findings are important for clinical practice since outside of the hospital setting, clinicians may see dengue patients toward the beginning of their illness and utilize that information to decide whether their patient has dengue or another febrile illness. The results of these models should be extended for the development of prediction algorithms to aid clinicians in diagnosing suspected dengue.
This study was not without its limitations. Some participants migrated out of the study area or withdrew from the study; however, our retention rate was approximately 95% per year , suggesting that any bias from loss to follow-up would be minimal. It is also possible that we did not capture all symptomatic dengue cases. However, in yearly participant surveys, only an average of 2–3% of participants reported having attended a health-care provider outside of the study or having an illness and not attending any medical provider , and approximately 20-fold more laboratory-confirmed dengue cases were captured in the cohort study than by the National Surveillance System . Unfortunately, due to the low number of severe dengue cases, this study did not have sufficient statistical power to compare severe dengue cases to DF cases using GEE models, and these low numbers may have influenced the lack of significant association of signs of severe dengue with testing dengue-positive. For this study, we used the 1997 WHO classification scheme for disease severity. In 2009, the WHO updated its guidelines for classification of dengue disease severity , ; it would be interesting to re-analyze the data in a future study using the new classification scheme. Studies of the usefulness and applicability of the revised guidelines have been recently performed , .
In summary, this study is one of the few cohort studies to provide early data on the full clinical spectrum of pediatric dengue. Though we found significantly increased odds for association of several clinical signs and symptoms with testing dengue-positive, these increases were more modest for the early phase of illness compared to the course of illness, suggesting that caution should be taken when using the results from the entire course of illness to develop prediction algorithms. Non-parametric methods such as decision tree analysis overcome some of the limitations of traditional logistic regression models and have recently been applied to develop algorithms for prediction of dengue diagnosis and disease severity , , . These and other data-adaptive approaches such as Super Learner  that are less subject to bias should be further explored to develop prediction algorithms for early identification of dengue cases and improved clinical management.
We thank our study team based at the Centro de Salud Sócrates Flores Vivas, the Hospital Infantil Manuel de Jesús Rivera, the Sustainable Sciences Institute, and the National Virology Laboratory in the Centro Nacional de Diagnóstico y Referencia, for their dedication and excellent work to ensure high-quality medical attention and study performance, tireless data entry, top-notch laboratory work, and stellar database management and support, particularly Magaly Amador, Sonia Arguello, William Avilés, Yahoska Buitrago, José Ramon Cisneros, Douglas Elizondo, Carolina Flores, Nicole Fitzpatrick, Gamaliel Gutierrez, Samantha Hammond, Jaqueline Herrera, Brenda López, Roger López, Juan Carlos Matute, Julia Medina, Juan Carlos Mercado, Berman Moraga, Mirtha Monterrey, Azucena Munguia, Federico Narvaez, Grethel Navas, Andrea Nuñez, Sergio Ojeda, Zoila Orozco, Leonel Pérez, Maria Angeles Pérez, Miguel Reyes, Carlos Romero, Crisanta Rocha, Cinthia Saborio, Saira Saborio, Leyla Saenz, Nery Sanchez, Sheyla Silva, Yolanda Tellez, Maria José Vargas, Ubania Vargas, and other study personnel. We are especially thankful to Dr. Alcides Gonzalez for his continued support over the years. Finally, we are indebted to the children, who participated in the study, and their parents.
Conceived and designed the experiments: HHB OO AG KS EH. Performed the experiments: OO GK. Analyzed the data: HHB OO AG KS. Contributed reagents/materials/analysis tools: AB GK EH. Wrote the paper: HHB KS AG EH.
- 1. World Health Organization (2009) Dengue: Guidelines for diagnosis, treatment, prevention and control. Geneva: WHO Press. World Health Organization2009Dengue: Guidelines for diagnosis, treatment, prevention and controlGenevaWHO Press
- 2. Gibbons RV, Vaughn DW (2002) Dengue: An escalating problem. BMJ 324: 1563–1566.RV GibbonsDW Vaughn2002Dengue: An escalating problem.BMJ32415631566
- 3. Guzman MG, Halstead SB, Artsob H, Buchy P, Farrar J, et al. (2010) Dengue: A continuing global threat. Nat Rev Microbiol 8: S7–16.MG GuzmanSB HalsteadH. ArtsobP. BuchyJ. Farrar2010Dengue: A continuing global threat.Nat Rev Microbiol8S716
- 4. World Health Organization (2000) Strengthening implementation of the global strategy for dengue fever/dengue haemorrhagic fever prevention and control. Geneva: WHO Press. World Health Organization2000Strengthening implementation of the global strategy for dengue fever/dengue haemorrhagic fever prevention and controlGenevaWHO Press
- 5. Zavala-Velazquez JE, Yu XJ, Walker DH (1996) Unrecognized spotted fever group rickettsiosis masquerading as dengue fever in Mexico. Am J Trop Med Hyg 55: 157–159.JE Zavala-VelazquezXJ YuDH Walker1996Unrecognized spotted fever group rickettsiosis masquerading as dengue fever in Mexico.Am J Trop Med Hyg55157159
- 6. Watt G, Jongsakul K, Chouriyagune C, Paris R (2003) Differentiating dengue virus infection from scrub typhus in Thai adults with fever. Am J Trop Med Hyg 68: 536–538.G. WattK. JongsakulC. ChouriyaguneR. Paris2003Differentiating dengue virus infection from scrub typhus in Thai adults with fever.Am J Trop Med Hyg68536538
- 7. Ellis RD, Fukuda MM, McDaniel P, Welch K, Nisalak A, et al. (2006) Causes of fever in adults on the Thai-Myanmar border. Am J Trop Med Hyg 74: 108–113.RD EllisMM FukudaP. McDanielK. WelchA. Nisalak2006Causes of fever in adults on the Thai-Myanmar border.Am J Trop Med Hyg74108113
- 8. Balmaseda A, Hammond SN, Perez L, Tellez Y, Saborio SI, et al. (2006) Serotype-specific differences in clinical manifestations of dengue. Am J Trop Med Hyg 74: 449–456.A. BalmasedaSN HammondL. PerezY. TellezSI Saborio2006Serotype-specific differences in clinical manifestations of dengue.Am J Trop Med Hyg74449456
- 9. Potts JA, Gibbons RV, Rothman AL, Srikiatkhachorn A, Thomas SJ, et al. (2010) Prediction of dengue disease severity among pediatric Thai patients using early clinical laboratory indicators. PLoS Negl Trop Dis 4: e769.JA PottsRV GibbonsAL RothmanA. SrikiatkhachornSJ Thomas2010Prediction of dengue disease severity among pediatric Thai patients using early clinical laboratory indicators.PLoS Negl Trop Dis4e769
- 10. Clark DV, Mammen MP Jr, Nisalak A, Puthimethee V, Endy TP (2005) Economic impact of dengue fever/dengue hemorrhagic fever in Thailand at the family and population levels. Am J Trop Med Hyg 72: 786–791.DV ClarkMP Mammen JrA. NisalakV. PuthimetheeTP Endy2005Economic impact of dengue fever/dengue hemorrhagic fever in Thailand at the family and population levels.Am J Trop Med Hyg72786791
- 11. Potts JA, Thomas SJ, Srikiatkhachorn A, Supradish PO, Li W, et al. (2010) Classification of dengue illness based on readily available laboratory data. Am J Trop Med Hyg 83: 781–788.JA PottsSJ ThomasA. SrikiatkhachornPO SupradishW. Li2010Classification of dengue illness based on readily available laboratory data.Am J Trop Med Hyg83781788
- 12. Low JG, Ong A, Tan LK, Chaterji S, Chow A, et al. (2011) The early clinical features of dengue in adults: Challenges for early clinical diagnosis. PLoS Negl Trop Dis 5: e1191.JG LowA. OngLK TanS. ChaterjiA. Chow2011The early clinical features of dengue in adults: Challenges for early clinical diagnosis.PLoS Negl Trop Dis5e1191
- 13. Ramos MM, Tomashek KM, Arguello DF, Luxemburger C, Quinones L, et al. (2009) Early clinical features of dengue infection in Puerto Rico. Trans R Soc Trop Med Hyg 103: 878–884.MM RamosKM TomashekDF ArguelloC. LuxemburgerL. Quinones2009Early clinical features of dengue infection in Puerto Rico.Trans R Soc Trop Med Hyg103878884
- 14. Binh PT, Matheus S, Huong VT, Deparis X, Marechal V (2009) Early clinical and biological features of severe clinical manifestations of dengue in Vietnamese adults. J Clin Virol 45: 276–280.PT BinhS. MatheusVT HuongX. DeparisV. Marechal2009Early clinical and biological features of severe clinical manifestations of dengue in Vietnamese adults.J Clin Virol45276280
- 15. Gregory CJ, Santiago LM, Arguello DF, Hunsperger E, Tomashek KM (2010) Clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area–Puerto Rico, 2007-2008. Am J Trop Med Hyg 82: 922–929.CJ GregoryLM SantiagoDF ArguelloE. HunspergerKM Tomashek2010Clinical and laboratory features that differentiate dengue from other febrile illnesses in an endemic area–Puerto Rico, 2007-2008.Am J Trop Med Hyg82922929
- 16. Chadwick D, Arch B, Wilder-Smith A, Paton N (2006) Distinguishing dengue fever from other infections on the basis of simple clinical and laboratory features: Application of logistic regression analysis. J Clin Virol 35: 147–153.D. ChadwickB. ArchA. Wilder-SmithN. Paton2006Distinguishing dengue fever from other infections on the basis of simple clinical and laboratory features: Application of logistic regression analysis.J Clin Virol35147153
- 17. Wilder-Smith A, Earnest A, Paton NI (2004) Use of simple laboratory features to distinguish the early stage of severe acute respiratory syndrome from dengue fever. Clin Infect Dis 39: 1818–1823.A. Wilder-SmithA. EarnestNI Paton2004Use of simple laboratory features to distinguish the early stage of severe acute respiratory syndrome from dengue fever.Clin Infect Dis3918181823
- 18. Potts JA, Rothman AL (2008) Clinical and laboratory features that distinguish dengue from other febrile illnesses in endemic populations. Trop Med Int Health 13: 1328–1340.JA PottsAL Rothman2008Clinical and laboratory features that distinguish dengue from other febrile illnesses in endemic populations.Trop Med Int Health1313281340
- 19. Deparis X, Murgue B, Roche C, Cassar O, Chungue E (1998) Changing clinical and biological manifestations of dengue during the dengue-2 epidemic in French Polynesia in 1996/97–description and analysis in a prospective study. Trop Med Int Health 3: 859–865.X. DeparisB. MurgueC. RocheO. CassarE. Chungue1998Changing clinical and biological manifestations of dengue during the dengue-2 epidemic in French Polynesia in 1996/97–description and analysis in a prospective study.Trop Med Int Health3859865
- 20. Kalayanarooj S, Vaughn DW, Nimmannitya S, Green S, Suntayakorn S, et al. (1997) Early clinical and laboratory indicators of acute dengue illness. J Infect Dis 176: 313–321.S. KalayanaroojDW VaughnS. NimmannityaS. GreenS. Suntayakorn1997Early clinical and laboratory indicators of acute dengue illness.J Infect Dis176313321
- 21. Kuan G, Gordon A, Aviles W, Ortega O, Hammond SN, et al. (2009) The Nicaraguan pediatric dengue cohort study: Study design, methods, use of information technology, and extension to other infectious diseases. Am J Epidemiol 170: 120–129.G. KuanA. GordonW. AvilesO. OrtegaSN Hammond2009The Nicaraguan pediatric dengue cohort study: Study design, methods, use of information technology, and extension to other infectious diseases.Am J Epidemiol170120129
- 22. Balmaseda A, Hammond SN, Tellez Y, Imhoff L, Rodriguez Y, et al. (2006) High seroprevalence of antibodies against dengue virus in a prospective study of schoolchildren in Managua, Nicaragua. Trop Med Int Health 11: 935–942.A. BalmasedaSN HammondY. TellezL. ImhoffY. Rodriguez2006High seroprevalence of antibodies against dengue virus in a prospective study of schoolchildren in Managua, Nicaragua.Trop Med Int Health11935942
- 23. Harris E, Videa E, Perez L, Sandoval E, Tellez Y, et al. (2000) Clinical, epidemiologic, and virologic features of dengue in the 1998 epidemic in Nicaragua. Am J Trop Med Hyg 63: 5–11.E. HarrisE. VideaL. PerezE. SandovalY. Tellez2000Clinical, epidemiologic, and virologic features of dengue in the 1998 epidemic in Nicaragua.Am J Trop Med Hyg63511
- 24. Fernández RJ, Vázquez S (1990) Serological diagnosis of dengue by an ELISA inhibition method (EIM). Mem Inst Oswaldo Cruz 85: 347–351.RJ FernándezS. Vázquez1990Serological diagnosis of dengue by an ELISA inhibition method (EIM).Mem Inst Oswaldo Cruz85347351
- 25. Reed LJ, Muench H (1938) A simple method of estimating fifty percent endpoints. Am J Hyg 27: 493–497.LJ ReedH. Muench1938A simple method of estimating fifty percent endpoints.Am J Hyg27493497
- 26. World Health Organization (1997) Dengue haemorrhagic fever: Diagnosis, treatment, prevention and control. Geneva: World Health Organization. World Health Organization1997Dengue haemorrhagic fever: Diagnosis, treatment, prevention and controlGenevaWorld Health Organization
- 27. Gutierrez G, Standish K, Narvaez F, Perez MA, Saborio S, et al. (2011) Unusual dengue virus 3 epidemic in Nicaragua, 2009. PLoS Negl Trop Dis 5: e1394.G. GutierrezK. StandishF. NarvaezMA PerezS. Saborio2011Unusual dengue virus 3 epidemic in Nicaragua, 2009.PLoS Negl Trop Dis5e1394
- 28. Balmaseda A, Standish K, Mercado JC, Matute JC, Tellez Y, et al. (2010) Trends in patterns of dengue transmission over 4 years in a pediatric cohort study in Nicaragua. J Infect Dis 201: 5–14.A. BalmasedaK. StandishJC MercadoJC MatuteY. Tellez2010Trends in patterns of dengue transmission over 4 years in a pediatric cohort study in Nicaragua.J Infect Dis201514
- 29. Wang CC, Lee IK, Su MC, Lin HI, Huang YC, et al. (2009) Differences in clinical and laboratory characteristics and disease severity between children and adults with dengue virus infection in Taiwan, 2002. Trans R Soc Trop Med Hyg 103: 871–877.CC WangIK LeeMC SuHI LinYC Huang2009Differences in clinical and laboratory characteristics and disease severity between children and adults with dengue virus infection in Taiwan, 2002.Trans R Soc Trop Med Hyg103871877
- 30. Hanafusa S, Chanyasanha C, Sujirarat D, Khuankhunsathid I, Yaguchi A, et al. (2008) Clinical features and differences between child and adult dengue infections in Rayong province, southeast Thailand. Southeast Asian J Trop Med Public Health 39: 252–259.S. HanafusaC. ChanyasanhaD. SujiraratI. KhuankhunsathidA. Yaguchi2008Clinical features and differences between child and adult dengue infections in Rayong province, southeast Thailand.Southeast Asian J Trop Med Public Health39252259
- 31. Kittigul L, Pitakarnjanakul P, Sujirarat D, Siripanichgon K (2007) The differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection. J Clin Virol 39: 76–81.L. KittigulP. PitakarnjanakulD. SujiraratK. Siripanichgon2007The differences of clinical manifestations and laboratory findings in children and adults with dengue virus infection.J Clin Virol397681
- 32. Hammond SN, Balmaseda A, Perez L, Tellez Y, Saborio SI, et al. (2005) Differences in dengue severity in infants, children, and adults in a 3-year hospital-based study in Nicaragua. Am J Trop Med Hyg 73: 1063–1070.SN HammondA. BalmasedaL. PerezY. TellezSI Saborio2005Differences in dengue severity in infants, children, and adults in a 3-year hospital-based study in Nicaragua.Am J Trop Med Hyg7310631070
- 33. Wichmann O, Hongsiriwon S, Bowonwatanuwong C, Chotivanich K, Sukthana Y, et al. (2004) Risk factors and clinical features associated with severe dengue infection in adults and children during the 2001 epidemic in Chonburi, Thailand. Trop Med Int Health 9: 1022–1029.O. WichmannS. HongsiriwonC. BowonwatanuwongK. ChotivanichY. Sukthana2004Risk factors and clinical features associated with severe dengue infection in adults and children during the 2001 epidemic in Chonburi, Thailand.Trop Med Int Health910221029
- 34. Suwandono A, Kosasih H, Nurhayati , Kusriastuti R, Harun S, et al. (2006) Four dengue virus serotypes found circulating during an outbreak of dengue fever and dengue haemorrhagic fever in Jakarta, Indonesia, during 2004. Trans R Soc Trop Med Hyg 100: 855–862.A. SuwandonoH. KosasihNurhayatiR. KusriastutiS. Harun2006Four dengue virus serotypes found circulating during an outbreak of dengue fever and dengue haemorrhagic fever in Jakarta, Indonesia, during 2004.Trans R Soc Trop Med Hyg100855862
- 35. Thomas L, Brouste Y, Najioullah F, Hochedez P, Hatchuel Y, et al. (2010) Predictors of severe manifestations in a cohort of adult dengue patients. J Clin Virol 48: 96–99.L. ThomasY. BrousteF. NajioullahP. HochedezY. Hatchuel2010Predictors of severe manifestations in a cohort of adult dengue patients.J Clin Virol489699
- 36. Cao XT, Ngo TN, Wills B, Kneen R, Nguyen TT, et al. (2002) Evaluation of the world health organization standard tourniquet test and a modified tourniquet test in the diagnosis of dengue infection in Viet Nam. Trop Med Int Health 7: 125–132.XT CaoTN NgoB. WillsR. KneenTT Nguyen2002Evaluation of the world health organization standard tourniquet test and a modified tourniquet test in the diagnosis of dengue infection in Viet Nam.Trop Med Int Health7125132
- 37. World Health Organization (2011) Comprehensive guidelines for prevention and control of dengue and dengue hemorrhagic fever. India: WHO Regional Office for South-East Asia. World Health Organization2011Comprehensive guidelines for prevention and control of dengue and dengue hemorrhagic feverIndiaWHO Regional Office for South-East Asia
- 38. Phuong CX, Nhan NT, Kneen R, Thuy PT, van Thien C, et al. (2004) Clinical diagnosis and assessment of severity of confirmed dengue infections in Vietnamese children: Is the world health organization classification system helpful? Am J Trop Med Hyg 70: 172–179.CX PhuongNT NhanR. KneenPT ThuyC. van Thien2004Clinical diagnosis and assessment of severity of confirmed dengue infections in Vietnamese children: Is the world health organization classification system helpful?Am J Trop Med Hyg70172179
- 39. Phuong HL, de Vries PJ, Nga TT, Giao PT, Hung le Q, et al. (2006) Dengue as a cause of acute undifferentiated fever in Vietnam. BMC Infect Dis 6: 123.HL PhuongPJ de VriesTT NgaPT GiaoQ. Hung le2006Dengue as a cause of acute undifferentiated fever in Vietnam.BMC Infect Dis6123
- 40. Karande S, Gandhi D, Kulkarni M, Bharadwaj R, Pol S, et al. (2005) Concurrent outbreak of leptospirosis and dengue in Mumbai, India, 2002. J Trop Pediatr 51: 174–181.S. KarandeD. GandhiM. KulkarniR. BharadwajS. Pol2005Concurrent outbreak of leptospirosis and dengue in Mumbai, India, 2002.J Trop Pediatr51174181
- 41. Standish K, Kuan G, Aviles W, Balmaseda A, Harris E (2010) High dengue case capture rate in four years of a cohort study in Nicaragua compared to national surveillance data. PLoS Negl Trop Dis 4: e633.K. StandishG. KuanW. AvilesA. BalmasedaE. Harris2010High dengue case capture rate in four years of a cohort study in Nicaragua compared to national surveillance data.PLoS Negl Trop Dis4e633
- 42. Barniol J, Gaczkowski R, Barbato EV, da Cunha RV, Salgado D, et al. (2011) Usefulness and applicability of the revised dengue case classification by disease: Multi-centre study in 18 countries. BMC Infect Dis 11: 106.J. BarniolR. GaczkowskiEV BarbatoRV da CunhaD. Salgado2011Usefulness and applicability of the revised dengue case classification by disease: Multi-centre study in 18 countries.BMC Infect Dis11106
- 43. Narvaez F, Gutierrez G, Perez MA, Elizondo D, Nunez A, et al. (2011) Evaluation of the traditional and revised WHO classifications of dengue disease severity. PLoS Negl Trop Dis 5: e1397.F. NarvaezG. GutierrezMA PerezD. ElizondoA. Nunez2011Evaluation of the traditional and revised WHO classifications of dengue disease severity.PLoS Negl Trop Dis5e1397
- 44. Tanner L, Schreiber M, Low JG, Ong A, Tolfvenstam T, et al. (2008) Decision tree algorithms predict the diagnosis and outcome of dengue fever in the early phase of illness. PLoS Negl Trop Dis 2: e196.L. TannerM. SchreiberJG LowA. OngT. Tolfvenstam2008Decision tree algorithms predict the diagnosis and outcome of dengue fever in the early phase of illness.PLoS Negl Trop Dis2e196
- 45. Lee VJ, Lye DC, Sun Y, Leo YS (2009) Decision tree algorithm in deciding hospitalization for adult patients with dengue haemorrhagic fever in Singapore. Trop Med Int Health 14: 1154–1159.VJ LeeDC LyeY. SunYS Leo2009Decision tree algorithm in deciding hospitalization for adult patients with dengue haemorrhagic fever in Singapore.Trop Med Int Health1411541159
- 46. van der Laan MJ, Polley EC, Hubbard AE (2007) Super learner. Statistical Applications in Genetics and Molecular Biology 6: Article 25.MJ van der LaanEC PolleyAE Hubbard2007Super learner.Statistical Applications in Genetics and Molecular Biology6Article 25