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Early Clinical and Laboratory Indicators of Acute Dengue Illness

S. Kalayanarooj, D. W. Vaughn, S. Nimmannitya, S. Green, S. Suntayakorn, N. Kunentrasai, W. Viramitrachai, S. Ratanachu-eke, S. Kiatpolpoj, B. L. Innis, A. L. Rothman, A. Nisalak, and F. A. Ennis
Bangkok Childrens Hospital, Bangkok, Department of Virology, Armed Forces Research Institute of Medical Sciences, Bangkok, and Kamphaeng Phet Provincial Hospital, Kamphaeng Phet, Thailand; Division of Infectious Diseases and Immunology, University of Massachusetts Medical Center, Worcester, Massachusetts; Department of Virus Diseases, Walter Reed Army Institute of Research, Washington, DC

A prospective observational study was conducted to identify early indicators of acute dengue virus infection. Children with fever for 72 h without obvious cause were studied at hospitals in Bangkok and Kamphaeng Phet, Thailand, until resolution of fever. Of 172 evaluable subjects (91% of enrollees), 60 (35%) had dengue, including 32 with dengue fever (DF) and 28 with dengue hemorrhagic fever (DHF). At enrollment, children with dengue were more likely than children with other febrile illnesses (OFI) to report anorexia, nausea, and vomiting and to have a positive tourniquet test, and they had lower total white blood cell counts, absolute neutrophil and absolute monocyte counts, and higher plasma alanine and aspartate (AST) aminotransferase levels than children with OFI. Plasma AST levels were higher in children who developed DHF than in those with DF. These data identify simple clinical and laboratory parameters that help to identify children with DF or DHF.

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The dengue viruses, a group of four closely related viruses in the Flavivirus family (dengue virus serotypes 1 4), are the most important aviviruses from the standpoint of human morbidity [1, 2]. It is estimated that as many as 100 million dengue virus infections occur annually, principally in tropical and subtropical areas inhabited by the mosquito vectors, Aedes aegypti and Aedes albopictus [3]. Most dengue virus infections in children have minimal or no symptoms and cannot be easily distinguished clinically from other viral infections [4]. Classic dengue fever (DF) is an acute illness characterized by fever, retroorbital headache, severe myalgias, and occasionally a rash, which lasts from 5 to 7 days [5]. In a small percentage of dengue infections, a more severe form of disease, known as dengue hemorrhagic fever (DHF), occurs. DHF is characterized by acute fever associated with a

Received 2 November 1996; revised 27 January 1997. Presented in part: 44th Annual Meeting of the American Society of Tropical Medicine and Hygiene, San Antonio, Texas, November 1995 (abstract 158). Written informed consent was obtained from the parents or guardians of all patients. The study protocol was approved by institutional review boards of the Ministry of Public Health (Thailand), Surgeon Generals Ofce of the Department of the Army, and University of Massachusetts Medical Center, and human experimentation guidelines of the US Department of Health and Human Services and the authors institutions were followed. Financial support: NIH (AI-34533) and US Army Medical Research and Materiel Command. The opinions contained herein are those of the authors and should not be construed as representing the ofcial policies of the National Institutes of Health, the Department of the Army, or the Department of Defense. Reprints or correspondence: Dr. Francis A. Ennis, Division Director, Division of Infectious Diseases and Immunology, University of Massachusetts Medical Center, 55 Lake Ave. N., Worcester, MA 01655.
The Journal of Infectious Diseases 1997;176:31321 1997 by The University of Chicago. All rights reserved. 00221899/97/76020002$02.00

hemorrhagic diathesis and a tendency to develop shock (dengue shock syndrome), which distinguish it from DF. Abnormal hemostasis and plasma leakage are the two pathophysiologic hallmarks of DHF, which are manifested clinically as thrombocytopenia and hemoconcentration (or pleural effusion). These characteristic features of DHF typically occur simultaneously with defervescence [5], while the early clinical features of DHF are indistinguishable from DF [6]. A clinical denition of DHF established by the World Health Organization (WHO) is based on the presence of high continuous fever, hemorrhagic manifestations (including at least a positive tourniquet test), hepatomegaly, thrombocytopenia (platelet count 100,000/mm3), and hemoconcentration (hematocrit increased by 20% above baseline value) [7]. The WHO denition further subdivides DHF into four grades on the basis of the presence of spontaneous bleeding and the presence and severity of shock. Several hundred thousand cases of DHF are reported to WHO annually, although this is likely to be an underestimate of the true burden of disease [1, 2]. Infection with one serotype of dengue virus elicits lifelong homotypic immunity, but heterotypic immunity is short-lived [8]. Some studies suggest that the frequency of occurrence of DHF is inuenced by characteristics of the infecting virus, such as the serotype and even the specic strain [9 12]. However, host immune responses are also important in the pathogenesis of DHF, because the risk for DHF is increased at least 15-fold during secondary dengue virus infections [13]. Various mechanisms have been postulated to explain this increased risk for DHF in secondary dengue infections, including antibody-dependent enhancement of infection [14], complement activation by virus-antibody complexes [15, 16], and T cell mediated immunopathology [17]. One study demonstrated that the ability of undiluted serum to enhance dengue

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virus infection in vitro correlated with the development of DHF during a subsequent dengue virus infection [18]. Patients with DHF have been shown to have elevated levels of the cytokines interferon-g and interleukin-2 as well as the soluble forms of CD8, CD4, and the interleukin-2 receptor a chain, which are markers of T cell activation [19]. Other studies have also shown that patients with DHF have depressed serum levels of complement components C3, C4, and C5 and elevated levels of C3a [15, 16]. However, the relative importance of each of these immunologic responses in the pathogenesis of DHF has not been established. In addition, previous studies were performed with children in the later stages of illness, when it is often impossible to isolate dengue virus [20], and these studies lacked a well-matched comparison group. The low virus isolation rate in patients with DHF in those studies has precluded an analysis of the inuence of viral factors, such as serotype and strain, on disease severity. Early clinical diagnosis of dengue virus infection can be difcult. The WHO clinical and laboratory criteria for DHF are usually not present in the rst few days of illness. One study reported that ushed face without coryza and a positive tourniquet test were early predictors of DHF in febrile patients [21]; however, a denitive diagnosis of DHF could be made only with the development of thrombocytopenia and plasma leakage, which usually occurs only 1 2 days before the onset of shock [5]. We therefore designed a prospective study of children early in the course of suspected dengue virus infection in order to identify early clinical and laboratory predictors of the risk of DHF before the critical stage of disease, that is, before defervescence and the onset of bleeding and plasma leakage. This study was also designed to identify immunologic or viral factors that initiate the pathogenetic cascade leading to plasma leakage in DHF, which will be reported elsewhere. This report describes the study design, demographic information, and the clinical and laboratory data from children with DHF, DF, or other (nondengue) febrile illnesses (OFI). Methods
Subject enrollment. Previously healthy children who presented to Bangkok Childrens Hospital and the more rural Kamphaeng Phet Provincial Hospital between 25 April and 14 December 1994 were eligible for entry into this study if they met the following entry criteria: age 6 months to 14 years, weight 6 kg, fever for 72 h, oral temperature 38.5C recorded in the clinic, and ushed face. Children meeting these criteria were examined by a study pediatrician for eligibility, and children who had a specic identiable cause of fever were excluded. For example, children with an initial clinical diagnosis of measles, sinusitis, tonsillitis, or gastroenteritis were not eligible; however, children with nausea or diarrhea could still be eligible for enrollment if the physician did not consider the clinical picture diagnostic of gastroenteritis. Subjects were also excluded if, at the time of evaluation, there was hypotension, anemia, malnutrition, or a history of chronic

medical illnesses, such as congenital heart disease or thalassemia. The study was organized to enroll up to 6 patients per week at each study site. Study protocol. At the time of enrollment, subjects and their parents were interviewed by a study nurse to collect demographic data and medical history, and a blood specimen, 720 mL, depending on the subjects weight, was obtained. All subjects were then admitted to the hospital ward for observation under the care of a pediatrician with experience in the care of patients with DHF. Each morning, a study nurse reviewed the medical record for the previous 24 h and recorded clinical and laboratory data on a standardized data collection form. A tourniquet test was performed by inating a blood pressure cuff on the upper arm to midway between the systolic and diastolic blood pressures for 5 min and then examining a 2.5-cm2 area using a standard template. The number of petechiae within the template, up to 20, was determined. The tourniquet test was performed on alternating arms each day until the subject was discharged or until 20 petechiae were recorded on any 1 day. A venous blood specimen was collected each morning, up to a maximum of ve blood draws during hospitalization. Vital signs were obtained by the ward nurses at least every 6 h. If two consecutive 6-h oral temperatures were 38C, a capillary tube hematocrit determination was performed by the ward nurses on a ngerstick blood specimen. This was repeated 6 and 12 h later if the temperature remained 38C. A right lateral decubitus chest radiograph was obtained on the next morning unless fever (38C) recurred. Other laboratory studies were done and medications or intravenous uids administered, at the discretion of the attending physician. Subjects were discharged from the study if an alternative diagnosis was reached during observation. Subjects were discharged from the hospital if they were clinically stable and afebrile for at least 24 h. Subjects were asked to return to the outpatient department 7 or 8 days after enrollment, at which time a follow-up blood specimen was obtained. Blood specimens for this study were obtained in EDTA-containing tubes and immediately placed on ice. A 1-mL aliquot of whole blood was sent to the hospital laboratory for complete blood count (T540 counter; Coulter, Hialeah, FL). The remainder of the blood specimen was transported to the research laboratory, where plasma and platelets were separated by an initial centrifugation at 300 g, followed by centrifugation at 800 g. Peripheral blood mononuclear cells were separated from the red blood cell pellet by dilution in medium (RPMI 1640), followed by centrifugation over Histopaque (Sigma, St. Louis). One aliquot of plasma was analyzed for alanine aminotransferase (ALT), aspartate aminotransferase (AST), and albumin (Clinical System Analyzer, model 700; Beckmann Instruments, Brea, CA). The remainder of the plasma was aliquoted and stored at 070C. Serologic and virologic studies. Plasma samples were tested for serologic evidence of acute dengue virus infection by IgM and IgG ELISA and hemagglutination-inhibition (HAI) assays [22]. Standard criteria were used to identify acute primary and acute secondary dengue virus infections. If the serologic studies were indenitefor example, with no convalescent blood specimen available or a high titer of anti-dengue antibody that showed no change in titerand virus isolation attempts (see below) were negative, the serologic diagnosis was recorded as unknown.

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Dengue virus isolation was attempted by intrathoracic inoculation of plasma samples into Toxorrhynchites splendens mosquitoes [22]. The rst 3 consecutive daily plasma samples from each subject were tested, if available. Study design and denition of DHF cases. Study day 1 was dened as the calendar day during which the subject was enrolled in the study, and days were counted consecutively afterward. Fever day 0 was dened as the calendar day during which the temperature fell and stayed 38C, and days before and after this point were numbered consecutively (fever days 01, 02, etc. occurred before defervescence and fever days /1, /2, etc. occurred after defervescence). Clinical and laboratory data were recorded by trained study nurses using standardized data collection forms. Hospital charts were reviewed by a study physician to identify systematic recording errors. Data from the standardized forms were entered into a computer database twice for validation, using FoxPro for Windows (Microsoft, Redmond, WA). Serologic test results were read by a study physician blinded to the clinical information. Chest radiographs were read by a radiologist blinded to the clinical and serologic information, and a pleural effusion index was calculated as 100 1 (maximum width of right pleural effusion)/(maximal width of right hemithorax). Subjects with serologic or virologic evidence (or both) of acute dengue virus infection were assigned a nal clinical diagnosis by two of us (S.K., S.N.) on the basis of a review of clinical and laboratory data. A clinical diagnosis of DHF was assigned following the WHO clinical denition [7] on the basis of the presence of plasma leakage and thrombocytopenia. The serologic data were not used to assign the clinical diagnosis. Evidence of plasma leakage could include a peak hematocrit value 20% above the value at the convalescent visit (study day 810), a pleural effusion demonstrated on the chest radiograph, or detection of ascites on physical examination. Our initial denition of thrombocytopenia was a platelet count nadir of 100,000/mm3; however, because some subjects did not have platelet counts available from the time of maximal plasma leakage, we assigned a diagnosis of DHF to some subjects if there was evidence of plasma leakage and the available platelet counts showed a signicant decrease from the value at the convalescent visit. Cases of DHF were graded from 1 to 4 according to WHO criteria [7]. Any subject with serologic or virologic evidence of acute dengue infection who did not meet the criteria for DHF was considered as having DF. Subjects were diagnosed as having OFI when all of the following criteria were met: There was no clinical evidence of bacterial infection, there was no serologic or virologic evidence of acute dengue infection, and serologic testing was considered adequate to exclude acute dengue virus infection (see above). Statistical analysis. Differences in clinical and laboratory data were analyzed on the basis of the nal diagnosis assigned. The analysis was carried out both by comparing all dengue infections (combined DF and DHF) with OFI and by comparing subjects with DHF with those with DF. Proportional data were tested using x2 analysis or Fishers exact test. Continuous data were tested using the t test; statistically signicant differences were conrmed using the Mann-Whitney rank sum test for data not showing a normal distribution. Statistical analyses were carried out using SigmaStat 2.0 for Windows (Jandel Software, San Rafael, CA).

Figure 1. No. of subjects enrolled, by month and nal diagnosis. Final diagnoses were dengue hemorrhagic fever (DHF), dengue fever (DF), or other, nondengue febrile illnesses (OFI).

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Results Subject demographics. Between 25 April and 14 December 1994, we enrolled a total of 189 subjects into the study, of whom 142 were enrolled in Bangkok and 47 in Kamphaeng Phet. Most of the study subjects were enrolled between May and September (gure 1). The overall male-to-female ratio was 1.1:1, and the mean age was 6.8 { 3.2 years. The average duration of fever before study entry was 1.7 { 0.8 days. The mean age of subjects enrolled in Kamphaeng Phet was signicantly higher than the mean age of subjects enrolled in Bangkok (8.6 { 3.0 vs. 6.2 { 3.0 years). There were no signicant differences in male-to-female ratio or duration of fever before study entry between the two study sites. Final diagnoses. Of the 189 study subjects, 3 were diagnosed with bacterial infections during the study and were withdrawn, one each with Haemophilus inuenzae meningitis, Salmonella gastroenteritis, and a urinary tract infection. Fourteen other subjects could not be assigned a denite serologic diagnosis: 10 were assigned a diagnosis of unknown because they did not have adequate plasma specimens to rule out acute dengue virus infection, and 4 had serologic results consistent with a recent dengue infection but no evidence of acute dengue virus infection (xed high titer in HAI assay). Of the remaining 172 subjects, 60 (35%) had serologic or virologic evidence (58 had both) of acute dengue virus infection. Fifty-ve of these subjects (93%) had serologic responses characteristic of secondary dengue virus infection. Between May and October, when most subjects were enrolled, and excluding subjects without a denite serologic diagnosis, the percentage of study subjects with acute dengue infection ranged from a high of 44% in October to a low of 14% in September (gure 1). Of the 60 subjects with serologic or virologic evidence (or both) of acute dengue virus infection, 32 (53%) were classied as DF according to the above denitions and 28 (47%) as DHF.

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Table 1. Clinical and laboratory measures of disease severity in the study population, according to nal diagnosis assigned.
Final diagnosis
Measure
Hemoconcentration,* no. (%) Elevation of hematocrit at peak, % increase over convalescent, mean (SD) Weight change, entry to fever day /1, kg, mean (SD) Pleural effusion, no. (%) Pleural effusion index, mean (range) Thrombocytopenia, no. (%)

OFI
19/105 (18) 10.5 (8.8) 00.3 (0.6) 17/105 (16) 0.29 (0 3.3) 5/112 (4)

DF
3/28 (11) 11.4 (7.0) 00.3 (0.6) 2/30 (7) 0.07 (0 1.3) 5/32 (16)

DHF
12/25 (48) 22.8 (15.7) 0.1 (0.8) 22/26 (84) 14.1 (0 78) 23/28 (82)

NOTE. DHF, dengue hemorrhagic fever; DF, dengue fever; OFI, other nondengue febrile illnesses. * Peak hematocrit during observation 20% above hematocrit at follow-up (convalescence). Follow-up hematocrit values were not available for all study subjects. Platelet nadir 100,000 cells/mL.

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Among the 28 subjects with DHF, there were 5 subjects with grade 1 DHF (18%), 14 subjects with grade 2 DHF (50%), 9 subjects with grade 3 DHF (32%), and no subjects with grade 4 DHF. All of the 4 subjects with primary dengue virus infections were categorized as DF. Table 1 describes the evidence of disease severity in children with DHF compared with those with DF and OFI. Subjects with DHF had evidence of hemoconcentration, pleural effusion, and thrombocytopenia, consistent with the clinical denition. These subjects also had a slight weight gain from entry to fever day /1, whereas subjects with DF or OFI lost a mean of 0.3 kg. Elevations of hematocrit to 20% above the convalescent value were seen in half of the subjects with DHF. The absence of hemoconcentration in the remainder was attributed to the infrequent (6-h) hematocrit determinations, the effort to increase the oral uid intake, and, in some cases, the administration of intravenous uid. Four subjects who were assigned a diagnosis of DHF on the basis of thrombocytopenia and clinical evidence of plasma leakage showed no evidence of pleural effusion on their decubitus chest radiograph. On review of these records, it was felt that the maximal plasma leakage occurred before defervescence and the chest radiograph. Five subjects who were assigned a diagnosis of DHF had a signicant decrease in platelet count but no platelet count 100,000/ mm3. Reassigning these cases as DF did not signicantly alter the results of the statistical analyses. Three subjects who were assigned a clinical diagnosis of DF had a peak hematocrit 20% above the convalescent value. None of the 3 had thrombocytopenia, although 1 had a platelet nadir of 107,000/mm3. In each case, the nding of hemoconcentration related to a single abnormally high or low hematocrit determination, which on review was felt to be inconsistent with the rest of the clinical data. Two other subjects were assigned a diagnosis of DF even though a pleural effusion was detected. Both of these subjects had a small pleural effusion index (0.9 and 1.3) and no evidence of hemoconcentration,

ascites, spontaneous bleeding, or thrombocytopenia. All 5 of these subjects with the diagnosis of DF had serologic responses indicative of secondary dengue virus infection, and reassigning the cases as DHF did not signicantly alter the results of the statistical analyses. Furthermore, it should be noted that 17 (6.6%) of 112 subjects with a diagnosis of OFI had small pleural effusions (the highest pleural effusion index in this group was 3.3). Comparison of clinical and laboratory features at study entry. Table 2 shows a comparison of selected clinical and laboratory features at entry into the study among those subjects eventually classied as OFI, DF, or DHF. Subjects with dengue virus infection (DF or DHF) were signicantly older and were more likely to report anorexia, nausea, and vomiting than subjects with OFI. The male-to-female ratio and history of headache, abdominal pain, or bleeding were not signicantly different between the groups. Subjects with dengue virus infection were also more likely to have a positive tourniquet test. Initial laboratory studies showed that there were no signicant differences in hematocrit at entry between children with dengue virus infection and those with OFI. The platelet, total white blood cell (WBC), absolute neutrophil, and absolute monocyte counts at entry were signicantly lower in subjects with dengue virus infection than in subjects with OFI. Plasma AST and ALT levels were signicantly higher in children with dengue virus infection than in children with OFI. The only statistically signicant differences in initial ndings between subjects who had DF and those who developed DHF were that the subjects who later developed DHF had lower platelet counts and higher plasma AST levels at study entry. Comparison of clinical and laboratory features during observation. Table 3 summarizes clinical and laboratory ndings during the period of inpatient observation. The average duration of fever was greater in subjects with dengue infection than in those with OFI, but there were no differences between subjects with DF and those with DHF. Shock occurs around

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Table 2. Clinical features of study subjects at entry, tabulated according to nal diagnosis.
Final diagnosis
Dengue (DF / DHF)
60 32:28 8.1 (3.0)* 1.8 (0.9) 51/60 (85)* 40/59 (68) 42/60 (70) 20/59 (34) 44/57 (77) 8/60 (13) 33/51 (65)* 22/51 (43)* 255 (104) 37 (3.8) 6211 (2504)* 4473 (2449)* 130 (223)* 76 (68)* 37 (30)* 44 (6)

Characteristic
N Sex, M:F Age, years Duration of fever before enrollment, days Anorexia, no. (%) Nausea, no. (%) Vomiting, no. (%) Abdominal pain, no. (%) Headache, no. (%) Bleeding manifestations, no. (%) Tourniquet test positive, no. (%) 10 petechiae/2.5 cm2 20 petechiae/2.5 cm2 Platelet count, 1103/mL Hematocrit, % WBC count, cells/mL Absolute neutrophil count, cells/mL Absolute monocyte count, cells/mL AST, U/mL ALT, U/mL Albumin

OFI
112 62:50 6.2 (3.0) 1.6 (0.8) 69/110 (63) 49/100 (49) 58/111 (52) 30/108 (28) 70/105 (67) 9/112 (8) 42/108 (39) 23/108 (21) 298 (104) 38 (3.6) 11,120 (5437) 8148 (4865) 324 (458) 41 (12) 23 (20) 46 (6)

DF
32 15:17 8.1 (3.2) 1.7 (0.9) 25/32 (78) 19/31 (61) 21/32 (66) 9/32 (28) 24/30 (80) 3/32 (9) 18/28 (64) 10/28 (36) 283 (94) 37 (4.5) 6155 (2493) 4390 (2480) 119 (189) 57 (29) 29 (16) 45 (6)

DHF
28 17:11 8.1 (2.6) 1.9 (0.8) 26/28 (93) 21/28 (75) 21/28 (75) 11/27 (41) 20/27 (74) 5/28 (18) 15/23 (65) 12/23 (52) 225 (106) 38 (2.9) 6277 (2566) 4572 (2455) 143 (261) 98 (91) 46 (38) 44 (6)

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NOTE. Pairwise comparisons of clinical and laboratory data were done using x2 or Fishers exact test for proportional data and t test for continuous data. We compared all subjects with dengue infections (combined dengue fever [DF] and dengue hemorrhagic fever [DHF]) against subjects with other febrile illnesses (OFI) and we compared subjects with DHF against subjects with DF. Values are expressed as mean (SD) except as noted. WBC, white blood cell; AST, aspartate aminotransferase; ALT, alanine aminotransferase. P *.01 vs. OFI; .05 vs. OFI; .05 vs. DF.

the time of defervescence in DHF [5]; therefore, we compared clinical and laboratory features prior to this time point in order to identify prognostic factors (table 3). The number of subjects included in this analysis differs from the total study population because many subjects were enrolled 2 days before defervescence occurred (gure 2). There were statistically signicant differences between subjects with dengue virus infection and those with OFI on fever days 02 and 01 (2 days and 1 day before defervescence, respectively) relative to a positive tourniquet test, platelet count, total WBC and absolute neutrophil counts, and plasma levels of AST and ALT. A statistically signicant decrease in platelet counts in subjects with DHF compared with those with DF was evident on fever day 01. There was a small but statistically signicant elevation in hematocrits in children with DHF compared with those with DF on fever day 02, which did not remain signicant on fever day 01. Predictive value of clinical and laboratory observations. We calculated the positive (PPV) and negative (NPV) predictive values of parameters that were signicantly different between subjects with dengue infection and those with OFI, based on the entry criteria and sample sizes in our study population. PPV and NPV were calculated separately on the basis of whether the nding was present at enrollment or whether it

was present at any time on or before fever day 01, which represents at least 1 day before defervescence (table 4). We found the tourniquet test (10 petechiae/2.5 cm2) to have a high NPV at entry (0.79) and during hospitalization before defervescence (0.83) but low PPV (0.44 and 0.58, respectively) in this population. Total WBC count, absolute neutrophil count, and plasma AST level were slightly better discriminators of dengue virus infection than the tourniquet test. We also examined the PPV and NPV of plasma AST for discriminating DHF from DF and OFI, since AST levels were signicantly higher in the subjects with DHF. With plasma AST levels from enrollment only, the PPV and NPV for diagnosis of DHF of AST 40 U/mL were 0.27 and 0.96, respectively, and the PPV and NPV of AST 60 U/mL were 0.52 and 0.91, respectively. With all values on or before fever day 01, the PPV and NPV of AST 40 U/mL were 0.31 and 1.00, respectively, and the PPV and NPV of AST 60 U/mL were 0.52 and 0.95, respectively. Discussion Excellent descriptions of the clinical spectrum of dengue virus infection have been reported [5, 23]. However, the design of this study is unique because of the prospective enrollment of

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Table 3. Clinical features of study subjects during observation, tabulated according to nal diagnosis.
Final diagnosis
Dengue (DF / DHF)
4.0 (1.6)* 41 19/30 (63) 13/30 (43) 209 (111)* 37 (5) 5320 (2122)* 3389 (1760)* 94 (197) 96 (127) 43 (45) 54 30/36 (83)* 19/36 (53)* 210 (120)* 38 (4) 4776 (2762)* 2619 (1510)* 94 (135) 90 (71)* 39 (29)*

Characteristic
Total duration of fever, days Fever day 02 N Tourniquet test positive, no. (%) 10 petechiae/2.5 cm2 20 petechiae/2.5 cm2 Platelet count, 1103/mL Hematocrit, % WBC count, cells/mL Absolute neutrophil count, cells/mL Absolute monocyte count, cells/mL AST, U/mL ALT, U/mL Fever day 01 N Tourniquet test positive, no. (%) 10 petechiae/2.5 cm2 20 petechiae/2.5 cm2 Platelet count, 1103/mL Hematocrit, % WBC count, cells/mL Absolute neutrophil count, cells/mL Absolute monocyte count, cells/mL AST, U/mL ALT, U/mL

OFI
2.8 (1.6) 35 14/33 (42) 4/33 (12) 288 (109) 37 (6) 8739 (4274) 5899 (3571) 266 (360) 46 (18) 27 (20) 60 19/54 (35) 10/54 (19) 292 (113) 37 (4) 9513 (5574) 6333 (4760) 227 (439) 43 (14) 25 (26)

DF
3.7 (1.0) 20 12/17 (71) 9/17 (53) 239 (111) 36 (5) 5265 (2030) 3177 (1473) 114 (200) 64 (46) 35 (18) 30 17/22 (77) 11/22 (50) 265 (104) 37 (4) 4892 (3091) 2698 (1603) 73 (131) 70 (56) 32 (21)

DHF
4.3 (1.2) 21 7/13 (54) 4/13 (31) 184 (106) 39 (4) 5371 (2255) 3590 (2012) 76 (197) 124 (166) 51 (59) 24 13/14 (93) 8/14 (57) 144 (105) 39 (4) 4631 (2342) 2519 (1412) 120 (139) 115 (81) 47 (35)

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NOTE. Pairwise comparisons of clinical and laboratory data were done using x2 or Fishers exact test for proportional data and t test for continuous data. We compared all subjects with dengue infections (combined dengue fever [DF] and dengue hemorrhagic fever [DHF]) against subjects with other febrile illnesses (OFI) and we compared subjects with DF against subjects with DHF. Values are expressed as mean (SD) except as noted. No. of subjects differs from overall study population because some subjects were enrolled after indicated fever day. WBC, white blood cell; AST, aspartate aminotransferase; ALT, alanine aminotransferase. P *.01 vs. OFI; .05 vs. OFI; .05 vs. DF; .01 vs. DF.

subjects with undifferentiated fever, close inpatient observation throughout the illness, and standardized observations using established case denitions. This permits a detailed comparison of clinical and laboratory features between children with documented dengue virus infection and those with nondengue febrile illnesses. This cohort also provides a useful population for studying virologic and immunologic events early in the course of dengue virus infection [22] (Green S, et al., unpublished data). The major ndings of the present study are that acute dengue virus infection was a frequent cause of fever in children in this population presenting to an outpatient clinic between May and December; that DHF, as demonstrated by plasma leakage, developed in almost half of symptomatic secondary dengue virus infections; and that specic clinical and laboratory features were more commonly associated with acute dengue virus infections, particularly a positive tourniquet test, a depression of total WBC, absolute neutrophil, and monocyte counts, and an elevation of plasma AST level. We found that 35% of the acute nonbacterial febrile illnesses in children enrolled in our study was associated with an acute

dengue virus infection. We enrolled only children with fever for 72 h, and we excluded children with specic clinically identiable infections, such as sinusitis, pharyngitis, or measles, so we would anticipate that the percentage of all febrile illnesses caused by acute dengue virus infection is somewhat lower than this gure. We also required the presence of facial ushing for enrollment in our study. An earlier study using these same entry criteria found evidence of acute dengue virus infection in almost 90% of subjects [21]. However, that study was performed during the peak 3 months of dengue activity in 1989. In comparison, the present study enrolled subjects from May to December, and the overall incidence of hospitalization for DHF in the study areas was much lower than usual during this period, suggesting low transmission rates (Vaughn DW, et al., unpublished data). The percentage of our study subjects with acute dengue virus infection also varied considerably from month to month (gure 1). Thus, during periods of high incidence of dengue virus infections, the frequency of dengue among undifferentiated febrile illnesses would be expected to exceed the rate we observed.

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Figure 2. No. of subjects enrolled, by nal diagnosis and fever day corresponding to day of enrollment. Final diagnoses were dengue hemorrhagic fever (DHF), dengue fever (DF), or other, nondengue febrile illnesses (OFI). Fever day 0 was calendar day during which fever abated (i.e., subjects enrolled on fever day 0 defervesced within 24 h of enrollment).

Few other studies are available describing the frequency of dengue in endemic settings; most studies have focused on dened outbreaks or have not reported the frequency of dengue infections among all febrile illnesses. Halstead et al. [24] reported that 29% of febrile illnesses in outpatients in Bangkok in 1962 1964 was associated with acute dengue virus infection, a rate similar to ours. That study included children with fever for 72 h, and ushed face was not an entry criterion. Taken together, these results show that dengue virus infection is a frequent cause of undifferentiated fever in children presenting to outpatient clinics in Bangkok and Kamphaeng Phet, Thai-

land. Multiple dengue virus serotypes have cocirculated in these areas for several decades [1]. These results may be more widely applicable, however, since the recent expansion of dengue virus infections in other areas, such as Central and South America, has created similar conditions elsewhere [11]. In this study, approximately half of the ill children with an acute dengue virus infection developed DHF. This gure is much higher than reported in the previously cited study of outpatients in Bangkok in 1962 1964 [24], but it is similar to the nding of Burke et al. [25] among symptomatic dengue virus infections in children in 1980. There are several possible explanations for the differences observed. In our study, 93% of dengue virus infections were secondary dengue virus infections, compared with 66% in the study by Halstead et al. [24]. It has been clearly demonstrated that the risk of DHF is much higher in secondary dengue virus infections [12, 25]. We also performed detailed observations on all subjects, including decubitus chest radiographs; thus, we likely identied plasma leakage in some subjects that would not otherwise have been recognized. The 30-year difference in study periods might also have affected the results. The number of reported cases of DHF in the region was lower during the 1960s than during the present study period. Although this is probably related at least in part to improved recognition, changes in the circulating dengue viruses may also be possible. The differences in entry criteria between our study and that of Halstead et al. [24], noted above, might also have inuenced the frequency of DHF among subjects with acute dengue virus infection, but the similarity between our results and those of Burke et al. [25] suggests that this is not likely to be the major explanation. One focus of our study design was to identify clinical and laboratory observations that could distinguish acute dengue

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Table 4. Positive and negative predictive values of selected clinical and laboratory observations for identifying children with dengue virus infection.
Present at entry
Positive predictive value Negative predictive value

Present 1 days before defervescence

Positive predictive value Negative predictive value

Finding
Positive tourniquet test 10 petechiae/2.5 cm2 20 petechiae/2.5 cm2 AST 40 U/mL 60 U/mL Total WBC count 8000/mL 5000/mL Absolute neutrophil count 7000/mL 3000/mL

0.44 0.49 0.50 0.81 0.54 0.84 0.46 0.75

0.79 0.75 0.83 0.75 0.85 0.75 0.91 0.73

0.58 0.67 0.61 0.79 0.59 0.76 0.55 0.69

0.83 0.75 0.85 0.70 0.87 0.78 1.00 0.77

NOTE. AST, aspartate aminotransferase; WBC, white blood cell.

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infections from other febrile illnesses at an early stage, before plasma leakage developed in patients with DHF. Such indicators could be used to establish clinical algorithms for the identication of high-risk patients for close monitoring. We found that a positive tourniquet test, leukopenia, neutropenia, monocytopenia, and elevations of plasma AST levels could be helpful in this regard. The tourniquet test is a simple clinical procedure that reects capillary fragility and has been found to be positive in nearly all cases of DHF [5]. An earlier study found that a positive tourniquet test combined with a ushed face and no coryza, which were used as enrollment criteria in that study, had a sensitivity of 90%, a specicity of 97%, and a positive predictive value of 97% for diagnosis of dengue virus infection [21]. It should be noted that the majority of patients in that study had DHF. In the present study, in which only half of the subjects with dengue virus infections had DHF, we found that the tourniquet test was often positive in dengue virus infection, but we also found that one-third of children with other, nondengue febrile illnesses had a positive tourniquet test, yielding lower specicity and a correspondingly lower PPV (table 4). Differences in the design of these studies, noted above, are likely to explain the differences in ndings. Other characteristics of the tourniquet test result, such as the size of petechiae, were not recorded, and we were thus unable to compare these characteristics between subjects with dengue virus infections and those with OFI. Leukopenia is also well described as a feature of dengue infection and is felt to relate to bone marrow suppression by dengue virus [26, 27]. Our study expands on these observations to show that the presence of leukopenia, especially affecting the neutrophil and monocyte lineages, was useful for distinguishing dengue infections from other febrile illnesses in this population. We also saw an increased percentage of atypical lymphocytes (data not shown), as has been described [28, 29]. However, neither the percentage nor absolute number of atypical lymphocytes in our subjects with dengue virus infection was signicantly higher than in subjects with OFI prior to fever day 0, the day of defervescence, so this nding was not an early indicator. Other investigators have reported that elevations of AST and ALT levels are common in dengue infections, both DF and DHF [5, 30], but our study is the rst to show that these elevations are clearly more common in the early stages of dengue infections than in nondengue febrile illnesses. In particular, a normal plasma AST level was a strong negative predictor to exclude DHF. This observation must be applied cautiously in other regions, where other endemic diseases, such as viral hepatitis, typhoid fever, and leptospirosis, may cause hepatic injury and elevated AST levels. Some investigators have suggested that the clinical denition of DHF established by WHO [7] may not appropriately categorize all patients. In particular, hemoconcentration may be blunted if there is early administration of intravenous uids, if

there is excessive blood loss due to hemorrhage, or if hematocrit is not measured frequently. Because our study was organized to perform detailed observations, including a decubitus chest radiograph, at the phase of plasma leakage, these concerns can be put into some perspective. We found that a requirement of 20% increase in hematocrit over the recovery value identied only 48% of cases classied as DHF. However, plasma leakage was still identied in these cases, most often by the nding of a pleural effusion and also by the clinical observation of ascites. This is particularly notable since a minority of subjects with DHF in our study reached severity grade 3 or 4, so that many of our subjects might not have been hospitalized or recognized as having DHF if they had not been included in this study. Our data therefore support the position that plasma leakage and thrombocytopenia are critical features that distinguish DHF from DF [5]. At the same time, these data support the recommendation that other clinical ndings besides hemoconcentration be considered [7]. Since this study relied on clinical observations and relatively simple laboratory tests, the results should be readily applicable to developing countries, where most dengue virus infections occur. In addition, this cohort of subjects, with detailed clinical and virologic data, may be useful for identifying critical viral and immunologic factors leading to plasma leakage in DHF.

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Acknowledgments

We thank the Armed Forces Research Institute of Medical Sciences research nurses (Nathada Plavooth, Pranom Vangnai, Somnuk Lumjiak, Somalvadee Saravasee, Sumetha Hengprasert, and Wipa Chawachalasai) for their dedication in data and blood collection; Somkiat Changnak, Choompun Manomuth, Aree Na-nongkhai, Pranee Saisang, Somsamai Tapana, Weerasak Yeepoo, and Songdej Sangsri for technical assistance; Tipawan Kungvanrattana and Warinda Sriskham for data entry; Christine Kozik for auditing the clinical data; Chitchai Hemachudha for database management; Pantipa Patanawin for interpretation of the chest radiographs; Rapin Snitbhan for coordinating specimen collection; Robert Lew for assistance with data analysis; and the patients and families who participated in this study.

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