Kinetics of severe dengue virus infection and development of gut pathology in mice.

IMPORTANCE: Dengue virus, an arbovirus, causes an estimated 100 million symptomatic infections annually and is an increasing threat as the mosquito range expands with climate change. Dengue epidemics are a substantial strain on local economies and health infrastructure, and an understanding of what drives severe disease may enable treatments to help reduce hospitalizations. Factors exacerbating dengue disease are debated, but gut-related symptoms are much more frequent in severe than mild cases. Using mouse models of dengue infection, we have shown that inflammation and damage are earlier and more severe in the gut than in other tissues. Additionally, we observed impairment of the gut mucus layer and propose that breakdown of the barrier function exacerbates inflammation and promotes severe dengue disease. This idea is supported by recent data from human patients showing elevated bacteria-derived molecules in dengue patient serum. Therapies aiming to maintain gut integrity may help to abrogate severe dengue disease.

Effects of chloroquine or hydroxychloroquine treatment on non-SARS-CoV2 viral infections: A systematic review of clinical studies.

Chloroquine (CQ) and hydroxychloroquine (HCQ) have been used as antiviral agents for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) infection. We performed a systematic review to examine whether prior clinical studies that compared the effects of CQ and HCQ to a control for the treatment of non-SARS-CoV2 infection supported the use of these agents in the present SARS-CoV2 outbreak. PubMed, EMBASE, Scopus and Web of Science (PROSPERO CRD42020183429) were searched from inception through 2 April 2020 without language restrictions. Of 1766 retrieved reports, 18 studies met our inclusion criteria, including 17 prospective controlled studies and one retrospective study. CQ or HCQ were compared to control for the treatment of infectious mononucleosis (EBV, n = 4), warts (human papillomavirus, n = 2), chronic HIV infection (n = 6), acute chikungunya infection (n = 1), acute dengue virus infection (n = 2), chronic HCV (n = 2), and as preventive measures for influenza infection (n = 1). Survival was not evaluated in any study. For HIV, the virus that was most investigated, while two early studies suggested HCQ reduced viral levels, four subsequent ones did not, and in two of these CQ or HCQ increased viral levels and reduced CD4 counts. Overall, three studies concluded CQ or HCQ were effective; four concluded further research was needed to assess the treatments' effectiveness; and 11 concluded that treatment was ineffective or potentially harmful. Prior controlled clinical trials with CQ and HCQ for non-SARS-CoV2 viral infections do not support these agents' use for the SARS-CoV2 outbreak.

High titres of IgM-bound circulating immune complexes and erythrocytic oxidative damage are indicators of dengue severity.

Global incidence of dengue has drastically increased in the last few years. Despite the global morbidity and mortality associated with dengue infection, mechanisms of immune control and viral pathogenesis are poorly explored. Pancytopenias, along with increased oxidative stress, are salient clinical findings in severe dengue patients. Previously, we demonstrated significant differences of circulating immune complexes (CICs) among severe and non-severe dengue patients. Accordingly, here we sought to determine the contributory role of affinity-purified antibody-bound CICs in dengue severity. To characterize intracellular oxidative stress induced by antibody-bound CICs, 5-(and-6)-chloromethyl-2'-7'-dichlorodihydrofluorescein diacetate (DCFDA) was measured by flow cytometry. At the same time, CICs sensitized healthy red blood cells (RBC) and patients' RBC morphology was determined by scanning electron microscopy and flow cytometry analysis. Erythrophagocytosis and ferritin levels were further determined in severe and non-severe dengue patients. Our results showed that the severe patients had high titres of immunoglobulin (Ig)M-bound CICs (P < 0·0001) in their sera, increased intracellular oxidative stress (P < 0·0001), high ferritin levels (P < 0·0001), altered morphology of RBC and finally enhanced erythrophagocytosis. This study shows for the first time that RBC morphology is altered in severe dengue patients. Taken together, this study suggests that the enhanced IgM-bound CICs could contribute to the increased oxidative stress and act directly on RBC destruction of severe dengue patients, and is an important pathophysiological determinant. Hence, IgM-bound CICs can serve as an important laboratory parameter to monitor dengue infection progression.

A novel predictor of severe dengue: The aspartate aminotransferase/platelet count ratio index (APRI).

To evaluate risk factors for the development of dengue into severe dengue in Guangdong. A retrospective analysis of clinical data from 212 dengue patients between June and October 2014. A total of 174 (82.1%) patients in our study had classic dengue, of which 38 (17.9%) had severe diseases. The frequencies of jaundice, pleural effusion, ascites, and vaginal bleeding were significantly different between the two groups (P < 0.05). The routine laboratory test results for alanine aminotransferase, aspertate aminotransferase, albumin, leukocyte count, platelet count, activated partial prothrombin time, prothrombin time, and aspartate aminotransferase/platelet count ratio index showed a significant association with severe dengue (P < 0.01). The areas under the receiver operating characteristic curves (AUC) were 0.727 (95% CI 0.662-0.78), 0.699 (95%CI 0.632-0.760), 0.634 (95%CI 0.565-0.698), 0.757 (95%CI 0.694-0.813), 0.775 (95%CI 0.713-0.829), 0.713 (95%CI 0.647-0.773), 0.719 (95%CI 0.730-0.843), and 0.785 (95%CI 0.724-0.893), respectively. The logistic regression analysis identified three factors, including high WBC (OR 1.52), prolonged PT (OR 1.745). and high APRI (OR 1.05) may be associated with the discrimination criteria to identify patients with and without severe diseases. The combination of the three factors (WBC, PT, and APRI) showed better AUC (0.877) and OR (1.52) scores. Our study indicates that laboratory tests such as WBC, PT, and APRI, helped identify patients at risk of developing severe dengue. The APRI was identified as a valuable predictor of patients with severe dengue. Combining the WBC, PT, and APRI scores allowed a better prediction of severe dengue.

Association between dengue severity and plasma levels of dengue-specific IgE and chymase.

There is no definitive predictor of dengue severity, and this has led to a very large number of unnecessary hospitalizations worldwide. Although mast cell mediators are believed to a play role in dengue severity, the lack of precise kinetic data demands further research on early predictors. We enrolled 111 patients with confirmed dengue and 85 with "other febrile illness" (OFI) in a hospital-based prospective study in Vietnam. Dengue patients were classified as level 1, 2, or 3 based on the clinical intervention received. Blood samples were collected from each patient every day (pre- and post-defervescence) and after discharge. Plasma chymase, total IgE, and dengue-specific IgE were measured. Dengue-specific IgE levels showed an increasing trend during the course of illness and remained high even at post-discharge, although no significant difference was observed among severity levels. Total IgE showed no such trend. The specific IgE/total IgE ratio (S/T ratio) remained constantly higher in level 3 patients compared to other levels, with a significant difference at some time points. The S/T ratio of acute phase samples (before defervescence) tended to increase with increasing severity (level 1 < 2 < 3), and was significantly higher in level 3 patients than in level 1 and OFI patients. As an early predictor of severity allowing level 3 patients to be distinguished from other dengue patients, the S/T ratio achieved a sensitivity of 75% and specificity of 68%. We describe the kinetic profiles of IgEs, their ratio, and chymase levels at different severity levels. The S/T ratio was found to be associated with dengue severity, suggesting that it could potentially be used as an early predictor of severity.

Genotypic variability analysis of DENV-1 in Mexico reveals the presence of a novel Mexican lineage.

Here, we report for the first time the circulation of dengue virus type 1 (DENV-1) belonging to the lineage IV of genotype V (African American genotype) based on phylogenetic analysis of nucleotide sequences from 10 DENV-1-positive samples obtained in Mexico between 2012 and 2014. Our data revealed that the lineages III and IV of DENV-1 genotype V were found circulating during the same period, probably explaining the rise in the number of cases of severe dengue during that period.

Endothelial Cell Sensitization by Death Receptor Fractions of an Anti-Dengue Nonstructural Protein 1 Antibody Induced Plasma Leakage, Coagulopathy, and Mortality in Mice.

The mechanisms leading to the life-threatening dengue hemorrhagic fever (DHF) remain elusive. DHF preferentially occurs during secondary dengue infections, suggesting that aberrant immune responses are involved in its development. We previously demonstrated that the autoantibodies elicited by dengue virus (DENV) nonstructural protein 1 (NS1; anti-NS1 Igs) induce plasma leakage and mortality in mice with warfarinized anticoagulant suppression. However, the involved pathogenic Ig fractions of anti-NS1 Igs remain unclear. In this study, the autoreactive Igs in patients with DHF and in NS1-immunized rabbits crossreacted with TNF-related apoptosis-inducing ligand receptor 1 (death receptor [DR]4). Challenges with the DENV in a subcytotoxic dose sensitized endothelial cells to apoptosis. Treatments with the autoantibodies induced proapoptotic activities and suppressed the surface expression of endothelial anticoagulant thrombomodulin. Combined treatments comprising the DENV and DR4 affinity-purified fractions of anti-NS1 IgGs (anti-NS1-DR4 Ig), but not preimmune control IgGs, in subcytotoxic doses led to apoptosis in endothelial cells. Treatments with the anti-NS1-DR4 Ig led to plasma leakage, coagulopathy, and morality in mice with warfarinized anticoagulant suppression. These results suggest that DR4-induced endothelial cell sensitization through NS1-elicited autoantibodies exacerbates anticoagulant suppression, vascular injury, and plasma leakage. Detecting and blocking anti-DR Igs in patients may be novel strategies for managing severe DENV infection.

Risk factors and clinical outcome of profound thrombocytopenia in adult patients with DENV infections.

Severe thrombocytopenia is common in dengue virus (DENV) infections. However, studies focusing on the role of profound thrombocytopenia (PT) (nadir platelet counts ≤ 20 000/mm3) in DENV infections are scarce. This study aims to identify the associated features and outcome of DENV patients with PT. It involves 237 adult hospitalized patients who were DENV PCR positive. The presence of comorbidity (AOR = 4.625; 95% CI = 1.113-19.230), higher admission hematocrit (AOR = 1.213; 95% CI = 1.067-1.379), lower admission albumin (AOR = 0.870; 95% CI = 0.766-0.988) and lower admission platelets (AOR = 0.980; 95% CI = 0.969-0.991) was associated with platelets ≤ 20 000/mm3 in multivariate logistic regression. PT was not affected by DENV serotypes, coinfections and secondary DENV infections. Patients with PT had significantly higher risk of experiencing warning signs (AOR = 3.709, 95% CI = 1.089-12.634) and longer hospital stay (AOR = 1.943, 95% CI = 1.010-3.774). However, severe dengue disease, hemorrhagic manifestations and need for intensive care were not significantly associated with PT.

Association of BAK1 single nucleotide polymorphism with a risk for dengue hemorrhagic fever.

BACKGROUND: Dengue hemorrhagic fever (DHF) is a severe life-threatening form of dengue infection. Low platelet count is one of the characteristic clinical manifestations in patients with severe dengue. However, little is known about genetic factors in the host that cause low platelet count in patients with dengue. METHODS: A previous genome-wide association study of hematological and biochemical traits identified single nucleotide polymorphisms (SNPs) associated with low platelet count in healthy subjects. To examine the possible association of these SNPs with DHF, 918 Thai patients with dengue [509 patients with DHF and 409 with dengue fever (DF)] were genotyped for five SNPs: rs5745568 in BAK1, rs6141 in THPO, rs6065 in GP1BA, rs739496 in SH2B3, and rs385893 in RCL1. In addition, rs4804803 in CD209, that has been reported to be associated with dengue infection, was also genotyped to examine if rs4804803 affects the association detected in this study. RESULTS: The allele frequencies of each SNP were compared between the DHF and DF groups. Among the five SNPs, the G allele of rs5745568 in BAK1 was significantly associated with a risk for DHF [P = 0.006 and crude odd ratio (95 % confidence interval) = 1.32 (1.09-1.60)]. The association of this allele with DHF was also significant in a logistic regression analysis adjusted for age, sex, hospital (i.e., geographic region), immune status (i.e., primary or secondary infection), and virus serotype [P = 0.016 and adjusted odd ratio (95 % confidence interval) = 1.29 (1.05-1.58)]. The result was not influenced by rs4804803 [P = 0.0167 and adjusted OR (95 % CI) = 1.29 (1.05-1.58)]. No other SNPs including rs4804803 showed significant association. CONCLUSIONS: The low-level constitutive production of platelets caused by the G allele of rs5745568 seems to increase the risk of bleeding in dengue infection. Our results suggest that BCL-2 homologous antagonist/killer (BAK) protein, encoded by BAK1, plays a crucial role in the pathogenesis of DHF.

Association Between Increased Vascular Nitric Oxide Bioavailability and Progression to Dengue Hemorrhagic Fever in Adults.

In a prospective longitudinal adult study, vascular nitric oxide bioavailability measured as reactive hyperemia index was significantly higher at enrollment in patients who developed dengue hemorrhagic fever (DHF) (n = 11), compared with the non-DHF group (n = 63) and those with other febrile illnesses (n = 25) (P = .01). After adjustment for age, fever day, and body mass index, enrollment reactive hyperemia index was associated with a 4-fold increased risk for DHF, and predicted DHF with an area under the receiver operating curve of 0.86. Increased vascular nitric oxide in dengue is associated with increased vascular permeability and impaired homeostasis and may have utility as a predictor of DHF.

Immunopathogenesis of dengue hemorrhagic fever: contribution to the study of human liver lesions.

Dengue infection is an important tropical disease worldwide. The host immune response has been studied in order to better understand lesion mechanisms. It was performed an immunohistochemical study in 14 specimens of liver from patients with dengue hemorrhagic fever (DHF) to characterize cytokines and some factors present in liver lesions and their possible role in the pathogenesis of hepatic injury. Portal tract and hepatic acinus presented high expression of TLR2, TLR3, IL6, and granzyme B. Hepatic acinus also presented iNOS, IL18, and TGF-beta. Cells expressing IL12, IL13, JAk1, STAT1, and NF-κB were rarely visualized. Treg cells foxp3+ were absent. TLR2 and TLR3 seem to participate in cellular activation and cytokine production. Cytotoxic response seems to play a role. Although TGF-beta promotes the activation of Foxp3+ regulatory T cells, IL6 can significantly suppresses their generation. The expression of Treg cells is diminished probably as a result of the high frequency of these cytokines. Both cytokines play a role in the increased vascular permeability and edema observed in dengue liver specimens, with consequent plasma leakage and severity of the disease. It was observed a regular expression of IL-18 in hepatocytes and lymphocytes of the inflammatory infiltrate in portal tract, which reflects the acute inflammatory response that occurs in the liver and contributes to hepatic injury. At least in part, the increased number of cells expressing IL-18 could play a role of "up" regulation of FasL and correlate to the phenomenon of apoptosis, a mechanism of destruction of hepatocytes in DHF.

Extensive haemorrhagic necrosis of liver is an unpredictable fatal complication in dengue infection: a postmortem study.

BACKGROUND: Dengue infection carries a potential risk of death despite stringent management of plasma leak and haemorrhage. It appears that the extent of liver dysfunction determines the outcome. METHODS: We present a postmortem study of five patients, died of dengue shock syndrome who had markedly elevated liver enzymes and irreparable circulatory failure. RESULTS: All were females with a median age of 46 years (range 20-50 years). All had positive NS1 and IgM. Clinically, one patient developed severe degree of hepatic encephalopathy whilst three patients developed uncontrollable bleeding manifestations. Dengue virus was detected in three liver specimens by reverse transcription PCR. Histology of the liver revealed massive necrosis with haemorrhages in these patients with evidence of micro and macrovesicular steatosis with significant periportal inflammatory infiltrate. No significant ischaemic changes or necrosis was observed in the other organs. CONCLUSIONS: Severe haemorrhagic necrosis of the liver was the cause of death in these patients probably due to direct viral infection. Predilection for severe liver disease remains unknown. Therefore, it is prudent to think beyond plasma leak as the main pathology of dengue infection and attempts should be made to develop other treatment modalities to prevent and manage unforeseen fatal complications of dengue infection.

Protection from secondary dengue virus infection in a mouse model reveals the role of serotype cross-reactive B and T cells.

The four dengue virus (DENV) serotypes cause dengue fever and dengue hemorrhagic fever/dengue shock syndrome. Although severe disease has been associated with heterotypic secondary DENV infection, most secondary DENV infections are asymptomatic or result in classic DF. The role of cross-reactive immunity in mediating cross-protection against secondary heterotypic DENV infection is not well understood. DENV infection of IFN-α/ß and IFN-γ receptor-deficient (AG129) mice reproduces key features of human disease. We previously demonstrated a role in cross-protection for pre-existing cross-reactive Abs, maintained by long-lived plasma cells. In this study, we use a sequential infection model, infecting AG129 mice with DENV-1, followed by DENV-2 6-8 wk later. We find that increased DENV-specific avidity during acute secondary heterotypic infection is mediated by cross-reactive memory B cells, as evidenced by increased numbers of DENV-1-specific cells by ELISPOT and higher avidity against DENV-1 of supernatants from polyclonally stimulated splenocytes isolated from mice experiencing secondary DENV-2 infection. However, increased DENV-specific avidity is not associated with increased DENV-specific neutralization, which appears to be mediated by naive B cells. Adoptive transfer of DENV-1-immune B and T cells into naive mice prior to secondary DENV-2 infection delayed mortality. Mice depleted of T cells developed signs of disease, but recovered after secondary DENV infection. Overall, we found that protective cross-reactive Abs are secreted by both long-lived plasma cells and memory B cells and that both cross-reactive B cells and T cells provide protection against a secondary heterotypic DENV infection. Understanding the protective immunity that develops naturally against DENV infection may help design future vaccines.

Secondary infection as a risk factor for dengue hemorrhagic fever/dengue shock syndrome: an historical perspective and role of antibody-dependent enhancement of infection.

Today, dengue viruses are the most prevalent arthropod-borne viruses in the world. Since the 1960s, numerous reports have identified a second heterologous dengue virus (DENV) infection as a principal risk factor for severe dengue disease (dengue hemorrhagic fever/dengue shock syndrome, DHF/DSS). Modifiers of dengue disease response include the specific sequence of two DENV infections, the interval between infections, and contributions from the human host, such as age, ethnicity, chronic illnesses and genetic background. Antibody-dependent enhancement (ADE) of dengue virus infection has been proposed as the early mechanism underlying DHF/DSS. Dengue cross-reactive antibodies raised following a first dengue infection combine with a second infecting virus to form infectious immune complexes that enter Fc-receptor-bearing cells. This results in an increased number of infected cells and increased viral output per cell. At the late illness stage, high levels of cytokines, possibly the result of T cell elimination of infected cells, result in vascular permeability, leading to shock and death. This review is focused on the etiological role of secondary infections (SI) and mechanisms of ADE.

Dengue virus-induced hemorrhage in a nonhuman primate model.

Lack of a dengue hemorrhagic animal model recapitulating human dengue virus infection has been a significant impediment in advancing our understanding of the early events involved in the pathogenesis of dengue disease. In efforts to address this issue, a group of rhesus macaques were intravenously infected with dengue virus serotype 2 (strain 16 681) at 1 x 10(7) PFU/animal. A classic dengue hemorrhage developed 3 to 5 days after infection in 6 of 6 animals. Blood chemistry appeared to be normal with exception of creatine phosphokinase, which peaked at 7 days after infection. A modest thrombocytopenia and noticeable neutropenia concomitant with slight decrease of hemoglobin and hematocrit were registered. In addition, the concentration of D-dimer was elevated significantly. Viremia peaked at 3 to 5 days after infection followed by an inverse relationship between T and B lymphocytes and a bimodal pattern for platelet-monocytes and platelet-neutrophil aggregates. Dengue virus containing platelets engulfed by monocytes was noted at 8 or 9 days after infection. Thus, rhesus macaques inoculated intravenously with a high dose of dengue virus produced dengue hemorrhage, which may provide a unique platform to define the early events in dengue virus infection and help identify which blood components contribute to the pathogenesis of dengue disease.

Elevated levels of vitamin D and deficiency of mannose binding lectin in dengue hemorrhagic fever.

BACKGROUND: Altered plasma concentrations of vitamin D and mannose binding lectin (MBL), components of innate immunity, have been shown to be associated with the pathogenesis of viral infections. The objective of the present study was to find out whether plasma concentrations of MBL and vitamin D are different in patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). THE RESULTS: The plasma concentrations of vitamin D and MBL were assessed in 48 DF cases, 45 DHF cases and 20 apparently healthy controls using ELISA based methods. Vitamin D concentrations were found to be higher among both DF and DHF cases as compared to healthy controls (P < 0.005 and P < 0.001). Vitamin D concentrations were not different between DF and DHF cases. When the dengue cases were classified into primary and secondary infections, secondary DHF cases had significantly higher concentrations of vitamin D as compared to secondary DF cases (P < 0.050). MBL concentrations were not significantly different between healthy controls and dengue cases. MBL concentrations were observed to be lower in DHF cases as compared to DF cases (P < 0.050). Although MBL levels were not different DF and DHF cases based on immune status, the percentage of primary DHF cases (50%) having MBL levels lower than 500 ng/ml were less compared to primary DF cases (P = 0.038). CONCLUSIONS: The present study suggests that higher concentrations of vitamin D might be associated with secondary DHF while deficiency of MBL may be associated with primary DHF.

Expanded dengue syndrome: subacute thyroiditis and intracerebral hemorrhage.

BACKGROUND: Although most symptomatic dengue infections follow an uncomplicated course, complications and unusual manifestations are increasingly being reported due to rising disease burden. Expanded dengue syndrome is a new entity added into World Health Organization (WHO) classification system to incorporate this wide spectrum of unusual manifestations. We report a case of expanded dengue syndrome with subacute thyroiditis and intracerebral hemorrhage. This is the first case report of thyroiditis in dengue infection. CASE PRESENTATION: A 20 years old man presented with fever, myalgias, arthralgias, retro-orbital pain, vomiting and gum bleeding during a large dengue outbreak in Lahore, Pakistan. On 7th day of illness patient became afebrile, but he developed severe headaches, unconsciousness followed by altered behavior. On 9th day of illness patient developed painful neck swelling accompanied by fever, tremors, palpitations, hoarseness of voice and odynophagia. Examination revealed acutely swollen, tender thyroid gland along with features of hyperthyroidism. Laboratory evaluation revealed stable hematocrit, thrombocytopenia and leukopenia. Patient had seroconverted for anti-dengue IgM antibodies on the 10th day of illness. A non-contrast Computed Tomogram (CT) of the brain showed right frontal lobe hematoma. Thyroid profile showed increased free T3 and T4 and low TSH. Technetium thyroid scan showed reduced tracer uptake. He was diagnosed as having subacute thyroiditis and treated with oral prednisolone and propranolol. Follow up CT brain showed resolving hematoma. Patient's recovery was uneventful. CONCLUSION: Subacute thyroiditis may develop during the course of dengue fever and should be included as a manifestation of expanded dengue syndrome. It should be suspected in patients with dengue fever who develop painful thyroid swelling and clinical features of hyperthyroidism.

Timing of CD8+ T cell responses in relation to commencement of capillary leakage in children with dengue.

Immune activation is a feature of dengue hemorrhagic fever (DHF) and CD8+ T cell responses in particular have been suggested as having a role in the vasculopathy that characterizes this disease. By phenotyping CD8+ T cells (CD38+/HLA-DR+, CD38+/Ki-67+, or HLA-DR+/Ki-67+) in serial blood samples from children with dengue, we found no evidence of increased CD8+ T cell activation prior to the commencement of resolution of viremia or hemoconcentration. Investigations with MHC class I tetramers to detect NS3(133-142)-specific CD8+ T cells in two independent cohorts of children suggested the commencement of hemoconcentration and thrombocytopenia in DHF patients generally begins before the appearance of measurable frequencies of NS3(133-142)-specific CD8+ T cells. The temporal mismatch between the appearance of measurable surface activated or NS3(133-142)-specific CD8+ T cells suggests that these cells are sequestered at sites of infection, have phenotypes not detected by our approach, or that other mechanisms independent of CD8+ T cells are responsible for early triggering of capillary leakage in children with DHF.

Multicentre prospective study on dengue classification in four South-east Asian and three Latin American countries.

OBJECTIVE: To evaluate the existing WHO dengue classification across all age groups and a wide geographical range and to develop a revised evidence-based classification that would better reflect clinical severity. METHODS: We followed suspected dengue cases daily in seven countries across South-east Asia and Latin America and then categorised them into one of three intervention groups describing disease severity according to the overall level of medical and nursing support required. Using a pre-defined analysis plan, we explored the clinical and laboratory profiles characteristic of these intervention categories and presented the most promising options for a revised classification scheme to an independent group of WHO dengue experts for consideration. Potential warning signs were also evaluated by comparing contemporaneous data of patients who progressed to severe disease with the data of those who did not. RESULTS: A total of 2259 patients were recruited during 2006-2007 and 230 (13%) of the 1734 laboratory-confirmed patients required major intervention. Applying the existing WHO system, 47/210 (22%) of patients with shock did not fulfil all the criteria for dengue haemorrhagic fever. However, no three-tier revision adequately described the different severity groups either. Inclusion of readily discernible complications (shock/severe vascular leakage and/or severe bleeding and/or severe organ dysfunction) was necessary to devise a system that identified patients requiring major intervention with sufficient sensitivity and specificity to be practically useful. Only a small number of subjects (5%) progressed to severe disease while under observation; several warning signs were identified, but much larger studies are necessary to fully characterize features associated with disease progression. CONCLUSIONS: Based on these results, a revised classification system comprised of two entities, 'Dengue' and 'Severe Dengue', was proposed and has now been incorporated into the new WHO guidelines.

Infection-associated hemophagocytic syndrome among patients with dengue shock syndrome and invasive aspergillosis: a case series and review of the literature.

The authors report four autopsy cases of previously healthy children with dengue shock syndrome complicated with infection-associated hemophagocytosis and invasive aspergillosis. Hemophagocytosis is confirmed by histopathology of autopsied reticuloendothelial organs. All four children were identified to have invasive aspergillosis by histopathology and three cases were positive on fungal culture for Aspergillus spp. Regarding the cause of death among the four children without pre-existing underlying disease, three cases were directly ascribable to invasive aspergillosis and the remaining case was ascribed to dengue shock syndrome. The transmigration of preexisting fungi from the respiratory mucosa damaged by the dengue shock process is postulated as the pathogenesis of invasive aspergillosis. The main predisposing factor was found to be prolonged dengue shock syndrome. We reviewed the clinicopathologic features and therapeutic management of infection-associated hemophagocytic syndrome in patients with dengue shock syndrome and invasive aspergillosis.