ORIGINAL STUDIES

Natural History of Plasma Leakage in Dengue Hemorrhagic Fever

A Serial Ultrasonographic Study
Anon Srikiatkhachorn, MD,* Anchalee Krautrachue, MD, Warangkana Ratanaprakarn, MD, Lawan Wongtapradit, MD, Narong Nithipanya, MD, Siripen Kalayanarooj, MD, Ananda Nisalak, MD, Stephen J. Thomas, MD, Robert V. Gibbons, MD, Mammen P. Mammen, Jr., MD, Daniel H. Libraty, MD,* Francis A. Ennis, MD,* Alan L. Rothman, MD,* and Sharone Green, MD*
Background: Although plasma leakage is the major cause of mortality and morbidity in patients with dengue hemorrhagic fever (DHF), a detailed assessment of the natural course of this process is still lacking. We employed serial ultrasound examination to delineate the locations and the timing of plasma leakage and to evaluate the usefulness of ultrasound in detecting plasma leakage in DHF. Method: Daily ultrasound examinations of the abdomen and right thorax were performed in 158 suspected dengue cases to detect ascites, thickened gall bladder wall and pleural effusions. Cases were classied into dengue fever (DF), DHF or other febrile illness (OFI) based on serology and evidence of plasma leakage including hemoconcentration and pleural effusion detected by chest radiograph. Results: Ultrasonographic evidence of plasma leakage was detected in DHF cases starting from 2 days before defervescence and was detected in some cases within 3 days after fever onset. Pleural effusion was the most common ultrasonographic sign of plasma leakage (62% of DHF cases one day after defervescence). Thickening of the gallbladder wall and ascites were detected less frequently (43% and 52% of DHF cases respectively) and resolved more rapidly than pleural effusions. The size of pleural effusions, ascites and gall bladder wall thickness in DHF grade I and II were smaller than those of grade III patients. Ultrasound detected plasma leakage in 12 of 17 DHF cases who did not meet the criteria for signicant hemoconcentration. Conclusions: Ultrasound examinations detected plasma leakage in multiple body compartments around the time of defervescence. Ultrasonographic signs of plasma leakage were detectable before changes in hematocrits. Ultrasound is a useful tool for detecting plasma leakage in dengue infection.
Accepted for publication January 4, 2007. From the *University of Massachusetts Medical School, Worcester, Massachusetts; Queen Sirikit National Institute of Child Health, Bangkok, Thailand; and Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand. Address for correspondence: Center for Infectious Disease and Vaccine Research, University of Massachusetts Medical School, 55 Lake Avenue North, Room S5-326, Worcester, MA 01655. E-mail: anon. srikiatkhachorn@umassmed.edu. Copyright 2007 by Lippincott Williams & Wilkins ISSN: 0891-3668/07/2604-0283 DOI: 10.1097/01. inf.0000258612.26743.10

Key Words: dengue hemorrhagic fever, pleural effusion, ascites, gall bladder wall thickening (Pediatr Infect Dis J 2007;26: 283290)

engue virus (DV) infections are a signicant health threat to populations living in tropical and subtropical regions.1 Infection with any of the 4 serotypes of DV can produce a broad spectrum of illness, ranging from asymptomatic infection to severe life-threatening illness.2 Symptomatic dengue illness is typically classied into dengue fever (DF), a self limited febrile illness, or a more severe form, dengue hemorrhagic fever (DHF), which is characterized by plasma leakage into the chest and abdominal cavities and bleeding diathesis. Although it has been well established that hypotension secondary to plasma leakage typically occurs during the 48 hours period after defervescence, the onset, the course and the anatomic pattern of plasma leakage leading to compromised circulation have not been delineated in detail. Studies have suggested that ultrasonogram of the chest and abdomen might be useful for detecting signs of plasma leakage in DHF patients. Several sonographic ndings have been reported including uid in the chest and the abdomen, thickening of the gallbladder wall and pericardial effusion.39 In most studies ultrasound examinations were performed only once and therefore these studies failed to yield important information on the kinetics of plasma leakage. In addition, due to the retrospective nature and the lack of nondengue cases in most previous studies, the sensitivity and the specicity of various ultrasound ndings in differentiating DHF cases from DF or other febrile illnesses (OFI) remain unknown. To study the course of plasma leakage in DHF, we performed daily chest and abdominal ultrasonographic studies in children prospectively recruited for suspected DV infection. Patients with DF and OFI were included in the study. The ability to objectively follow evidence of plasma leakage in these patients provides signicant insights into the pattern of plasma leakage in this disease.

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MATERIALS AND METHODS

Patients. Children less than 15 years of age who had fever (38C) for less than 72 hours without an obvious focus of infection were recruited from June 2004 to November 2005. Patients were admitted for observation and treatment according to World Health Organization guidelines.2 DV infections were conrmed by RT-PCR to detect dengue genome in plasma obtained on the day of admission, and by serology of paired acute and convalescence plasma.10 12 Patients with positive dengue RT-PCR were observed until one day after defervescence or longer depending on their clinical course. Daily complete blood count, plasma albumin and AST levels were obtained. Ultrasonograms and a right lateral decubitus chest radiogram were obtained as described below. Clinical classication of dengue cases was assigned according to the World Health Organization criteria by 2 reviewers who were blinded to the ultrasound results. Percent hemoconcentration was calculated according to the formula: % Hemoconcentration (peak hematocrit surrounding defervescence) (admission hematocrit or minimum hematocrit before defervescence)/(admission hematocrit or minimum hematocrit before defervescence) 100. A diagnosis of DHF was assigned if a patient fullled all of the following clinical criteria: (1) fever, (2) liver enlargement, (3) hemorrhagic manifestations including positive tourniquet test or skin or mucosal bleeding, (4) evidence of plasma leakage including shock and/or ascites or pleural effusion detected by clinical examination or right lateral decubitus chest radiogram, or hemoconcentration. Laboratory criteria include (1) thrombocytopenia (platelet count below 100,000/mm3) and (2) hemoconcentration dened as a 20% or more increase in hematocrit from base-line value.2 DHF cases were classied according to their severity as follows: DHF grade I, patients with DHF without spontaneous bleeding or hypotension; DHF grade II, DHF patients with spontaneous bleeding; DHF grade III, DHF cases with hypotension with pulse pressure of 20 mm Hg or less; DHF grade IV, DHF cases with profound shock and unobtainable blood pressure.2 The day of defervescence (temperature less than 38C) was dened as fever day 0. Days before defervescence were dened as fever day 1, 2, etc and those after defervescence were dened as fever day 1, 2 respectively. The study was approved by the Institutional Review Boards of the Queen Sirikit National Institute of Child Health, the Thai Ministry of Public Health, the US Army Surgeon General and the University of Massachusetts Medical School. Written informed consent was obtained from the legal guardian of each participant. Ultrasonogram. Ultrasound examinations were performed daily with a portable ultrasound scanner (GE Logiq Book) using a 2.5 MHz convex transducer. The studies were performed by a team of 4 radiologists who were blinded to the laboratory results of the study participants. Ultrasound procedures were carried out according to a predetermined protocol and the results recorded on a standardized form. To detect pleural effusion, longitudinal scans of the right hemithorax at the midclaivular and the midaxillary line and a transverse scan of the right upper abdominal quadrant were

performed in the supine position. A longitudinal scan at the right midaxillary line in an upright position was performed after subjects were in an upright position for at least 3 minutes. The vertical dimensions of the uid collection were determined by measuring the distance between the top of the dome of the diaphragm and the base of the lung (Fig. 1 A, B available online only). Measurements of anterior gallbladder wall thickness were carried out without fasting (Fig. 1C, D available online). We examined the hepatorenal pouch and retrovesicular area in a supine position for ascites. Fluid present in the hepatorenal pouch was recorded as present or absent (Fig. 1E, F available online). Quantitative measurements of the uid collected behind the urinary bladder were taken by measuring the anteroposterior and transverse diameter of the uid in a transverse scan of the retrovesicular space (Fig. 1G, H available online). To evaluate the interoperator variability in interpreting the ultrasonogram, unmarked ultrasound records were read by a different ultrasonographer at the end of the recruitment season and the 2 readings were compared. The concordance rate for the presence or absence of an ultrasound nding was above 90%. Chest Radiogram. A right lateral decubitus chest radiogram was obtained one day after defervescence (fever day 1). The magnitude of a pleural effusion was determined semiquantitatively as pleural effusion index (PEI) using the following formula: PEI (vertical dimension of the uid/width of the right pleural cavity) 100.10 The chest radiograms were read blinded after the completion of the recruitment season. Statistical Analysis. Variables are reported as mean (SE) or number (%) as appropriate. Analysis of categorical variables was performed using 2. Continuous variables were analyzed using multivariate analysis. Correlations were analyzed using Spearman analysis for nonparametric test and Pearson analysis for parametric test. A P value 0.05 was considered signicant. All analyses were performed using SPSS (version 12) statistical program.

RESULTS
Characteristics of Study Subjects. Table 1 shows the baseline characteristics of the study patients. There were 82 patients with DV infection with 61 and 21 cases classied as DF and DHF, respectively. All the DHF cases met all 4 clinical criteria including fever, hepatomegaly, hemorrhagic tendency and clinical and/or radiologic evidence of plasma leakage. All had platelet counts below 100,000/mm3. There were 27, 10, 7 and 36 cases with dengue serotypes 1, 2, 3 and 4, respectively (Table 2). The PCR was negative in 2 cases with positive serology. Eleven dengue-infected patients had primary infections based on the serologic response; all had DF. The remainder of the dengue cases had secondary DV infections. Among the DHF cases, there were 2, 14 and 5 cases of DHF grade I, II and III, respectively. Seventy-six participants were categorized as OFI. There were no differences in hematocrits or plasma albumin levels between groups at admission, but
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TABLE 1. Baseline Characteristic of Patients

Clinical Diagnosis Number of cases Age, y (mean SE) Sex (M/F) Albumin (g/dL) (mean SE) Hematocrit (%) (mean SE) Platelet ( 103 count/mm3) AST (mean SE) Fever duration at presentation (mean SE) Fever day (mean SE) DF 61 9.58 (0.34) 37/24 4.2 (0.1) 37.5 (0.4) 186 (8.5)* 59 (11) 1.9 (0.1) 2.5 (0.2) DHF 21 10.31 (0.53) 10/11 4.2 (0.1) 38.4 (0.8) 165 (22)* 85 (11) 2.4 (0.1) 2.2 (0.3) OFI 76 9.16 (1.1) 43/33 4.2 (0.1) 37.1 (0.3) 259 (8) 36 (2) 1.6 (0.1) 1.8 (0.2)

*Average platelet count was lower in DF and DHF groups than OFI group (P 0.01). AST was elevated in DF and DHF patients compared with patients with other febrile illness (P 0.002). Mean durations of fever at presentation were greater in DHF group compared with DF and OFI groups (P 0.045). There were no differences in hematocrits or plasma albumin concentrations at the time of presentation in any of these groups. DF, dengue fever; DHF, dengue hemorrhagic fever; OFI, other febrile illness.

TABLE 2. The Serotypes of Virus and the Patterns of Antibody Response in Dengue Virus Infected Cases
Primary Antibody Response DF Dengue 1 Dengue 2 Dengue 3 Dengue 4 Not identified 5 3 1 1 1 DHF 0 0 0 0 0 Secondary Antibody Response DF 10 6 5 29 0 DHF 12 1 1 6 1

Virus Serotypes

DV infected patients had higher AST levels but lower platelet counts than the patients with OFI (Table 1). Temporal Sequence of Ultrasonographic Signs During DV Infection. To study the development of plasma leakage early in the course of the disease, we analyzed the ultrasound ndings on the day of presentation when the duration of fever was less than 72 hours. Although few patients in any diagnostic group had positive ultrasound ndings at that time, retrovesicular uid and pleural effusion were detected in a higher proportion of DHF patients compared with DF and OFI patients (Table 3). Of the 3 DHF cases with ascites detected by ultrasound examinations, only one had ascites detected by physical examination. The initial ultrasound ndings detected in non-DHF patients were absent on subsequent examinations while the ndings in DHF were persistent

(Fig. 2A, B). No differences in plasma albumin levels, pulse pressure and changes in hematocrit between DHF cases and DF and OFI cases were detected during the initial 24 hours of hospitalization (Fig. 2C, D, E). However, hemoconcentration became signicantly greater in DHF cases compared with non-DHF cases during the subsequent 24 hours (Fig. 2D). Chest ultrasonograms revealed pleural effusion in 16%, 33% and 62% of DHF patients on fever day 1, 0 and 1 respectively (Fig. 3A). The percentage of DHF cases with a thickened gallbladder wall (dened as more than 2 mm) was 11%, 19% and 43% on fever day 1, 0 and 1 respectively (Fig. 3B). Some DF and OFI cases had a thickened gallbladder wall which was not found on subsequent examinations. The temporal pattern of ascites was similar to the pattern of pleural effusion with uid detected in the hepatorenal pouch in 6%, 29%, 42% of DHF cases, and in the retrovesicular space in 17%, 47%, 52% of DHF cases on fever day 1, 0 and 1 respectively (Fig. 3D, E). The sensitivity, specicity, positive and negative predictive value of ultrasound scans in diagnosing DHF are shown in Table 4. Detection of Pleural Fluid by Chest Ultrasound and Chest Roentgenogram. Pleural effusions detected by chest roentgenograms are generally considered a sign of plasma leakage in DHF. To compare chest roentgenograms and chest ultrasound scans in detecting plasma leakage, we performed right lateral decubitus chest roentgenograms and upright chest ultrasound scans on all patients with conrmed DV infections and forty-two OFI patients (as controls) on fever day 1. Right lateral decubitus chest roentgenogram detected pleural effusions in 100% of DHF cases, but also in 9% of DF cases and 19% of OFI cases (Table 5). The sizes of the pleural effusions, expressed as PEI, were signicantly larger in DHF cases than in DF and OFI cases (DHF: 15.5% 3.1%, DF: 0.2% 0.1%, OFI: 0.4% 0.1%, P 0.001, Table 5). The sizes of pleural effusions detected in DF and OFI cases were not different (P 0.97). Chest ultrasound scans detected uid in 62%, 1.6% and 0% of DHF, DF and OFI cases respectively. The vertical dimension of pleural uid detected by ultrasound showed a good correlation with the size of the effusion determined by right lateral decubitus chest roentgenogram (r2 0.49, P 0.001 Fig. 4A). The sensitivity and specicity of the right lateral decubitus chest roentgenogram and upright chest ultrasound in diagnosing DHF one day after defervescence are shown in Table 4. We analyzed the relationship between the sizes of pleural effusions and the severity of illness in patients with DHF. The magnitude of pleural effusions determined either

TABLE 3. The Number and the Percentage of Cases With Positive Ultrasound Findings on the Day of Presentation
Clinical Diagnosis DF DHF OFI Total Cases 61 21 76 Fluid Detected by Chest Ultrasound Scan 1 (1.6) 2 (9.5)* 0 (0) Gall Bladder Wall Thickness 2 mm 3 (4.9) 1 (4.2) 0 (0) Retrovesicular Fluid 4 (6.5) 3 (14) 4 (5.2) Hepatorenal Pouch Fluid 1 (1.6) 1 (4.8) 0 (0)

*Higher proportion of DHF cases had positive chest ultrasound findings compared to the other 2 groups. P 0.006 by Pearson 2 test. Values in parentheses indicate % values. DF, dengue fever; DHF, dengue hemorrhagic fever; OFI, other febrile illness.

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FIGURE 2. Time course of the ultrasound findings, clinical and laboratory parameters in suspected dengue cases with positive ultrasound examination at presentation, (A) vertical dimension of pleural fluids and (B) anteroposterior retrovesicular fluid. Empty circle: DHF, filled circle: non-DHF. Each line represents an individual case. (C) Minimal pulse pressure, (D) changes in hematocrit and (E) plasma albumin concentrations (DHF: empty circles, DF: filled circles, OFI: triangles). *P 0.025, comparing between DHF and non-DHF cases.

by right lateral decubitus chest roentgenograms or by upright chest ultrasound scans was signicantly larger in DHF grade III than in grade I and II (Table 5). In addition, there were signicant correlations between the size of pleural uids, detected by either chest ultrasound or right lateral decubitus chest roentgenogram, and the degree of hemoconcentration, measured as percent change in hematocrit (Fig. 4B, 4C). Gallbladder Wall Thickness. Serial measurements revealed a progressive increase in the mean gallbladder wall thickness in DHF patients (Fig. 5A). The gallbladder wall thickness was signicantly greater in DHF cases than in DF or OFI cases on fever day 1 (1.2 0.4, 0.7 0.1 and 0.7 0.1 for DHF, DF and OFI respectively, P 0.026) and thereafter. The

FIGURE 3. Percentages of patients with various ultrasound findings indicative of plasma leakage during the course of illness: (A) pleural effusion, (B) gallbladder wall thickness over 2 mm, (C) fluid in the hepatorenal pouch, (D) retrovesicular fluid. Empty circle: DHF, filled circle: DF, filled triangle: OFI.

timing of the onset and peak gallbladder wall thickening varied between individual cases as shown in Figure 5B. In 4 cases where the duration of the hospital stay was long enough to allow the resolution of gallbladder wall edema to be observed, the duration of gallbladder wall edema ranged between 2 and 4 days. The mean maximum wall thicknesses in DHF grade I, II and III patients were 2.7 2.2 mm (n 2), 2.1 0.5 mm (n 14) and 4.6 0.6 mm (n 5), respectively. The mean maximum wall thickness was significantly higher in DHF grade III patients than in others (P 0.01, Fig. 5C). Furthermore, the gallbladder wall thickness correlated with the degree of hemoconcentration, plasma albumin and AST concentrations (Fig. 5D, E, F). Quantitative Measurement of Ascites. The mean anteroposterior dimension of the retrovesicular uids measured in millimeters was signicantly larger in DHF patients than in DF patients on fever day 1 (DHF: 1.7 1.2 mm, DF: 0.1 0.1 mm, P 0.03), and thereafter (Fig. 6A). Similar patterns were observed with the transverse and the longitudinal dimensions (data not shown). Figure 6B shows the course of retrovesicular uid accumulation in individual DHF patients. The mean maximum transverse dimension of patients with DHF grade III was signicantly larger than those of DHF grade I and II patients (DHF grade III: 56 4 mm, DHF grade I: 29 29 mm, DHF grade II: 20 8 mm, P 0.024). No signicant differences were found in the sizes of the uid measured in other dimensions between DHF patients with different clinical severity (data not shown). The sensitivity, specicity, positive and negative predictive value of the hepatorenal pouch uid and retrovesicular uid on fever day 1 in diagnosing DHF are shown in Table 4. Correlations Between Plasma Leakage at Different Locations. Pleural effusion detected either by right lateral decubitus chest roentgenogram or chest ultrasound was the most common nding of plasma leakage (100%) in this series, followed by gallbladder wall thickening (62%), and retrovesicu 2007 Lippincott Williams & Wilkins

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TABLE 4. Sensitivities, Specificities, Positive and Negative Predictive Values of Right Lateral Decubitus Chest Roentgenograms and Various Ultrasound Scans in Diagnosing DHF
Sensitivity (%) Right lateral decubitus chest roentgenogram (fever day 1) Upright chest ultrasound (fever day 1) Maximum gallbladder wall thickness 2 mm Hepatorenal pouch fluid (fever day 1) Retrovesicular fluid (fever day 1) 100 62 62 42 52 Specificity (%) 76 99 92 100 92 Positive Predictive Value (%) 61 93 54 100 55 Negative Predictive Value (%) 100 94 94 91 92

The specificity, sensitivity, positive and negative predictive value of right lateral decubitus chest radiograms, chest ultrasonograms and abdominal ultrasound in diagnosing DHF were calculated based on fever day 1 findings in 139 study subjects (21, 61, 57 cases of DHF, DF and OFI respectively). Nineteen OFI patients did not stay until defervescence and did not have chest radiograph performed. Right lateral decubitus chest radiograms were positive in 21 DHF, 5 DF and 8 OFI cases. Upright chest ultrasound detected fluid in 13 DHF, 1 DF, 0 OFI cases. Fluid in the hepatorenal pouch was detected in 9 DHF, 0 DF, 0 OFI cases. Retrovesicular fluid was detected in 11 DHF, 7 DF and 2 OFI cases. The specificity, sensitivity and the predictive values of the gall bladder wall thickness over 2 mm at anytime during the course of the hospitalization were calculated based on findings in 21 DHF, 61 DF, 76 OFI cases. Thirteen, 9, and 2 cases of DHF, DF and OFI cases had thickened gall bladder by this criteria. The formula used to calculate these parameters are (1) sensitivity (%) (number of DHF cases with positive findings/the number of DHF cases) 100, (2) specificity (%) (number of DF cases with negative findings number of OFI cases with negative findings)/the number of DF OFI cases 100, (3) positive predictive value (%) (number of DHF cases with positive findings/the number all cases with positive findings) 100, (4) negative predictive value (number of DF cases with negative findings number of OFI cases with negative findings)/the number all cases with negative findings 100.

lar uid (52%). Among the 21 DHF cases, 10 cases had all 3 ultrasound ndings of plasma leakage, 3 cases had 2 ultrasound ndings and 3 cases had only one ultrasound ndings. Five DHF cases (1 DHF grade I and 4 DHF grade II cases) exhibited no ultrasound evidence of plasma leakage and had a small pleural effusion detected only by right lateral decubitus chest roentgenograms (mean PEI 4.7% 1.02%). There was a signicant correlation between the sizes of pleural effusions (detected either by right lateral decubitus chest roentgenograms or ultrasound) and the gallbladder wall thickness (R2 0.29, P 0.01) and the sizes of the retrovesicular uids (R2 0.23, P 0.03).

DISCUSSION
We performed the rst prospective study employing serial ultrasonography to examine the anatomic and the temporal patterns of plasma leakage in DV infected patients. Our studies demonstrate that signs of plasma leakage were detected in some DHF patients early in the course of the illness but in most patients these ndings were detected around the time of defervescence. This is consistent with the clinical

observation that circulatory consequences of plasma leakage are usually observed within 48 hours after defervescence.2 Signicantly higher proportion of patients who were eventually diagnosed with DHF exhibited ultrasonographic signs of leakage at the time of clinical presentation than DF and OFI cases. The ultrasound ndings were detected one day before peak hemoconcentration and clinical signs of plasma leakage in 20% of DHF cases with at least 10% increase in hematocrit, indicating that ultrasound studies may be useful in early detection of plasma leakage. Serial hematocrit determination has been routinely used to detect hemoconcentration as a sign of plasma leakage. A 20% increase in hematocrit over baseline is generally accepted as indicative of plasma leakage.2 However, since baseline hematocrit values are rarely available at the time patients present for evaluation, the hematocrit can be used to guide management only by reference to normal values in the population. In previous studies, the hematocrit obtained at convalescence was used as the baseline hematocrit.13 Since our study subjects were enrolled early, preleakage hematocrits were available in most cases. Further, we have ob-

TABLE 5. The Number and the Percentage of Cases With Pleural Effusion, and the Sizes of the Effusions Detected by Either Right Lateral Decubitus Chest Roentgenogram or Upright Chest Ultrasound Scan on Fever Day 1
Right Lateral Decubitus Chest Roentgenogram Clinical Severity (total cases) Positive (case (%)) 8 (19) 5 (9) 21 (100) 2 (100) 14 (100) 5 (100) PEI % (mean (SE)) 0.4 (01) 0.2 (0.1) 15.5 (3.1)* 16.8 (68) 10.0 (2.9) 33.7 (4.2) Upright Chest Ultrasound Positive (case (%)) 0 (0%) 1 (1.6%) 13 (62%) 1 (50%) 7 (46%) 5 (100%) Vertical Dimension, mm (mean (SE)) 0 1 23.9 (6.3) 10.3 (10.4) 19.5 (7.7) 49.4 (12.1)

OFI (42) DF (61) DHF (21) DHF I (2) DHF II (14) DHF III (5)

*Different from DF and OFI (P 0.001). Different from DHF grade I and II (P 0.05). PEI, pleural effusion index; OFI, other febrile illness; DF, dengue fever; DHF, dengue hemorrhagic fever; DHF I, II, III, dengue hemorrhagic fever grade 1, 2, 3.

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FIGURE 4. Correlations between the sizes of pleural effusions determined by upright chest ultrasound or right lateral decubitus chest roentgenogram (A), and between these parameters and percent changes in hematocrit (B and C).

served that in some patients the lowest hematocrit readings were detected during the illness preceding the rise in hematocrits. The percent changes in hematocrits derived from these values therefore more closely reect degrees of hemoconcentration than values derived using hematocrits obtained at convalescence as baseline values in these cases. Our nding may reect a signicant proportion of DHF cases with spontaneous hemorrhage in our study. It also underscores the need to take into consideration, in addition to the nadir and the peak hematocrit readings, the relative timing of these readings and associated factors such as blood loss and

uid therapy, in making clinical classication. In cases where the degree of hemoconcentration is difcult to ascertain, a direct demonstration of uid leakage by ultrasound might help differentiate cases with borderline changes in hematocrits (10 20%), which would not have been correctly classied using hematocrits alone as criteria. In this series, 10 out of 61 cases of DF had an increase in hematocrit between 10 20%, but none had pleural effusion detected by chest ultrasound. In contrast, among 17 cases of DHF who had less than a 20% increase in hematocrit, all but 5 exhibited at least one ultrasonographic nding over the clinical course. Patients who

FIGURE 5. Time course of gallbladder wall thickening. (A) Mean gallbladder wall thickness in DF, DHF and OFI patients. Empty circle: DHF, filled circle: DF, filled triangle: OFI. *P .01 for comparison of DHF and non-DHF patients. (B) Gallbladder wall thickness of individual DHF patients over the course of the illness. Dotted, gray, and black lines indicate patients with DHF grade 1, 2 and 3 respectively. (C) Mean maximum gallbladder wall thickness in patients with OFI, DF and DHF. *P 0.001 for comparison of DHF and either DF or OFI cases, **P 0.01 for comparison of DHF grade III and either grade I or II cases. (DF) Correlation between peak gallbladder wall thickness and percent change in hematocrit (D), plasma albumin levels (E) or AST levels (F).

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FIGURE 6. Time course of retrovesicular fluid. (A) The mean of the fluid size in the anteroposterior dimension in DF, DHF and OFI patients over the course of the illness. Empty circle: DHF, filled circle: DF, filled triangle: OFI. *P .03, **P .01 for comparison of DHF and non-DHF patients. (B) Time course of retrovesicular fluid in individual DHF cases. Dotted, gray, and black lines indicate patients with DHF grade 1, 2 and 3 respectively.

had negative ultrasound ndings had small pleural effusions detected only by right lateral decubitus chest roentgenogram. Pleural uid detected by chest roentgenogram has been interpreted as evidence of plasma leakage. Our study demonstrates that small pleural effusions detected by lateral decubitus chest roentgenogram are a relatively common nding in nondengue febrile patients and in DF patients, conrming an earlier report.10 This indicates that a small pleural effusion in itself is not a reliable marker of plasma leakage as a criterion for clinical classication of dengue cases. Small pleural effusion, similar to the small and transient ascitic uid and thickened gall bladder wall observed in non-DHF patients, may reect mild local serosal inammation occurring as a nonspecic reaction in febrile illness, distinct from increased vascular permeability in DHF. Chest ultrasound has been reported to be more sensitive than upright chest roentgenograms in detecting pleural uid in DHF cases.7 We found that chest ultrasonogram was less sensitive than right lateral decubitus chest roentgenograms in detecting small pleural effusions. However, this lower sensitivity was associated with a higher specicity for DHF. The sizes of the effusions obtained by ultrasound correlated well with those determined by right lateral decubitus chest roentgenogram, and with disease severity and the degree of hemoconcentration. Thickening of the gallbladder wall has been reported in conditions with hypoalbuminemia and ascites,14 and in several viral infections including DHF.6 8,15,16 Although ageadjusted normal values of GBW thickness are not well established, the denition of thickened gallbladder wall has generally been accepted to be above 3 mm.17,18 Based on a receiver operating characteristic curve analysis of our data, we found that a cut-off of 2 mm yielded a better sensitivity in discriminating DHF cases (data not shown). Consistent with previous reports, we found a statistically signicant difference between maximum gallbladder thickness of patients with DHF grade III and those of DHF grades I and II.6 The gallbladder wall thickness also correlated with changes in hematocrits and plasma albumin, and with AST values. Similar correlations have been reported in hemorrhagic fever with renal syndrome.19 Ascites is another sign of plasma leakage in DHF patients. The minimum amount of ascites required for
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detection varied according to the location in the abdomen and the examination position.20 22 We detected uid in Morrison pouch and the pelvic cavity at comparable frequency in DHF cases. The incidence of ascites in DHF in our series is comparable to that previously reported.57,23 Fluid was detected in the pelvic cavity in some DF and OFI patients as well. However, those uid collections were minimal in size and were often detected only once during illness. Over half of the DHF cases in this series had all 3 ultrasonographic signs of plasma leakage; pleural effusion, thickened gallbladder wall and ascites. Pleural effusions occurred mostly on fever day 1, in contrast to gallbladder wall thickening and ascites, which were more variable in onset, ranging from fever day 2 to 1. The earlier onset of gallbladder wall thickening compared with the onset of pleural effusions is consistent with a previous report.8 The rapid resolution of gall bladder wall thickening and ascites suggests that the underlying mechanism is a transient increase in permeability rather than inammation. This is consistent with the absence of tissue inammation at autopsy in DHF and the rapid recovery in most DHF patients.24 Because of the lack of clinical or laboratory indices that will predict which patients will develop DHF, close monitoring of clinical signs and hematocrits are required to detect plasma leakage. Although we have demonstrated that ultrasound examinations can directly detect plasma leakage before signicant hemoconcentration occurs, signicant accumulation of uid is still required before it became detectable by ultrasound. A parameter capable of detecting plasma leakage in a timely manner is still needed. Because of the relative small number of severe DHF cases in this series, the diagnostic value of various parameters in differentiating DHF cases with cardiovascular compromise cannot be derived. In addition, the relatively small number of DHF cases limits the statistical power to detect differences in ultrasound ndings in patients with different virus serotypes. Additional studies which include more severe DHF cases will be required to answer these questions. Nevertheless, our results indicate that chest and abdominal ultrasonographic studies are useful in detecting plasma leakage in dengue infected individuals. Among various ultrasound examinations, upright chest ultrasound appeared to be the most sensitive and yielded superior

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positive and negative predictive value for detecting plasma leakage in DHF and provides a quantitative measurement that correlates with clinical severity.

ACKNOWLEDGMENTS
The authors thank the arbovirology and molecular sections of the Armed Forces Research Institute of Medical Sciences for diagnostic testing; Dr. Suchitra Nimmannitya for reviewing the clinical diagnoses; doctors and nurses of Queen Sirikit National Institute of Child Health and the staff of the Armed Forces Research Institute of Medical Sciences for patient care and sample collection. This work is supported by National Institutes of Health Grant NIH-P01AI34533 and the Military Infectious Disease Research Program. The opinions or assertions contained herein are the private ones of the authors and are not to be construed as ofcial or reecting the view of the U.S. Government. REFERENCES
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