Early CI S. Kalayanarooj, D. W, Vaughn, S. Nimmanr 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 al and Laboratory Indicators of Acute Dengue Illness Banglok Chinen’ Hospitl, Bangkok, Departmen of Virology, Armed Forces Research Insite of Medical Sciences, Bankok. and Kamphaeng Phet Provincial Hospital, Kanphacne Pht, Thailand: "Division of Infections Diseases end Immunology, Univers of Massochusets Modical Center, Worcester, Massochusets: Dspartment ‘of Virus Diseases, Walter Reed Army Inte of Research Washingion. DC AA prospective observational study was conducted to identify early indicators of acute deng virus infection, Children with fever for <72 b 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 hemorshagie fever (DHF). At enrollment, children with dengue were more likely than children with ‘other febrile illnesses (OF to report anorexia, nausea, and vomiting and tohave a postive tourniquet test, and they had lower toral 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 ‘aa dentfy simple clinical and laboratory parameters that help to identity children with DF oor DHF, The dengue viruses, a group of four closely related viruses in the Flavivirus family (dengue virus serotypes 1-4), ane the ‘most important flaviviruses from the standpoint of human mor- bidity [1, 2) Is estimated that as many as 100 million dengue virus infections oceur annually, principally in topical and sub- tropical areas inhabited by the mosquito vectors, Aedes aezypi and Aedes albopictus (3}. Most dengue virus infections in chil dren have minimal or no symptoms and cannot be easily distin guished clinically from other viral infections (4), Classic den- gue fever (DF) is an acute illness characterized by fever, retroorbital headache, severe myalgias, and occasionally arash which lass from 5 to 7 days [5]. In a small percentage of dengue infections, a more severe form of disease, known as dengue hemorthagic fever (DHF), occurs. DHF is characterized by acute fever associated with a Ressved 2 November 1996; eis 27 January 1997 Presented in pr: 4th Annual Meeting of he American Society of Tropical Madiine a yen, San Antoni, esas, November 1995 (asst 158). ‘Weis formed coca! we bined fom the pret or mace of i ratients The sy prone! wan apprved hy instal review toads of the Minty of Poe Hah (Than, Surgon General Office of the Departnct of the Ann, and University of Mastochusts Medical Contr tnd aman experimcnition pds of he US Deparment of Health and Human Services an the author” instations were flowed Financial soppot: NIH (AL34533) and US Army Medical Reseach and Materiel Command The options coninod heen are dhse of de autor and shoul no be ona representing he fii pois of the Nationa Insittes of Heath the Deparment of the Army, o the Departnet of Defense Reps or erespondenee: Dr. Frans A. Enns, Division Diet. Divi son of lfecious Dscases and Inmusalogs, University of Massachusots Modi Cente, 55 Lake Ave. N., Worse, MA O16SS ‘The outnl of Into Dhan 997 176313-21 ‘nn 1998977762 MSH. 00 hhemorthagic diathesis and a tendency to develop shock (dengue shock syndrome), which distinguish it from DF. Abnormal hhemostasis and plasma leakage are the two pathophysiologic hallmarks of DHE, which are manifested clinically as thrombo- eytopenia 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). clinical definition of DHF established by the World Health Organization (WTO) is based ‘on the presence of high continuous fever, hemorsbagic manifes- tations (including at least a positive tourniquet test), hepato- megaly, thrombocytopenia (platelet count <100,000/mm), and hemoconcentration (hematocrit increased by =20% above baseline value) [7]. The WHO definition further subdivides DIF into four grades on the basis ofthe presence of spontanc- fous bleeding and the presence and severity of shock. Several hhundred 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 vitus elicits lifelong hhomotypie immunity, but heterotypic immunity is short-lived [8]. Some studies suggest thatthe frequency of occurrence of DHF is influenced by characteristics ofthe infecting virus, such as the serotype and even the specific strain [9-12]. However, host immune responses are also important in the pathogenesis of DHE, because the risk for DHF is increased atleast 15-Fold uring secondary dengue virus infections [13] Various mechanisms have been postulated to explain this eased tisk for DHF in secondary dengue infections, includ ing antibody-dependent enhancement of infection [14], eom- plement netivation by virus-antibody complexes (15, 16}. and T cell-mediated immunopathology [17]. One study demon- strated that the ability of undiluted serum to enhance dengue 1202 Jeqe120 21 uo ean8 4a 0a Kalayanarj et al virus infection in vitro correlated with the development of DHF during a subsequent dengue virus infection [18]. Patients with DIF have been shown to have elevated levels ofthe eytokines interferon-y and interleukin-2 as well as the soluble forms of CDS, CDS, and the interleukin-2 receptor «chain, which are ‘markers of T cell activation [19]. Other studies have also shown that patients with DHF have depressed serum levels of comple- ‘ment 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 audition, 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 1 well-matched comparison group. The low virus isolation rate in patients with DHF in those studies has precluded an analysis, of the influence of viral factors, such as serotype and strain, con disease severity. Early clinical diagnosis of dengue virus infection can be difficult, The WHO clinical and laboratory erteria for DH are usually not present in the frst few days of illness. One study reported that flushed face without coryza and a positive tourniquet test were early predictors of DHF in febrile patients [21]; however, @ definitive diagnosis of DHF could be made only with the development of thrombocytopenia and plasma Jeakage, which usually occurs only 12 days before the onset of shock [5]. We therefore designed a prospective study of chiklren early in the course of suspected dengue virus infection in order t0 ‘identify early clinical and laboratory predictors of the risk of DHF before the critical stage of disease, that is, before defer~ vescence and the onset of bleeding and plasma leakage. This study was also designed to identify immunologic or viral fac- tors 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 (OF, Methods Subjecr enrollment. Previously healthy children who presented to Bangkok Children’s Hospital and the more rural Kamphaeng. Phot Provincial Hospital tween 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 72 h, oral temperature =38.5°C recorded in the clinic, and ‘ushed face. Children meeting these criteria were examined by a study pediatrician for eligibility, and childzon who had a specific ‘Mentifiable cause of fever were excluded. For example, children ‘with an ini elinical diagnosis of measles, sinusitis, tonsils, or gastroenteritis were not eligible: however, children with nausea or diathea could still be eligible for enrollment if the physician did not consider the clinical picture diagnostic of gastroenteritis, Subjects were also excluded if, atthe time of evaluation, there was hypotension, ancmia, malnutition, or a history of chronic {ND 1997;176 (Avent) medical illnesses, such as congenital heart disease or thalassemia, The study was organized to encoll up to 6 patients per week at each study ste 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, 7-20 mL, pending on the subject's weight, was obtained, All subjects were then admitted tothe hospital ward for observation under the care of a pediatrician with experience in the care of patients with DHE. Each moming, a study nurse reviewed the medical rovord for the previous 24 h and recorded clinical and laboratory data on a ssandandized data collection form, A tourniquet test was performed by inflating a blood pressure cuff on the upper arm to midway between the systolic and diastolic blood pressures for $ min and then examining a 2S-cm? area using a standaed template. The ‘number of petechiae within the cemplate, up to 20, was determined, The tourniquet test was performed on altemating arms each day until the subject was discharged or until >20 petechiae were r= corded on any I day. A venous blond specimen was collected each ‘orig, up to. maximum of five blood draws during hospitaliza- Vital signs wore obtained by the want nurses at least every 6 h If two consecutive 6-H oral temperatures were <38°C, a capil lary sube hematocrit determination was performed by the ward nurses on a fingerstick blood specimen. This was repeated 6 and 12h later if the temperature remained <38°C. A ight lateral ddcubitus chest radiograph was obtained on the next morning un- less fever (38°C) recurred. Other laboratory studies were done and medications or intravenous fluids administered, at the disers ysician. Subjects were discharged from the study if an alternative diagnosis was reached during observation, Subjects were discharged from the hospital i they were clinically stable and afebile for atleast 24h, Subjects were asked to return to the outpatient department 7 8 days afer enrollment, at whieh time a follow-up blood specimen was obtained Blood specimens for this stuly were obtained in EDTA-con- taining tubes and immediately placed on ice. A I-mL aliquot of \whole blood was sen tothe hospital laboratory for eomplete blood count (TS40 counter; Coulter, Hialeah, FL). The remainder ofthe blood specimen was transported to the research laboratory, where plasma and platelets were separated by an intial centrifugation at 300 g, followed by centrifugation at 800 g. Peripheral blood mononuclear eels were separated fom the fed 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 amino- transferase (AST), and albumin (Clinieal System Analyzer, model 700; Beckmann Instruments, Brea, CA), The remainder of the plasma was aliquoted and stored at ~70°C Serologic and virologic stules. Plasma samples were tested for serologic evidence of acute dengue virus infection by IpM and IgG ELISA and hemagglutination-inhibtion (HAI assays (22) Standard criteria were used to identify acute primary and acute secondary dengue virus infections. Ifthe serologic studies wer inite—for example, with no convalescent blood specimen available or a high titer of anti-dengue antibody that showed no change in tter—and virus isolation attempts (see below) were negative the serologic diagnosis was recorded as unknown, tion ofthe attendND 1997176 (August) Dengue virus isolation was attempted by inathoracie inocula- tion of plasma samples into Toxarrhynchitessplendens mosquitoes [22]. The frst 3 consecutive daily plasma samples from each sub= ject were tested, if available Study design and definition of DHF cases. Study day 1 was defined as the calendar day during which the subject was enrolled ‘inthe study and days were counted consecutively afterward, Fever day 0 was defined asthe calendar day during which the temperature fell and stayed <38°C, and days before and after this point were numbered consecutively (fever days ~1, ~2, ete. occurred before defervescence and fever days +1, +2, et occurred after deferves- Clinical and laboratory data were recorded by trained study nurses using standardized data collection forms. Hospital chars were reviewed by a study physician to identify systematic r= cording errors, Data from the standardized forms were entered Jo a computer database twice for validation, using FoxPro for Windows (Microsoft, Redmond, WA), Serologic test results were read by’a study physician blinded tthe linical information. Chest radiographs were read hy a radiologist blinded to the clinical and serologi information, and a pl sion index was calculated as 10% (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 inal clinical diagnosis by two of us (S.K., SN.) on the basis of review of clinical and laboratory data. A clinical diagnosis of DHF was assigned follow= ing the WHO clinical definition [7] on the basis of the presence of plasma leakage and thrombocytopenia. The serologic data were not used to assign the clinial di e of plasma leak- aye could include a peak hematocrit value >20% above the value at the convalescent visit (study day 8-10), a pleural effusion dem- ‘onstrated on the chest radiograph, or detection of ascites on physi cal examination, Out inital definition of thromboeytepenia was a platelet count nadir of =100,000mm’; however, because some subjects didnot have platl ‘maximal plasma leakage, we assigned a diagnosis of DHF to some subjects iTthere was evidence of plasma leakage and the available platelet counts showed a significant decrease from the value atthe convalescent visit. Cases of DHF were graded fom 1 10 4 ae- cording to WHO eriteta (7. Any subject with serologic o vtologie evidence of acute dengie ‘counts available from the time of infection who did not meet the criteria for DHF was considered as having DF. Subjects were diagnosed a having OFL when all ofthe following criteria were met: There was no clinical evidence of acteral infection, thre was no sorologic or virologic evidence of acute dengue infection, and serologic testing was considered adequate to exclude acute dengue virus infection (see above), Differences in clinical and laboratory data were analyzed on the basis ofthe final diagnosis assigned. The analysis was eared out both by comparing all dengue infections (combined DF and DHF) with OFT and by comparing subjects \with DHF with those with DF. Proportional data were tested using 1 analysis or Fisher's exact test, Continuous data were tested using the tes; statistically significant differences were confirmed using the Mann-Whitney rank sum test for data not showing normal distribution. Statistical analyses were carried out using SigmaStat 2.0 for Windows (Jandel Software, San Rafael, CA). Saristical analysis ary Inictrs of Acute Dengue Hlness us DHF BDF CIOF! mlOtherlunk. 35 30 25 20 15 10 # subjects enrolled ‘Apr June Aug = Oct ec. Month Figure 1. No.of subjects envlled, by month and final diagnosis, Final diagnoses were dengue hemorshagie fever (DHF), dengue fever (DF), or ater, nondengue Febrile illnesses (OF. Results Subject demographics, Between 25 Apriland {4 December 1994, wwe 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 tnd September (figure 1). The overall male-to-female ratio was Ls, 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 signifi cantly higher than the mean age of subjects enrolled in Bangkok (8.6 + 30 vs. 6.2 + 3.0 years). There were no significant differences in male-to-female ratio or duration of fever before study entry between the two study sites Final diagnoses. OF the 189 study subjeets, 3 were diag- nosed with bacterial infections during the study and were with drawn, one each with Haemophilus inluensae meningitis, Sal ‘monella gastroenteritis, and a urinary tract infection. Fourteen other subjects could not be assigned a definite serologic diagno- sis: 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 (fixed high titer in HAL assay). OF the remaining 172 subjects, 60 (35%) had serologic or virologic evidence (58 had both) of acute dengue virus infection, Fifty-five of these subjects (93%) had serologic responses characteristic of sec- ondary dengue virus infection, Between May and October when most subjects were enrolled, and excluding subjects with- out a definite serologic diagnosis, the percentage of study sub- jects with acute dengue infection ranged from a high of 44% in October toa low of 14% in September (figure 1) OF the 60 subjects with serologic or virologic evidence (oF both) of acute dengue virus infection, 32 (53%) were classified as DF according tothe above definitions and 28 (47%) as DHF. 1202 Jeqe120 21 uo ean8 4a 0a6 Kalayanaro) etal ‘Table final diagnosis assigned Elevation of materi at peak increase over contlecont, mean (SD) Weight change, eny to fever day +1, ke mean (SD) Pour ffison 0.) Pleura ffision inex, mea ngs) {ND 1997;176 (Avent) Clinical and laboratory measures of dissase severity inthe stady population, acconding to Final diagnosis ress) 321228 (48) 0s 6s) 4g 28087) =03100 03.06) ‘1s Imes) 2307) 222684) 0290-33) DUTO-13) 1410-78) siz S82 (16) 232862) NOTE, DHE, dengue homage fe: DF, dengue fever; OF, her nondenge feb ines * Peak hemor daring cbsrvation =20% shove Hematocrit low-up (comalescens),Fllow-p eral, values were act avalible forall say abject "Platter nar = 100.00 ee Among the 28 subjects with DHF, there were 5 subjects with grade | 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 infee- tions were categorized as DI Table I describes the evidence of discase severity in children with DHF compared with those with DF and OFI, Subjects \ith DHF had evidence of hemoconcentation, pleural effusion and thrombocytopenia, consistent with the clinical definition These subjects also ad a slight weight gain from entry to fever day +1, whereas subjeets with DF or OFL lost a mean of 03 ke, Elevations of hematocrit to 20% above the convalescent value were seen in half of the subjets with DHF. The absence of hemoconcentrtion in the remainder was attributed to the infiequent (64) hematocrit determinations, the effort 10 ine crease the oral Hid intake, and, in some eases, the administra tion of intravenous fhid. Four subjects who were assigned a diagnosis of DHF on the bass of thrombocytopenia and clinical evidence of plasma leakage showed no evidence of pleural effusion on their decubitus chest radiograph. On review of these recon, 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 signif- cant deerease in platelet count but no platelet count 100,006 rm Reassigning these cases as DF did not significantly alter the results of the statistical analyses Three subjects who were assigned a clinica 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'mm", In each case, the finding of hemo- concentration related toa single abnormally high or low hema- tocrit determination, which on review was felt tobe inconsis- tent withthe rest ofthe clinical data. Two other subjects were assigned a diagnosis of DF even though a pleural effusion was detected, Both ofthese subjects had a small pleural effusion index (0.9 and 1.3) and no evidenee of hemoconcentration, ascites, spontaneous bleeding, oF thrombocytopenia. All $ of these subjects withthe diagnosis of DF had serologic responses Indicative of secondary dengue virus infection, and reassigning the eases as DHF did not significantly alter the results of the statistical analyses, Furthermore, it should be noted that 17 (6.6%) of 112 subjects with a diagnosis of OFT had small pleural effusions (the highest pleural effusion index in this {group was 3.3). Comparison of clinical and laboratory features at study en- fay, Table 2 shows a comparison of selected clinical and labo- ratory features at entry into the study among those subjects eventually classified as OF, DF, or DHF. Subjects with dengue Virus infection (DF or DHF) were significantly older and were ‘more likely to report anorexia, nausea, and vomiting than sub- jects with OFI, The male-to-female ratio and history of head- ache, abdominal pain, or bleeding were not significantly ditfer- ent between the groups. Subjects with dengue virus infection were also more likely to have a postive tourniquet test. Initial laboratory studies showed that there were no significant der ences 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 monoeyte counts at entry were significantly lower in subjects with dengue virus infection than in subjects with OFT. Plasma AST and ALT. levels were significantly higher in children with dengue virus infection than in children with OFI, The only statistically sig- int differences in initial findings 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 ob- servation, Table 3 summarizes clinical and laboratory find- ings during the period of inpatient observation. The average duration of fever was greater in subjeets with dengue infection than in those with OFI, but there were no differences between sjects with DF and those with DHF. Shock occurs aroundND 1997176 (August) Table 2. Clinical features of study subjects at enty, ary Inictrs of Acute Dengue Hlness 37 bulated according to final diagnosis, Fina diagnosis Dengue Charstritic ont (wr + Duy br bur Sex, ME eas ss sar nna Ape, years «200, 51.0" 162) seo Duration of fever before ellen, days 10 (08) Ess) 1703) 909 Aono, 20-8) a0 63) S160 859° 283208) 2628 03) Nausea, 10.) 3910089) 59 68) 1931 @) 212875) Vomiting. no (6) switt (2) 26000) 2132 6), 2128 75) Aoinal pain no (%) 50108 (28) 239 8 932 2) texan Headache, 9.0) swi0s (67) us7 7 240 60) 2027 7 leding manifestations, no (5) one) 00 (13) 3320) 528 18) Tourniquet tes postive, 6. (0) "10 petshie2$ em 4210809) 3351 6594 W928 (0H 1523 (5) $320 pechige?§ em? 2u10s al) 1028.60) 1223 62) Platelet ant, 10 298 104) 208) 225 (106) Hematocrit % 3865) 373.8) a3) 382% WBC coun, cellu 11,120 6837) cant asaye 61352095) 272560) solute neuropil cou, cells S138 868) 473 (435)¢ 4380 2480) 457 2585) ‘Nolte manne can, els i. 324 458) 130 223" rig as) 13 Gory AST, UimL 4102) 76 68)" S709) on one ALT, Unt, sen 37 G0" 298) 4668) Abin 4o 410) 450) #0 NOTE, Pairwise compurions of clinical and labore dts were done axing x or Fishers ext test for propria data et for soniows dita We compare al sijets wth dengue infection Combined dengue fever [DF] and deus heretic fever [DHF gas sbjets with fhe fee ness (OF) and we compu subjects with DHF against subjects widh DE, Values ae expel as mean (SD) excep as nat. WBC, White Boel, AST, aspartate inoranafrce, ALT, alate amintanstonse P*=01 vs, OFI; +205 vx OF 05 vx 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 deferves- cence occurred (figure 2). There were statistically significant differences between subjects with dengue virus infection and those with OFT on fever days ~2 and ~1 (2 days and 1 day before defervescence, respectively) relative to a positive tourni- quet test, platelet count, total WBC and absolute neutrophil counts, and plasma levels of AST and ALT. A statistically significant decrease in platelet counts in subjects with DHF compared with those with DF was evident on fever day —1 ‘There was a small but statistically significant elevation in hema- tocrits in children with DHF compared with those with DF on fever day —2, which did not remain significant on fever day —1 Predictive value of elinical and laboratory observations We calculated the positive (PPV) and negative (NPV) pre- dictive values of parameters that were significantly different between subjects with dengue infection and those with OFI, based on the entry criteria and sample sizes in our study popula tion, PPV and NPV were calculated separately on the basis of Whether the finding was present at enrollment or whether it was present at any time on or before fever day —1, which represents at least | day before defervescence (table 4). We found the tourniquet test (=10 petechiae25 cm?) to have a high NPV at entry (0.79) and during hospitalization before defervescence (0.83) but low PPV (0.4 and 0.88, respectively) in this population. Total WBC count, absolute neutrophil count, and plasma AST level were slightly better discriminators of dengue vir infection than the tourniquet test. We also examined the PPV and NPV of plasma AST for discriminating DHF from DF and OFF, since AST levels were signiticanly higher in the subjects with DHF. With plasma AST levels from enrollment ony, the PPV and NPV for diagno- sSisof DHF of AST >40 Um. were 0.27 and 0.96, respectively, and the PPV and NPV of AST >60 Uiml wore 0.2 and 031, respectively. With all values on or before fever day —1, the PPV and NPV of AST >40 Ulm. were 0.31 and 1.00, respec- tively, and the PPV and NPV of AST >60 Uim. 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as Kalayanarj et al Table 3. Clinical features of study subjects during observation, tabulatd according to final {ND 1997;176 (Avent) Final diagnosis Dengue Charstritic on (or + Duy be bur Total dean of ever days 2H) 006 370g 4302) over day 2 “ournigust st postive, po. (4) ‘SIO patch’ on 433 «4 19130 (63) wary 713 64) 20 ptechige25 em? 43312) 1330 47 9117133) abc, Pale count, XU? 288 (105) 20 ut avant 14 (106) emir 3706) 3713) 36 way WHC coun, lp. 73927) ss20 2122)" 24s 2030) 311 @2ss) Aso newrphil oun, els 5999 G71) 3399 700)" s17 4473) 3590 2012) ‘Able monocyte count cell. 266 G40) 9897! 14200) 7497) AST, Wim 4618) sour! a0) 124466), ALT, Ui, 2700) Bus" sas) 5169) Fever day “1 N @ s x0 4 Tournigus et positive, 0.8) “10 petsshine2S ome 19154 05) 3036 83)¢ wan 14 03) 2920 petecie2.S em? Tost 9) 1936153)" 1122 (80) $147) Place coum, X10 iL 2213) 210 (209° 24s (104) 4 cosy? a7) se) 7a wa ‘WBC coun, cellu 9513 57) a6 2760) 4892 6091) 4631, 2348) Absoltenevtrpil count, cells 6333 740) 2619 S10)" 2698 1603) 25191812) Absolute monoeyte count, ellpL 227439) 91138) BB), 1204139), AST, Unk Bu) soar 2086) us (a ALT, Um, 2526) 9097" zen 4705) NOTE, Pairssecompurions of lncl an labore data were done axing x” or Fishers ext test for proprio dat and et for cons das {We compar all jet wth dengue infections combined dengue fever [DF] and dengue hemohaic fever [DHF] aga! mje wither re illness (FI and we compared subjects with DF against subjects with DHF. Vals ar expend as mean (SD) esse at ned, No of subjects dfs fom overall Study population because some subjcs were called alr inicaied fever ay WBC, Wh source. P*=01 vs, OFI; "= 05 vs, OF 05 vs. DF; 0 DF subjects with undifferentiated fever, close inpatient observation throughout the illness, and standardized observations using es- tablished case definitions. This permits a detailed comparison of clinical and laboratory features between children with docu- mented dengue virus infection and these with nondengue fe- brile 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, etal, unpub lished data). The major findings 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 see- ondary dengue virus infections; and that specific 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 nonbacteral febrile illnesses {in children enrolled in our study was associated with an acute bod call AST, ssarae-anibotutsfense: ALT, alanine dengue virus infection, We enrolled only children with fever for <72 h, and we excluded children with specific clinically identifiable infections, such as sinusitis, pharyngitis, or mea- sles, $0 we would anticipate thatthe percentage of all febrile illnesses caused by acute dengue virus infection is somewhat Tower than this figure. We also required the presence of facial flushing for enrollment in our study. An eatlier 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, ct al, unpublished data). The percentage of our study subjects with acute dengue virus infection also varied considerably from ‘month to month (figure 1). Thus, during periods of high inci- dence of dengue virus infections, the frequeney of dengue among undifferentiated febrile ilinesses would be expected to exceed the rate We observedND 1997176 (August) Im DHF SDF DOFI 40 35 25 15 10 # subjects enrolled 8 3 $543 240 Fever day Figure 2. No, of subjects enrolled, by final diagnosis and Fever day corresponding to day of enrollment. Final diagnoses were denave hemorzhagie fever (DHF), dengue fever (DF), of other, sondengve febrile illnesses (OFT), Fever day (1 was calendar day during which fever abated (ie, subjects enrolled on Tever day 0 defervesced within 24h of enollmen), Fow other studies are available describing the frequency of dengue in endemic settings: most studies have focused on de- fined outbreaks or have not reported the frequency of dengue infections among all febrile illnesses. Halstead et al. [24] re ported 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 fushed 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 ary Inictrs of Acute Dengue Hlness n9 land. Multiple dengue virus serotypes have cocirculated in these areas for several decades [1]. These results may be more widely applicable, however, sine the recent expansion of den- jue virus infections in other areas, sueh as Central and South America, has ereated similar conditions elsewhere [11] In this study, approximately half of the ill children with an acute dengue Virus infection developed DHF. This figure is much higher than reported in the previously cited study of ‘outpatients in Bangkok in 1962-1964 [24], but it is similar to the finding 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 infec- tions (12, 25]. We also performed detailed observations on all subjects, including decubitus chest radiographs; thus, we likely identified plasma leakage in some subjects that would not other- wise 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 influenced the frequency of DHF among subjects with acute dengue virus infection, but the similarity between our results and those of Burke etal. [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 and negative predictive values of selected clinical and laboratory’ observations for children with dengue virus infestion. Positive Nepaive Positive Negative Finding rodicive ve _prodicine value predictive value pri value Posie tought fest 0 pacchiae2 em? oa on ose ons 220 peeing 2 em! oe 075 oer 075 ast >40 UML 030 oss oot oss $0 Lim ost 075 079 on n000iuL ost oss ns9 os Absolute neutrophil count 7h. oe os oss 00 00 os ons ro on NOTE, AST, sprite aminotanterss: WHC, white blo! el 2 i20 Kalayanaro) etal infections fom other febrile illnesses at an early stage, before plasma leakage developed in patients with DHF. Such indica tors could be used to establish elinial algorthins for the identi- fication of high-risk patients for close monitoring. We found that a positive tourniquet test, leukopenia, neutropenia, mono- eytopenia, and elevations of plasma AST levels could be help- ful in this regard, “The tourniquet testi a simple clinical procedure that reflects capillary fragility and has been found to be positive in nearly all eases of DHF (5]. An eater study found that a positive toumiquet test combined with a flushed face and no coryza, which were used as enrollment criteria in that study, had sensitivity of 90%, a specificity of 97%%, and a positive pre- dictive 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 balf of the subjects with dengue virus infections had DHE, we found that the tourniquet test was often positive in dengue virus infection, but we also found that one-third of children with other, nonden- gue febrile illnesses had a positive toumiquet test, yielding lower specificity and a correspondingly lower PPV (table 4). Differences in the design of these studies, noted above, are likely to explain the differences in findings, Other characteris ties ofthe tourniquet test result, such as the size of petechine, 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 numberof atypical Iympho- eytes in our subjects with dengue virus infection was signi cantly higher than in subjects with OF] prior to fever day 0, the day of defervescence, so this finding 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 first to show that these elevations are clearly more common in the early stages of dengue infections than in nondengue febrile illnesses. In partic ular, anormal 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 thatthe clinical definition of DHF established by WHO [7] may not appropriately eatego- rize all patients. In particular, hemoconcentration may be blunted if there is early administration of intravenous fluids, if {ND 1997;176 (Avent) there is excessive bload loss due to hemorrhage, or if hemato- crit is not measured frequently. Because our study was orga- nized 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% inerease in hematocrit over the recovery value identified only 48% of cases classified as DHF. However, plasma leakage \was still identified in these cases, most often by the finding of «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 leakave and thrombocytopenia are critical features that distinguish DHF from DF [5]. At the same time, these data support the recom ‘mendation that other elinial findings besides hemoconcentra- tion 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 DI Acknowledgments We thank the Armed Forces Research Institute of Medical Sci= ences research nurses (Nathada Plavooth, Pranom Vangnai, Som- uk Lumjiak, Somalvadee Saravasee, Sumetha Hengpraset, and \Wipa Chawachalasai) for their dedication in data and blood eole tion: Somikiat Changnak, Choompun Manomuth, Aree No-no kai, Prance Saisang, Somsamai Tapana, Weerasak Yeepoo, and Songdej Sangst for technical assistance: Tipawan Kungvanrattana and Warinda Sriskham for data entry: Christine Kozik for auditing the clinical data; Chitehai Hemachasha for database management Pantipa Patanawin for interpretation of the chest radiographs; Rapin Snitbhan for coondinating specimen collection; Robert Lew for asistance with data analysis; and the patients and families who Participated inthis study 1 Halen SI, The XX contr dengue pandemic: ned ie srilnce ‘nd eescash, World Heskh Stat 1992:45292-, 2. 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