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Summary Background The ability to accurately predict early progression of dengue to severe disease is crucial for patient triage and clinical management. Previous systematic reviews and meta-analyses have found significant heterogeneity in predictors of severe disease due to large variation in these factors during the time course of the illness. We aimed to identify factors associated with progression to severe dengue disease that are detectable specifically in the febrile phase. Methods We did a systematic review and meta-analysis to identify predictors identifiable during the febrile phase associated with progression to severe disease defined according to WHO criteria. Eight medical databases were searched for studies published from Jan 1, 1997, to Jan 31, 2020. Original clinical studies in English assessing the association of factors detected during the febrile phase with progression to severe dengue were selected and assessed by three reviewers, with discrepancies resolved by consensus. Meta-analyses were done using random-effects models to estimate pooled effect sizes. Only predictors reported in at least four studies were included in the meta-analyses. Heterogeneity was assessed using the Cochrane Q and I2 statistics, and publication bias was assessed by Egger's test. We did subgroup analyses of studies with children and adults. The study is registered with PROSPERO, CRD42018093363. Findings Of 6643 studies identified, 150 articles were included in the systematic review, and 122 articles comprising 25 potential predictors were included in the meta-analyses. Female patients had a higher risk of severe dengue than male patients in the main analysis (2674 [16·2%] of 16 481 vs 3052 [10·5%] of 29 142; odds ratio [OR] 1·13 [95% CI 1·01–1·26) but not in the subgroup analysis of studies with children. Pre-existing comorbidities associated with severe disease were diabetes (135 [31·3%] of 431 with vs 868 [16·0%] of 5421 without; crude OR 4·38 [2·58–7·43]), hypertension (240 [35·0%] of 685 vs 763 [20·6%] of 3695; 2·19 [1·36–3·53]), renal disease (44 [45·8%] of 96 vs 271 [16·0%] of 1690; 4·67 [2·21–9·88]), and cardiovascular disease (nine [23·1%] of 39 vs 155 [8·6%] of 1793; 2·79 [1·04–7·50]). Clinical features during the febrile phase associated with progression to severe disease were vomiting (329 [13·5%] of 2432 with vs 258 [6·8%] of 3797 without; 2·25 [1·87–2·71]), abdominal pain and tenderness (321 [17·7%] of 1814 vs 435 [8·1%] of 5357; 1·92 [1·35–2·74]), spontaneous or mucosal bleeding (147 [17·9%] of 822 vs 676 [10·8%] of 6235; 1·57 [1·13–2·19]), and the presence of clinical fluid accumulation (40 [42·1%] of 95 vs 212 [14·9%] of 1425; 4·61 [2·29–9·26]). During the first 4 days of illness, platelet count was lower (standardised mean difference −0·34 [95% CI −0·54 to −0·15]), serum albumin was lower (−0·5 [–0·86 to −0·15]), and aminotransferase concentrations were higher (aspartate aminotransferase [AST] 1·06 [0·54 to 1·57] and alanine aminotransferase [ALT] 0·73 [0·36 to 1·09]) among individuals who progressed to severe disease. Dengue virus serotype 2 was associated with severe disease in children. Secondary infections (vs primary infections) were also associated with severe disease (1682 [11·8%] of 14 252 with vs 507 [5·2%] of 9660 without; OR 2·26 [95% CI 1·65–3·09]). Although the included studies had a moderate to high risk of bias in terms of study confounding, the risk of bias was low to moderate in other domains. Heterogeneity of the pooled results varied from low to high on different factors. Interpretation This analysis supports monitoring of the warning signs described in the 2009 WHO guidelines on dengue. In addition, testing for infecting serotype and monitoring platelet count and serum albumin, AST, and ALT concentrations during the febrile phase of illness could improve the early prediction of severe dengue. Funding Wellcome Trust, National Institute for Health Research, Collaborative Project to Increase Production of Rural Doctors, and Royal Thai Government.

Malaria fever is extreme overall wellbeing face an issue. Malaria fever keeps on being a peril to the ascendant nations. Epidemiological data from various territories of Pakistan is missing to intently appraise the event of various sorts of intestinal sickness.

Background Major histocompatibility complex class I chain–related (MIC) A and B (MICA and MICB) are polymorphic stress molecules recognized by natural killer cells. This study was performed to analyze MIC gene profiles in hospitalized Thai children with acute dengue illness. Methods MIC allele profiles were determined in a discovery cohort of patients with dengue fever or dengue hemorrhagic fever (DHF) (n = 166) and controls (n = 149). A replication cohort of patients with dengue (n = 222) was used to confirm specific MICB associations with disease. Results MICA*045 and MICB*004 associated with susceptibility to DHF in secondary dengue virus (DENV) infections (odds ratio [OR], 3.22; [95% confidence interval (CI), 1.18–8.84] and 1.99 [1.07–2.13], respectively), and MICB*002 with protection from DHF in secondary DENV infections (OR, 0.41; 95% CI, .21–.68). The protective effect of MICB*002 against secondary DHF was confirmed in the replication cohort (OR, 0.43; 95% CI, .22–.82) and was stronger when MICB*002 is present in individuals also carrying HLA-B*18, B*40, and B*44 alleles which form the B44 supertype of functionally related alleles (0.29, 95% CI, .14–.60). Conclusions Given that MICB*002 is a low expresser of soluble proteins, these data indicate that surface expression of MICB*002 with B44 supertype alleles on DENV-infected cells confer a protective advantage in controlling DENV infection using natural killer cells.

Without an effective vaccine or specific treatment for dengue, the ability to accurately predict early progression to severe disease relies on patient triage and clinical management. Previous systematic reviews and meta-analyses have found significant heterogeneity in predictors associated with severe disease, due to large variations of these factors during the time-course of the illness. Our study aimed to identify the factors associated with progression to severe dengue disease, which are detectable specifically in the febrile phase. We conducted a systematic review and meta-analysis to identify predictors associated with disease progression identifiable during the febrile phase. Eight medical databases were searched for studies published from January 1997 to February 2018. Relevant studies were selected and assessed by three reviewers with discrepancies resolved by consensus. Meta-analyses were performed using random-effects models to estimate pooled effect sizes. Heterogeneity and publication bias were also assessed. Of 5,293 studies identified, 98 were included in the meta-analyses. The presence of pre-existing comorbidities (diabetes mellitus, hypertension and renal disease) were associated with severe dengue. Vomiting, abdominal pain and tenderness, spontaneous and mucosal bleeding, and clinical fluid accumulation were clinical features associated with progression to severe disease. During the first four days of illness, platelet count, and serum albumin were lower among individuals who progressed to severe disease whilst aminotransferase levels were higher. Dengue virus serotype 2 and secondary infection were also associated with severe disease. This analysis supports the monitoring of the warning signs described in the 2009 WHO guidelines. In addition, testing for infecting serotype and monitoring platelet count, serum albumin, AST and ALT during the febrile phase of illness would improve the early prediction of severe dengue. Funding Statement: The authors state: "There was no funding source for this study." SS acknowledges funding from the Collaborative Project to Increase Production of Rural Doctor and Royal Thai Government Scholarship. ID ackowledges research funding from an Imperial College Junior Research Fellowship, a Wellcome Trust Sir Henry Dale Fellowship, Janssen Pharmaceutica and joint Centre funding from the UK Medical Research Council and Department for International Development. AH is a National Institute for Health Research (NIHR) Senior Investigator. She also acknowledges the support of the NIHR Health Protection Research Unit in Healthcare Associated Infections and Antimicrobial Resistance at Imperial College London in partnership with Public Health England (PHE), in collaboration with The Sanger Institute, the University of Cambridge Veterinary School and Imperial College Health Partners Declaration of Interests: The authors declare no competing interests. Ethics Approval Statement: We followed the protocol described in the Preferred Reporting Items for Systematic Reviews and Meta-analyses for Protocols (PRISMA-P) guidelines and the steps discussed in the guide to systematic review and meta-analysis of prognostic factor studies by Riley et al . Our protocol was registered with PROSPERO (CRD42018093363).

BACKGROUND: Follicular helper T cells (T(FH)) are specialized CD4 T cells required for B-cell help and antibody production. METHODS: Given the postulated role of immune activation in dengue disease, we measured the expansion and activation of T(FH) in the circulation (peripheral T(FH) [pT(FH)]) collected from Thai children with laboratory-confirmed acute dengue virus (DENV) infection. RESULTS: We found significant expansion and activation of pT(FH) subsets during acute infection with the highest frequencies of activated pT(FH) (PD1(hi) pT(FH) and PD1(+)CD38(+) pT(FH)) detected during the critical phase of illness. Numbers of activated pT(FH) were higher in patients with secondary compared with primary infections and in patients with more severe disease. We also found a positive correlation between the frequencies of activated pT(FH) and the frequencies of plasmablasts. CONCLUSIONS: To our knowledge, this is the first ex vivo analysis of pT(FH) activation during acute DENV infection. Overall, our study supports the model that pT(FH) contribute to disease evolution during the critical stage of illness.

BACKGROUND Human leukocyte antigen (HLA) supertypes are groups of functionally related alleles that present structurally similar antigens to the immune system. OBJECTIVES To analyze HLA class I supertype associations with clinical outcome in hospitalized Thai children with acute dengue illness. METHODS Seven hundred sixty-two patients and population-matched controls recruited predominantly in Bangkok were HLA-A and -B typed. HLA supertype frequencies were compared and tested for significant dengue disease associations using logistic regression analyses. Multivariable models were built by conducting forward stepwise selection procedures. RESULTS In the final logistic regression model, the HLA-B44 supertype was protective against dengue hemorrhagic fever (DHF) in secondary infections (odds ratio [OR] = 0.46, 95% confidence interval [CI], .30-.72), while the HLA-A02 supertype (OR = 1.92, 95% CI, 1.30-2.83) and the HLA-A01/03 supertype (OR = 3.01, 95% CI, 1.01-8.92) were associated with susceptibility to secondary dengue fever. The B07 supertype was associated with susceptibility to secondary DHF in the univariate analysis (OR = 1.60, 95% CI, 1.05-2.46), whereas that was not retained in the final model. CONCLUSIONS As the HLA-B44 supertype is predicted to target conserved epitopes in dengue, our results suggest that B44 supertype-restricted immune responses to highly conserved regions of the dengue proteome may protect against secondary DHF.

Dengue causes more human morbidity globally than any other vector-borne viral disease. Recent research has led to improved epidemiological methods that predict disease burden and factors involved in transmission, a better understanding of immune responses in infection, and enhanced animal models. In addition, a number of control measures, including preventative vaccines, are in clinical trials. However, significant gaps remain, including the need for better surveillance in large parts of the world, methods to predict which individuals will develop severe disease, and immunologic correlates of protection against dengue illness. During the next decade, dengue will likely expand its geographic reach and become an increasing burden on health resources in affected areas. Licensed vaccines and antiviral agents are needed in order to effectively control dengue and limit disease.

Background: AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA. Objective: To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance. Study design: A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011–2013. Results and conclusion: The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.

A fundamental mystery for dengue and other infectious pathogens is how observed patterns of cases relate to actual chains of individual transmission events. These pathways are intimately tied to the mechanisms by which strains interact and compete across spatial scales. Phylogeographic methods have been used to characterize pathogen dispersal at global and regional scales but have yielded few insights into the local spatiotemporal structure of endemic transmission. Using geolocated genotype (800 cases) and serotype (17,291 cases) data, we show that in Bangkok, Thailand, 60% of dengue cases living200 meters apart come from the same transmission chain, as opposed to 3% of cases separated by 1 to 5 kilometers. At distances200 meters from a case (encompassing an average of 1300 people in Bangkok), the effective number of chains is 1.7. This number rises by a factor of 7 for each 10-fold increase in the population of the "enclosed" region. This trend is observed regardless of whether population density or area increases, though increases in density over 7000 people per square kilometer do not lead to additional chains. Within Thailand these chains quickly mix, and by the next dengue season viral lineages are no longer highly spatially structured within the country. In contrast, viral flow to neighboring countries is limited. These findings are consistent with local, density-dependent transmission and implicate densely populated communities as key sources of viral diversity, with home location the focal point of transmission. These findings have important implications for targeted vector control and active surveillance.

The global burden of dengue and its geographic distribution have increased over the past several decades. The introduction of dengue in new areas has often been accompanied by high case-fatality rates. Drawing on the experience in managing dengue cases at the Queen Sirikit National Institute of Child Health in Bangkok, Thailand, this article provides the authors' perspectives on key clinical lessons to improve dengue-related outcomes. Parallels between this clinical experience and outcomes reported in randomized controlled trials, results of efforts to disseminate practice recommendations, and suggestions for areas for further research are also discussed.

Background Ho Chi Minh City and Bangkok are highly dengue endemic. The extent to which disease patterns are attributable to local versus regional dynamics remains unclear. To address this gap we compared key transmission parameters across the locations. Methods and Principal Findings We used 2003–2009 age-stratified case data to inform catalytic transmission models. Further, we compared the spatial clustering of serotypes within each city. We found that annual case numbers were highly consistent across the two cities (correlation of 0.77, 95% CI: 0.74–0.79) as was the annual force of infection (correlation of 0.57, 95% CI: 0.46–0.68). Serotypes were less similar with serotype-specific correlations ranging from 0.65 for DENV1 to -0.14 for DENV4. Significant spatial clustering of serotypes was observed in HCMC at distances, Author Summary All four serotypes of dengue have circulated endemically throughout Southeast Asia for decades. However, despite the enormous burden of disease, there remains poor understanding of the similarity in disease patterns across the region. We analyzed data from over 100,000 cases of dengue from two of the largest cities in the region, Bangkok and Ho Chi Minh City between 2001 and 2009. We use basic statistical methods to reconstruct the annual probability of infection in the two cities during this time period using methods that are robust to differences in reporting mechanisms. We find that both the epidemic curves and annual probabilities of infection were highly correlated across the cities, however, serotype-specific correlations were far more variable. Finally, we used geocoded case homes from Ho Chi Minh to demonstrate that cases in the city clustered at spatial scales (

The immune response to dengue virus (DENV) infection is complex and not fully understood. Using longitudinal data from 181 children with dengue in Thailand who were followed for up to 3 years, we describe neutralizing antibody kinetics following symptomatic DENV infection. We observed that antibody titers varied by serotype, homotypic vs heterotypic responses, and primary versus postprimary infections. The rates of change in antibody titers over time varied between primary and postprimary responses. For primary infections, titers increased from convalescence to 6 months. By comparing homotypic and heterotypic antibody titers, we saw an increase in type specificity from convalescence to 6 months for primary DENV3 infections but not primary DENV1 infections. In postprimary cases, there was a decrease in titers from convalescence up until 6 months after infection. Beginning 1 year after both primary and postprimary infections, there was evidence of increasing antibody titers, with greater increases in children with lower titers, suggesting that antibody titers were boosted due to infection and that higher levels of neutralizing antibody may be more likely to confer a sterilizing immune response. These findings may help to model virus transmission dynamics and provide baseline data to support the development of vaccines and therapeutics.

Dengue is one of the most important mosquito-borne viral illnesses. The first DHF outbreak was reported from the Philippines in 1953. Initially it was endemic only in Southeast Asia and the Western Pacific regions. After about 50 years from the first outbreak, it spread globally to almost every continent including North and South America, Australia and Africa. The majority of cases during the 50s to 80s were children, but today the disease affects both children and adults of all age groups. The disease is caused by dengue viruses that have four serotypes: dengue 1, dengue 2, dengue 3 and dengue 4. Primary infection usually results in milder illness, while more severe disease occurs in cases of repeated infection with different serotypes. In this paper clinical manifestations and management of dengue/DHF/DSS are summarized.

Dengue virus infection causes a spectrum of clinical manifestations, usually classified according to the World Health Organization (WHO) guidelines into dengue fever (DF) and dengue hemorrhagic fever (DHF). The ability of these guidelines to categorize severe dengue illness has recently been questioned.We evaluated dengue case definitions in a prospective study at a pediatric hospital in Bangkok, Thailand, during 1994-2005. One thousand thirteen children were enrolled within the first 3 days after onset of fever and observed with standardized data collection. Cases were classified on the basis of application of the strict WHO criteria. All dengue virus infections were laboratory confirmed. We retrospectively grouped patients on the basis of whether they received significant intervention based on fluid replacement and/or requirements for blood transfusion.Eighty-five (58%) of 150 persons with DHF, 40 (15%) of 264 with DF, and 73 (12%) of 599 with other febrile illnesses (OFIs) received significant intervention. Sixty-eight percent of dengue cases requiring intervention met strict WHO criteria for DHF. In contrast, only 1% of OFI cases met WHO criteria for DHF. Plasma leakage and thrombocytopenia were the 2 components contributing to the specificity of the WHO case definition and identified dengue cases that required intervention. Hemorrhagic tendency did not reliably differentiate DF and DHF. In DF cases, thrombocytopenia and bleeding were associated with severity.Dengue illness is heterogeneous in severity, and severe clinical features occurred in patients whose cases were not characterized as DHF. The WHO case definition of DHF demonstrated sensitivity of 62% and specificity of 92% for identification of dengue illness requiring intervention, without the need for laboratory confirmation of dengue virus infection, in an area of endemicity.

Severe dengue virus (DENV) infection is characterized by a cascade of cytokine production, including the production of tumor necrosis factor-alpha (TNF-alpha) and lymphotoxin-alpha (LT-alpha). We have analyzed a variety of polymorphisms in the TNF and LTA genes of 435 ethnic Thais who had subclinical DENV infection, primary or secondary dengue fever (DF), or primary or secondary dengue hemorrhagic fever (DHF). The TNF -238A polymorphism marking the TNF-4,LTA-3 haplotype occurred in a significantly greater number of patients with secondary DHF (20 [15.2%] of 132) than patients with secondary DF (7 [4.1%] of 169) (P.001; P corrected by use of Bonferroni adjustment, .022; odds ratio, 4.13 [95% confidence interval, 1.59-11.17]). In a subset of patients, the LTA-3 haplotype was associated with in vivo intracellular production of LT-alpha and TNF-alpha during the acute viremic phase of infection. Two extended human major histocompatibility complex (MHC) haplotypes containing TNF-4 and LTA-3, together with HLA-B48, HLA-B57, and HLA-DPB1*0501, were detected only in patients with secondary DHF. These observations indicate that polymorphism in functionally distinct MHC-encoded proteins contributes to the risk of developing severe secondary DENV infection and warrants further investigation.

In response to a January 2005 report of dengue hemorrhagic fever (DHF) in Timor Leste, the World Health Organization sent a team to assist the National Hospital Guido Valadares (NHGV) in Dili with clinical case management and diagnostic support. The hospital reported 67 admissions including 8 deaths (case fatality rate approximately 12%) over the previous weeks with case histories clinically compatible with DHF. During the intervention, an additional 44 suspected dengue patients were admitted to the pediatric ward of NHGV. Among 41 patients with clinical diagnoses of dengue fever or DHF, 38 (93%) were laboratory confirmed. Although cause and effect cannot be definitely attributed, the case fatality rate decreased to 3.6% after the intervention with education about dengue management strategies.

Dengue is the most rapidly advancing vector-borne disease with an estimated 50 million dengue infections occurring annually. As a result of major demographic changes rapid urbanization on a massive scale global travel and environmental change the world - particularly the tropical world - faces enormous challenges from emerging infectious diseases. Dengue epitomizes these challenges. In the early years of the 21st century we are collectively failing to meet the threat posed by dengue as the disease spreads unabated and almost 40% of the worlds population now live at risk of contracting it. Because of the rapidly increasing public health importance of dengue the 2002 World Health Assembly Resolution (WHA55.17) urged greater commitment among Member States and WHO to dengue control; of particular significance is the 2005 Revision of the International Health Regulations (WHA58.3) which includes dengue fever as an example of a disease that may constitute an international public health emergency. (excerpt)

Why certain individuals progress to severe dengue disease is unknown. In this study, blood groups associated with dengue disease were investigated. ABO phenotypes were identified by use of serum from 399 patients with dengue-virus infection who participated in a cohort study. ABO blood-group frequencies were similar in primary versus secondary dengue-virus infections. However, in secondary infection, individuals with blood group AB were likely to have dengue hemorrhagic fever grade 3 than either grades 1 and 2 combined (corrected P value

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 classified 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 significant 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.

Background Infants born to dengue immune mothers acquire maternal antibodies to dengue. These antibodies, though initially protective, decline during the first year of life to levels thought to be disease enhancing, before reaching undetectable levels. Infants have long been studied to understand the interaction between infection and disease on an individual level. Methods/Findings Considering infants (cases, Author Summary Infants born to dengue immune mothers acquire maternal dengue antibodies. These antibodies, though initially protective, decline during the first year of life to levels thought to be disease enhancing, before reaching undetectable levels. We show that in this population, DENV-2 and DENV-4 almost exclusively cause disease in the presence of dengue antibody, despite infections occurring in others. We also observe serotype-specificity in the mean age of infant cases, consistent with differential waning of antibody to each serotype. These results highlight serotype-specificity in the way the immune response interacts with infection to cause disease. In addition, we show that the mean age of infant cases over time is informative about transmission in the whole population. Therefore, ongoing surveillance for dengue in infants could provide useful insights into dengue epidemiology, particularly after the introduction of a dengue vaccine targeting adults and older children.

Long-term observational studies can provide valuable insights into overall dengue epidemiology. Here, we present analysis of dengue cases at a pediatric hospital in Bangkok, Thailand, during a 40-year period from 1973 to 2012. Data were analyzed from 25,715 hospitalized patients with laboratory-confirmed dengue virus (DENV) infection. Several long-term trends in dengue disease were identified including an increase in mean age of hospitalized cases from an average of 7-8 years, an increase after 1990 in the proportion of post-primary cases for DENV-1 and DENV-3, and a decrease in the proportion of dengue hemorrhagic fever and dengue shock syndrome cases in primary and post-primary cases over time. Exploratory mechanistic analysis of these observed trends considered changes in diagnostic methods, demography, force of infection, and Japanese encephalitis vaccination as possible explanations. Thailand is an important setting for studying DENV transmission as it has a "mature" dengue epidemiology with a strong surveillance system in place since the early 1970s. We characterized changes in dengue epidemiology over four decades, and possible impact of demographic and other changes in the human population. These results may inform other countries where similar changes in transmission and population demographics may now or may soon be occurring.

A comparative analysis was performed of the 3′-untranslated region (UTR) of Dengue virus (DENV) sampled from Bangkok, Thailand, over a 30 year period and representing all four serotypes. Considerable genetic variation was observed both within and among serotypes. Notably, a full-length version of the critical 3′-long stable hairpin structure was absent from some isolates, suggesting the occurrence of complex structural interactions within the 3′-UTR, including the influence of upstream mutations. The Thai sequences were then combined with 61 globally sampled isolates of DENV taken from patients with either dengue fever or severe dengue disease. No consistent association was found between 3′-UTR secondary structure and the clinical outcome of DENV infection, although some evidence for a trend in this direction was observed in DENV-2. It was concluded that the 3′-UTR is not the sole determinant of DENV virulence in nature, although variation in secondary structure may greatly influence viral fitness.

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.

Dengue virus circulation and association with epidemics and severe dengue disease were studied in hospitalized children with suspected dengue at the Queen Sirikit National Institute of Child Health in Bangkok, Thailand, from 1973 to 1999. Dengue serology was performed on all patients and viral isolation attempted on laboratory-confirmed patients. Acute dengue was diagnosed in 15,569 children and virus isolated from 4,846. DEN-3 was the most frequent serotype in primary dengue (49% of all isolates), DEN-2 in secondary and in dengue hemorrhagic fever (37% and 35%, respectively). The predominant dengue serotype varied by year: DEN-1 from 1990-92, DEN-2 from 1973-86 and 1988-89; DEN-3 in 1987 and 1995-99; and DEN-4 from 1993-94. Only DEN-3 was associated with severe outbreak years. Our findings illustrate the uniqueness of each serotype in producing epidemics and severe disease and underscore the importance of long-term surveillance of dengue serotypes in understanding the epidemiology of these viruses.

Background The effect of prior dengue virus (DENV) exposure on subsequent heterologous infection can be beneficial or detrimental depending on many factors including timing of infection. We sought to evaluate this effect by examining a large database of DENV infections captured by both active and passive surveillance encompassing a wide clinical spectrum of disease. Methods We evaluated datasets from 17 years of hospital-based passive surveillance and nine years of cohort studies, including clinical and subclinical DENV infections, to assess the outcomes of sequential heterologous infections. Chi square or Fisher’s exact test was used to compare proportions of infection outcomes such as disease severity; ANOVA was used for continuous variables. Multivariate logistic regression was used to assess risk factors for infection outcomes. Results Of 38,740 DENV infections, two or more infections were detected in 502 individuals; 14 had three infections. The mean ages at the time of the first and second detected infections were 7.6 ± 3.0 and 11.2 ± 3.0 years. The shortest time between sequential infections was 66 days. A longer time interval between sequential infections was associated with dengue hemorrhagic fever (DHF) in the second detected infection (OR 1.3, 95% CI 1.2-1.4). All possible sequential serotype pairs were observed among 201 subjects with DHF at the second detected infection, except DENV-4 followed by DENV-3. Among DENV infections detected in cohort subjects by active study surveillance and subsequent non-study hospital-based passive surveillance, hospitalization at the first detected infection increased the likelihood of hospitalization at the second detected infection. Conclusions Increasing time between sequential DENV infections was associated with greater severity of the second detected infection, supporting the role of heterotypic immunity in both protection and enhancement. Hospitalization was positively associated between the first and second detected infections, suggesting a possible predisposition in some individuals to more severe dengue disease. Electronic supplementary material The online version of this article (doi:10.1186/s12889-015-1590-z) contains supplementary material, which is available to authorized users.

Little is known of the role of classical HLA-A and -B class I alleles in determining resistance, susceptibility, or the severity of acute viral infections. Appropriate paradigms for immunogenetic studies of acute viral infections are dengue fever (DF) and dengue hemorrhagic fever (DHF). Both primary and secondary infections with dengue virus (DEN) serotypes 1, 2, 3 or 4, can result in either clinically less severe DF or the more severe DHF. In secondary exposures, a memory response is induced in immunologically primed individuals, which can both clear the infecting dengue virus and contribute to its pathology. In a case-control study of 263 ethnic Thai patients infected with either DEN-1, -2, -3 or -4, we detected HLA class I associations with secondary infections, but not in immunologically naive patients with primary infections. HLA-A*0203 was associated with the less severe DF, regardless of the secondary infecting virus serotype. By contrast, HLA-A*0207 was associated with susceptibility to the more severe DHF in patients with secondary DEN-1 and DEN-2 infections only. Conversely, HLA-B*51 was associated with the development of DHF in patients with secondary infections, and HLA-B*52 was associated with DF in patients with secondary DEN-1 and DEN-2 infections. Moreover, HLA-B44, B62, B76 and B77 also appeared to be protective against developing clinical disease after secondary dengue virus infection. These results confirm that classical HLA class I alleles are associated with the clinical outcome of exposure to dengue virus, in previously exposed and immunologically primed individuals.

Dengue haemorrhagic fever (DHF), the most severe form of illness following infection with a dengue virus, is characterized by plasma leakage and a period of increased microvascular permeability. Monitoring of plasma volume and body fluid compartment shifts is an integral part of the clinical management of DHF, and is crucial to the performance of clinical research studies on DHF pathogenesis. Multifrequency bioelectrical impedance spectroscopy (BIS) was assessed as a non-invasive method to monitor body fluid compartment shifts in children participating in a prospective, hospital-based, study of dengue virus infections in Thailand. Over the 48 h surrounding defervescence, the extracellular water/intracellular water ratio (ECW/ICW) rose in children with dengue virus infections and correlated with increasing disease severity [DHF > intermediate dengue fever (DF)/DHF>DF]. Plasma leakage remained within the ECW compartment and was not directly measured by multifrequency BIS. Expansion of the ECW space in DHF appeared to be primarily due to diminished renal water clearance. During the course of dengue illness, multifrequency BIS did not improve on serial haematocrit and bodyweight determinations for monitoring plasma volume contraction and ECW expansion, respectively.

Dengue hemorrhagic fever (DHF), the most severe form of illness following infection with a dengue virus, is characterized by plasma leakage, thrombocytopenia, and hepatic inflammation. The interrelationships among virus burden, immune activation, and development of DHF were examined in 54 children with secondary dengue-3 virus infections participating in a prospective, hospital-based study. DHF was associated with higher mean plasma viremia early in illness and earlier peak plasma interferon-gamma levels. Maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia. Maximum plasma levels of interleukin (IL)-10 and soluble tumor necrosis factor receptor-II correlated with the degree of thrombocytopenia, independently of viremia levels. Hepatic transaminase elevation correlated with plasma soluble IL-2 receptor levels and not with viremia levels. Quantitative differences in virus burden and host immune responses, and the timing of type 1 cytokine responses, have differing influences on the severity of disease manifestations during secondary dengue-3 virus infections.

Lactate and lactate dehydrogenase (LDH) have been found to be elevated in cardiopulmonary failure, sepsis, shock and hepatic injury. Severe dengue hemorrhagic fever (DHF) patients also develop shock and experience a certain degree of hepatic injury, implicating that serum lactate and LDH may be elevated in Dengue shock syndrome (DSS).To determine serum lactate and LDH levels in dengue patients to see whether they can be used as predictors of severe dengue cases.A cross sectional study was conducted on suspected dengue patients admitted to the dengue ward, Queen Sirikit National Institute of Child Health (QSNICH), between May 2011 and February 2012. Laboratory tests were used to confirm dengue cases in the enrolled patients. Blood for serum lactate was drawn in patients every day after enrollment. Blood for LDH and liver function test (LFT) were drawn 3 times: enrollment day, day of leakage, and discharge day. Lactate and LDH levels are compared among dengue and non-dengue patients. Dengue fever (DF), DHF and DSS patients were classified according to the WHO 1997 dengue classification.253 patients were enrolled, comprising of 120 DF, 75 DH, 30 DSS, and 28 non-dengue patients. The majority of dengue patients had liver impairment, demonstrated by elevated aspartate aminotransferase (AST) (94.9%) and alanine aminotransferase (ALT) levels (68.6%) while non-dengue patients have minimal elevation. Serum lactate levels were not elevated in the early stages in dengue patients, but were elevated in non-dengue patients. The mean serum lactate levels in DSS patients increased towards the end of febrile phase and reached maximum values on Day 0 (2.2 U/L). On the other hand, serum lactate levels were found to be decreasing in the non-dengue group. The mean serum lactate levels on Day 0 was found to be different in DSS patients (2.26 U/L) compared to DF 1.63 U/L), DHF (1.79 U/L) and non-dengue patients (1.68 U/L) (p0.05). Mean serum LDH levels were elevated in the early stages of the disease in all groups of patients, but with different levels. Mean serum LDH levels was 709.2 in DF, 1,873 in DHF, 654.5 in DSS, and 434 IU in non-dengue patients. The mean LDH levels in dengue patients were500 IU, while it was500 IU in non-dengue patients. The increasing mean levels of LDH towards the end of febrile phase were only seen in DHF and DSS patients, but not in DF and non-dengue patients. The mean levels of LDH on Day 0 in DHF, DSS, DF and non-dengue patients are 1,060.7, 1,180.7, 787.2, and 423.8 IU, respectively.Serum lactate and LDH was found to be elevated in DHF and/or DSS patients. Lactate may be used as a predictor of DSS if the level is2 U/L on Day 0. LDH can be used to differentiate patients with or without dengue in the early febrile phase, if the level is500 IU. If the level of LDH is increased to approximately 1, 000 IU on Day 0, it may be a predictor of severe dengue infection or DHF and DSS with plasma leakage.

Background Dengue viral infections are prevalent in the tropical and sub-tropical regions of the world, resulting in substantial morbidity and mortality. Clinical manifestations range from a self-limited fever to a potential life-threatening plasma leakage syndrome (dengue hemorrhagic fever). The objective of this study was to assess the utility of near infrared spectroscopy (NIRS) measurements of muscle oxygen saturation (SmO2) as a possible continuous measure to detect plasma leakage in children with dengue. Methods Children ages 6 months to 15 years of age admitted with suspected dengue were enrolled from the dengue ward at Queen Sirikit National Institute for Child Health. Children were monitored daily until discharge. NIRS data were collected continuously using a prototype CareGuide Oximeter 1100 with sensors placed on the deltoid or thigh. Daily ultrasound of the chest and a right lateral decubitus chest x-ray the day after defervescence were performed to detect and quantitate plasma leakage in the pleural cavity. Results NIRS data were obtained from 19 children with laboratory-confirmed dengue. Average minimum SmO2 decreased for all subjects prior to defervescence. Average minimum SmO2 subsequently increased in children with no ultrasound evidence of pleural effusion but remained low in children with pleural effusion following defervescence. Average minimum SmO2 was inversely correlated with pleural space fluid volume. ROC analysis revealed a cut-off value for SmO2 which yielded high specificity and sensitivity. Conclusions SmO2 measured using NIRS may be a useful guide for real-time and non-invasive identification of plasma leakage in children with dengue. Further investigation of the utility of NIRS measurements for prediction and management of severe dengue syndromes is warranted.

T lymphocyte activation during dengue is thought to contribute to the pathogenesis of dengue hemorrhagic fever (DHF). We examined the T cell receptor Vbeta gene usage by a reverse transcriptase-polymerase chain reaction assay during infection and after recovery in 13 children with DHF and 13 children with dengue fever (DF). There was no deletion of specific Vbeta gene families. We detected significant expansions in usage of single Vbeta families in six subjects with DHF and three subjects with DF over the course of infection, but these did not show an association with clinical diagnosis, viral serotype, or HLA alleles. Differences in Vbeta gene usage between subjects with DHF and subjects with DF were of borderline significance. These data suggest that the differences in T cell activation in DHF and DF are quantitative rather than qualitative and that T cells are activated by conventional antigen(s) and not a viral superantigen.

Background: A number of commercial ELISA for dengue diagnosis have recently become available, though direct comparison between these assays have not been published. Objectives: The Venture Technologies Dengue IgM and IgG Dot Blot assays and the PanBio Dengue Duo IgM and IgG Capture ELISA were compared. Study Design: Paired sera from patients with dengue (n=20) and Japanese encephalitis (JE, n=10), and single sera from patients with typhoid (n=10), leptospirosis (n=10) and scrub typhus (n=10) were assayed according to the manufacturer’s instructions. Results: The Dot Blot IgM ELISA showed higher sensitivity than the PanBio IgM ELISA (100 vs. 95%), while the PanBio IgM ELISA showed higher specificity in JE (100 vs. 20%) and non-flavivirus infections (100 vs. 97%). Defining elevation of either IgM or IgG as a positive result, the Dot Blot and ELISA tests both showed 100% sensitivity in dengue infection, while the PanBio test showed superior specificity in JE (70 vs. 0%) and non-flavivirus infections (100 vs. 67%). Conclusions: Both assays are useful aids to the serological diagnosis of dengue infection. The clinical setting, user preference and local conditions will be important in determining which test is more appropriate.

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little data available, however, describing the kinetics of viral replication in humans with natural dengue virus (DV) infection. Standard procedures for measuring titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma samples based on reverse transcription-polymerase chain reaction (RT-PCR) using a mutant RNA target as a competitor. This technique was reproducible and accurate for samples containing any of the four DV serotypes, and could be applied to samples containing as few as 250 copies of RNA per reaction. We examined plasma viral RNA levels in 80 children with acute DV infection; sequential plasma samples were tested in 34 of these children. Plasma viral RNA levels ranged as high as 109 RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r = 0.69). Plasma viral RNA levels fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels between children with DHF and children with dengue fever, but peak viral RNA levels were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections. J. Med. Virol. 63:29–34, 2001. © 2001 Wiley-Liss, Inc.

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.