Diagnosis and Prognosis of Severe Dengue

Dengue has a broad spectrum of clinical symptoms, ranging from asymptomatic infection, undifferentiated fever and dengue fever (DF) to dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). DHF and DSS are the most severe forms of the disease, as they involve bleeding and hypovolemic shock and may lead to death if they are not diagnosed early and treated correctly. Every year, about 36 million cases of DF, of which about 2.1 million cases are DHF/DSS resulting in 21,000 deaths among children and young adults, are reported. Dengue not only has negative effects on public health worldwide but also poses significant economic burden. However, no licensed vaccines or (specific) anti-viral therapies for prevention or treatment of dengue are available. Thus, timely diagnosis and an accurate prognosis of severe dengue in the first few days of illness for clinical triage, treatment management, disease surveillance and research activities are vital and very helpful. We conducted three studies aiming to identify early markers of dengue, especially severe dengue and identify prognostic markers of severe dengue in Vietnamese children with clinically suspected dengue within the first few days of the illness. The results firstly showed that plasma collected within the first week of the illness could be used for dengue diagnosis as well as differentiation between primary and secondary dengue virus infections. Secondly, the research showed that plasma concentration of NS1 correlated with plasma viraemia and associated with DSS. NS1 concentrations in enrolment plasma of patients within the first four days of illness could be used for predicting DSS in patients with DENV-1 or DENV-2 infections. Thirdly, in the first three days of illness, we observed differences in clinical and haematological profiles between patients with DHF and those with DF or other febrile illnesses. Logistic models were then generated using the early clinical characteristics and/or haematological profiles to predict patients who will develop DHF. Our findings also showed that a greater virus burden leads to an augmented cascade of plasma concentrations of IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12p70, IL-13, IFNγ and TNFα in DHF patients compared to DF patients. This thesis shows the results and also points out predictive models that may be used to prognosticate of severe dengue among patients with clinically suspected dengue who are admitted to hospital within the first few days of the illness

Genetic variants of MICB and PLCE1 and associations with the laboratory features of dengue

Background: A previous genome-wide association study identified 2 susceptibility loci for severe dengue at MICB rs3132468 and PLCE1 rs3740360 and further work showed these mutations to be also associated with less severe clinical presentations. The aim of this study was to determine if these specific loci were associated with laboratory features of dengue that correlate with clinical severity with the aim of elucidating the functional basis of these genetic variants. Methods: This was a case-only analysis of laboratory-confirmed dengue patients obtained from 2 prospective cohort studies and 1 randomised clinical trial in Vietnam (Trial registration: ISRCTN ISRCTN03147572. Registered 24th July 2012). 2742 dengue cases were successfully genotyped at MICB rs3132468 and PLCE1 rs3740360. Laboratory variables were compared between genotypes and stratified by DENV serotype. Results: The analysis showed no association between MICB and PLCE1 genotype and early viraemia level, platelet nadir, white cell count nadir, or maximum haematocrit in both overall analysis and in analysis stratified by serotype. Discussion: The lack of an association between genotype and viremia level may reflect the sampling procedures within the included studies. The study findings mean that the functional basis of these mutations remains unclear. Trial registration: ISRCTN ISRCTN03147572. Registered 24th July 2012

Lovastatin for the Treatment of Adult Patients With Dengue: A Randomized, Double-Blind, Placebo-Controlled Trial.

BACKGROUND: Dengue endangers billions of people in the tropical world, yet no therapeutic is currently available. In part, the severe manifestations of dengue reflect inflammatory processes affecting the vascular endothelium. In addition to lipid lowering, statins have pleiotropic effects that improve endothelial function, and epidemiological studies suggest that outcomes from a range of acute inflammatory syndromes are improved in patients already on statin therapy. METHODS: Following satisfactory review of a short pilot phase (40 mg lovastatin vs placebo in 30 cases), we performed a randomized, double-blind, placebo-controlled trial of 5 days of 80 mg lovastatin vs placebo in 300 Vietnamese adults with a positive dengue NS1 rapid test presenting within 72 hours of fever onset. The primary outcome was safety. Secondary outcomes included comparisons of disease progression rates, fever clearance times, and measures of plasma viremia and quality of life between the treatment arms. RESULTS: Adverse events occurred with similar frequency in both groups (97/151 [64%] placebo vs 82/149 [55%] lovastatin; P = .13), and were in keeping with the characteristic clinical and laboratory features of acute dengue. We also observed no difference in serious adverse events or any of the secondary outcome measures. CONCLUSIONS: We found lovastatin to be safe and well tolerated in adults with dengue. However, although the study was not powered to address efficacy, we found no evidence of a beneficial effect on any of the clinical manifestations or on dengue viremia. Continuing established statin therapy in patients who develop dengue is safe.Chinese Clinical Trials Registration. ISRCTN03147572

Identification of Tuberculosis Susceptibility Genes with Human Macrophage Gene Expression Profiles

Although host genetics influences susceptibility to tuberculosis (TB), few genes determining disease outcome have been identified. We hypothesized that macrophages from individuals with different clinical manifestations of Mycobacterium tuberculosis (Mtb) infection would have distinct gene expression profiles and that polymorphisms in these genes may also be associated with susceptibility to TB. We measured gene expression levels of >38,500 genes from ex vivo Mtb-stimulated macrophages in 12 subjects with 3 clinical phenotypes: latent, pulmonary, and meningeal TB (n = 4 per group). After identifying differentially expressed genes, we confirmed these results in 34 additional subjects by real-time PCR. We also used a case-control study design to examine whether polymorphisms in differentially regulated genes were associated with susceptibility to these different clinical forms of TB. We compared gene expression profiles in Mtb-stimulated and unstimulated macrophages and identified 1,608 and 199 genes that were differentially expressed by >2- and >5-fold, respectively. In an independent sample set of 34 individuals and a subset of highly regulated genes, 90% of the microarray results were confirmed by RT-PCR, including expression levels of CCL1, which distinguished the 3 clinical groups. Furthermore, 6 single nucleotide polymorphisms (SNPs) in CCL1 were found to be associated with TB in a case-control genetic association study with 273 TB cases and 188 controls. To our knowledge, this is the first identification of CCL1 as a gene involved in host susceptibility to TB and the first study to combine microarray and DNA polymorphism studies to identify genes associated with TB susceptibility. These results suggest that genome-wide studies can provide an unbiased method to identify critical macrophage response genes that are associated with different clinical outcomes and that variation in innate immune response genes regulate susceptibility to TB

Corticosteroids for dengue - why don't they work?

BACKGROUND: Dysregulated immune responses may contribute to the clinical complications that occur in some patients with dengue. FINDINGS: In Vietnamese pediatric dengue cases randomized to early prednisolone therapy, 81 gene-transcripts (0.2% of the 47,231 evaluated) were differentially abundant in whole-blood between high-dose (2 mg/kg) prednisolone and placebo-treated patients two days after commencing therapy. Prominent among the 81 transcripts were those associated with T and NK cell cytolytic functions. Additionally, prednisolone therapy was not associated with changes in plasma cytokine levels. CONCLUSION: The inability of prednisolone treatment to markedly attenuate the host immune response is instructive for planning future therapeutic strategies for dengue

Genetic variants of MICB and PLCE1 and associations with non-severe dengue.

BACKGROUND: A recent genome-wide association study (GWAS) identified susceptibility loci for dengue shock syndrome (DSS) at MICB rs3132468 and PLCE1 rs3740360. The aim of this study was to define the extent to which MICB (rs3132468) and PLCE1 (rs3740360) were associated with less severe clinical phenotypes of pediatric and adult dengue. METHODS: 3961 laboratory-confirmed dengue cases and 5968 controls were genotyped at MICB rs3132468 and PLCE1 rs3740360. Per-allele odds ratios (OR) with 95% confidence intervals (CI) were calculated for each patient cohort. Pooled analyses were performed for adults and paediatrics respectively using a fixed effects model. RESULTS: Pooled analysis of the paediatric and adult cohorts indicated a significant association between MICB rs3132468 and dengue cases without shock (OR  =  1.15; 95%CI: 1.07 - 1.24; P  =  0.0012). Similarly, pooled analysis of pediatric and adult cohorts indicated a significant association between dengue cases without shock and PLCE1 rs3740360 (OR  =  0.92; 95%CI: 0.85 - 0.99; P  =  0.018). We also note significant association between both SNPs (OR  =  1.48; P  =  0.0075 for MICB rs3132468 and OR  =  0.75, P  =  0.041 for PLCE1 rs3740360) and dengue in infants. DISCUSSION: This study confirms that the MICB rs3132468 and PLCE1 rs3740360 risk genotypes are not only associated with DSS, but are also associated with less severe clinical phenotypes of dengue, as well as with dengue in infants. These findings have implications for our understanding of dengue pathogenesis

Methods to discriminate primary from secondary dengue during acute symptomatic infection

Abstract Background Dengue virus infection results in a broad spectrum of clinical outcomes, ranging from asymptomatic infection through to severe dengue. Although prior infection with another viral serotype, i.e. secondary dengue, is known to be an important factor influencing disease severity, current methods to determine primary versus secondary immune status during the acute illness do not consider the rapidly evolving immune response, and their accuracy has rarely been evaluated against an independent gold standard. Methods Two hundred and ninety-three confirmed dengue patients were classified as experiencing primary, secondary or indeterminate infections using plaque reduction neutralisation tests performed 6 months after resolution of the acute illness. We developed and validated regression models to differentiate primary from secondary dengue on multiple acute illness days, using Panbio Indirect IgG and in-house capture IgG and IgM ELISA measurements performed on over 1000 serial samples obtained during acute illness. Results Cut-offs derived for the various parameters demonstrated progressive change (positively or negatively) by day of illness. Using these time varying cut-offs it was possible to determine whether an infection was primary or secondary on single specimens, with acceptable performance. The model using Panbio Indirect IgG responses and including an interaction with illness day showed the best performance throughout, although with some decline in performance later in infection. Models based on in-house capture IgG levels, and the IgM/IgG ratio, also performed well, though conversely performance improved later in infection. Conclusions For all assays, the best fitting models estimated a different cut-off value for different days of illness, confirming how rapidly the immune response changes during acute dengue. The optimal choice of assay will vary depending on circumstance. Although the Panbio Indirect IgG model performs best early on, the IgM/IgG capture ratio may be preferred later in the illness course

A randomized, double-blind placebo controlled trial of balapiravir, a polymerase inhibitor, in adult dengue patients.

BACKGROUND: Dengue is the most common arboviral infection of humans. There are currently no specific treatments for dengue. Balapiravir is a prodrug of a nucleoside analogue (called R1479) and an inhibitor of hepatitis C virus replication in vivo. METHODS: We conducted in vitro experiments to determine the potency of balapiravir against dengue viruses and then an exploratory, dose-escalating, randomized placebo-controlled trial in adult male patients with dengue with <48 hours of fever. RESULTS: The clinical and laboratory adverse event profile in patients receiving balapiravir at doses of 1500 mg (n = 10) or 3000 mg (n = 22) orally for 5 days was similar to that of patients receiving placebo (n = 32), indicating balapiravir was well tolerated. However, twice daily assessment of viremia and daily assessment of NS1 antigenemia indicated balapiravir did not measurably alter the kinetics of these virological markers, nor did it reduce the fever clearance time. The kinetics of plasma cytokine concentrations and the whole blood transcriptional profile were also not attenuated by balapiravir treatment. CONCLUSIONS: Although this trial, the first of its kind in dengue, does not support balapiravir as a candidate drug, it does establish a framework for antiviral treatment trials in dengue and provides the field with a clinically evaluated benchmark molecule. CLINICAL TRIALS REGISTRATION: NCT01096576