Suchitra Nimmannitya

Dengue Viremia Titer, Antibody Response Pattern, and Virus Serotype Correlate with Disease Severity

Viremia titers in serial plasma samples from 168 children with acute dengue virus infection who were enrolled in a prospective study at 2 hospitals in Thailand were examined to determine the role of virus load in the pathogenesis of dengue hemorrhagic fever (DHF). The infecting virus serotype was identified for 165 patients (DEN-1, 46 patients; DEN-2, 47 patients; DEN-3, 47 patients, DEN-4, 25 patients). Patients with DEN-2 infections experienced more severe disease than those infected with other serotypes. Eighty-one percent of patients experienced a secondary dengue virus infection that was associated with more severe disease. Viremia titers were determined for 41 DEN-1 and 46 DEN-2 patients. Higher peak titers were associated with increased disease severity for the 31 patients with a peak titer identified (mean titer of 107.6 for those with dengue fever vs. 108.5 for patients with DHF, P=.01). Increased dengue disease severity correlated with high viremia titer, secondary dengue virus infection, and DEN-2 virus type.

Early Clinical and Laboratory Indicators of Acute Dengue Illness

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.

Dengue in the Early Febrile Phase: Viremia and Antibody Responses

A multicenter effort was begun in 1994 to characterize the pathophysiology of dengue using a study design that minimized patient selection bias by offering enrollment to all children with undifferentiated fever for <72 h. In the first year, 189 children were enrolled (age range, 8 months to 14 years). Thirty-two percent of these children had dengue infections (60 volunteers). The percentage of children with a secondary dengue infection was 93%, with only 4 (7%) having a primary dengue infection. The virus isolation rate from the plasma of children with dengue was 98%. Viremia correlated highly with temperature. All four dengue virus serotypes were isolated at both study sites. This study demonstrates that all four serotypes of dengue virus can cause dengue hemorrhagic fever, that all dengue patients as defined by serology experience viremia during the febrile phase, and that as fever subsides, so does viremia.

Early Immune Activation in Acute Dengue Illness Is Related to Development of Plasma Leakage and Disease Severity

T lymphocyte activation and increased cytokine levels have been described in retrospective studies of children presenting with dengue hemorrhagic fever (DHF). Serial plasma samples obtained in a prospective study of Thai children presenting with <72 h of fever were studied. Plasma levels of 80-kDa soluble tumor necrosis factor receptors (sTNFRs) were higher in children who developed DHF than in those with dengue fever (DF) or other nondengue febrile illnesses (OFIs) and were correlated with the degree of subsequent plasma leakage. Soluble CD8 and soluble interleukin-2 receptor levels were also elevated in children with DHF compared with those with DF. Interferon-gamma and sTNFR 60-kDa levels were higher in children with dengue than in those with OFIs. TNF-alpha was detectable more often in DHF than in DF or OFIs (P<.05). These results support the hypothesis that immune activation contributes to the pathogenesis of DHF. Further studies evaluating the predictive value of sTNFR80 for DHF are warranted.

Activation of T lymphocytes in dengue virus infections. High levels of soluble interleukin 2 receptor, soluble CD4, soluble CD8, interleukin 2, and interferon-gamma in sera of children with dengue.

It has been reported that the severe complication of dengue virus infection, dengue hemorrhagic fever (DHF) is much more commonly observed during secondary dengue virus infections than primary infections. In order to elucidate the role of T lymphocytes in the pathogenesis of DHF, we attempted to determine whether T lymphocytes are activated in vivo during dengue virus infections, by examining the levels of soluble IL-2 receptor (sIL-2R), soluble CD4 (sCD4), soluble CD8 (sCD8), interleukin-2 (IL-2) and interferon-gamma (IFN gamma) in the sera of 59 patients with DHF and 41 patients with dengue fever (DF). The levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma were significantly higher in the acute sera of patients with DHF than in the sera of healthy children (P less than 0.001 for all markers). The acute sera of patients with DF contained higher levels of sIL-2R, sCD4, IL-2, and IFN gamma than the sera of healthy children (P less than 0.001 for sIL-2R, IL-2, and IFN gamma; P less than 0.05 for sCD4), but did not have elevated levels of sCD8. The levels of sIL-2R (P less than 0.05), sCD4 (P less than 0.001), and sCD8 (P less than 0.001) were higher in DHF than in DF on days 3-4 after the onset of fever. The levels of IL-2 and IFN gamma in patients with DHF were highest 1 d before defervescence. There were no significant differences in the levels of sIL-2R, sCD4, sCD8, IL-2, and IFN gamma among grades 1, 2, and 3 of DHF. These results indicate (a) T lymphocytes are activated and produce IL-2 and IFN gamma in vivo during DHF and DF, (b) CD4+ T lymphocytes are activated in DHF and DF, and the level of activation is higher in DHF than in DF, and (c) activation of CD8+ T lymphocytes is evident in DHF, but not in DF.

Cross-protective immunity can account for the alternating epidemic pattern of dengue virus serotypes circulating in Bangkok

Dengue virus, the causative agent of dengue fever and its more serious manifestation dengue hemorrhagic fever, is widespread throughout tropical and subtropical regions. The virus exists as four distinct serotypes, all of which have cocirculated in Bangkok for several decades with epidemic outbreaks occurring every 8–10 years. We analyze time-series data of monthly infection incidence, revealing a distinctive pattern with epidemics of serotypes 1, 2, and 3 occurring at approximately the same time and an isolated epidemic of serotype 4 occurring in the intervening years. Phylogenetic analysis of virus samples collected over the same period shows that clade replacement events are linked to the epidemic cycle and indicates that there is an interserotypic immune reaction. Using an epidemic model with stochastic seasonal forcing showing 8- to 10-year epidemic oscillations, we demonstrate that moderate cross-protective immunity gives rise to persistent out-of-phase oscillations similar to those observed in the data, but that strong or weak cross-protection or cross-enhancement only produces in-phase patterns. This behavior suggests that the epidemic pattern observed in Bangkok is the result of cross-protective immunity and may be significantly altered by changes in the interserotypic immune reaction.

Early CD69 Expression on Peripheral Blood Lymphocytes from Children with Dengue Hemorrhagic Fever

Recent reports have demonstrated immune activation in dengue hemorrhagic fever (DHF) by cytokine and soluble receptor detection in blood. The goal of this study was to determine which cell types are activated and likely to be responsible for cytokine production. Whole blood specimens from 51 Thai children presenting within 72 h of fever onset and with detectable plasma dengue viral RNA were studied by flow cytometry. Absolute CD4 T cell, CD8 T cell, NK cell, and gammadelta T cell counts were decreased in children with DHF compared with those with dengue fever (DF) early in the course of illness. The percent of cells expressing CD69 was increased on CD8 T cells and NK cells in children who developed DHF more than in those with DF. These data directly demonstrate that cellular immune activation is present early in acute dengue and is related to disease severity.

Clade Replacements in Dengue Virus Serotypes 1 and 3 Are Associated with Changing Serotype Prevalence

ABSTRACT The evolution of dengue virus (DENV) is characterized by phylogenetic trees that have a strong temporal structure punctuated by dramatic changes in clade frequency. To determine the cause of these large-scale phylogenetic patterns, we examined the evolutionary history of DENV serotype 1 (DENV-1) and DENV-3 in Thailand, where gene sequence and epidemiological data are relatively abundant over a 30-year period. We found evidence for the turnover of viral clades in both serotypes, most notably in DENV-1, where a major clade replacement event took place in genotype I during the mid-1990s. Further, when this clade replacement event was placed in the context of changes in serotype prevalence in Thailand, a striking pattern emerged; an increase in DENV-1 clade diversity was associated with an increase in the abundance of this serotype and a concomitant decrease in DENV-4 prevalence, while clade replacement was associated with a decline in DENV-1 prevalence and a rise of DENV-4. We postulate that intraserotypic genetic diversification proceeds at times of relative serotype abundance and that replacement events can result from differential susceptibility to cross-reactive immune responses.

Elevated plasma interleukin-10 levels in acute dengue correlate with disease severity

Dengue viruses, of which there are four serotypes,are the most important arthropod-borne viral infec-tions in the world, accounting for more than 250,000cases of dengue hemorrhagic fever (DHF) and 10,000deaths annually [Monath, 1994]. Infection with dengueviruses can yield different clinical syndromes, includ-ing (1) undifferentiated febrile illness, seen more com-monly in children; (2) dengue fever (DF), a flu-like syn-drome characterized by high fever, headache, retro-orbital pain, myalgias, abdominal pain, nausea, andvomiting; and (3) dengue hemorrhagic fever (DHF), aplasma leak syndrome that, in its most severe form,can be life-threatening [Nimmannitya, 1987].Plasma leakage is a major clinical feature of DHFand tends to occur around the time of defervescence.We have been interested in the events that precede theperiod of plasma leakage to better define its etiology.We have found that dengue virus-specific CD4

Dengue Hemorrhagic Fever in Infants: Research Opportunities Ignored

The age distribution of cases of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS) in infants under the age of 1 year are reported from Bangkok, Thailand, and for the first time for Ho Chi Minh City, Vietnam; Yangon, Myanmar; and Surabaya, Indonesia. The four dengue viruses were isolated from Thai infants, all of whom were having a primary dengue infection. Progress studying the immunologically distinct infant DHF/DSS has been limited; most contemporary research has centered on DHF/DSS accompanying secondary dengue infections. In designing research results obtained in studies on a congruent animal model, feline infectious peritonitis virus (FIPV) infections of kittens born to FIPV-immune queens should be considered. Research challenges presented by infant DHF/DSS are discussed.