A new lineage nomenclature to aid genomic surveillance of dengue virus.

Dengue virus (DENV) is currently causing epidemics of unprecedented scope in endemic settings and expanding to new geographical areas. It is therefore critical to track this virus using genomic surveillance. However, the complex patterns of viral genomic diversity make it challenging to use the existing genotype classification system. Here, we propose adding 2 sub-genotypic levels of virus classification, named major and minor lineages. These lineages have high thresholds for phylogenetic distance and clade size, rendering them stable between phylogenetic studies. We present assignment tools to show that the proposed lineages are useful for regional, national, and subnational discussions of relevant DENV diversity. Moreover, the proposed lineages are robust to classification using partial genome sequences. We provide a standardized neutral descriptor of DENV diversity with which we can identify and track lineages of potential epidemiological and/or clinical importance. Information about our lineage system, including methods to assign lineages to sequence data and propose new lineages, can be found at: dengue-lineages.org.

Reemergence of Cosmopolitan Genotype Dengue Virus Serotype 2, Southern Vietnam.

We performed phylogenetic analysis on dengue virus serotype 2 Cosmopolitan genotype in Ho Chi Minh City, Vietnam. We document virus emergence, probable routes of introduction, and timeline of events. Our findings highlight the need for continuous, systematic genomic surveillance to manage outbreaks and forecast future epidemics.

Applying artificial intelligence and digital health technologies, Viet Nam.

Problem: Direct application of digital health technologies from high-income settings to low- and middle-income countries may be inappropriate due to challenges around data availability, implementation and regulation. Hence different approaches are needed. Approach: Within the Viet Nam ICU Translational Applications Laboratory project, since 2018 we have been developing a wearable device for individual patient monitoring and a clinical assessment tool to improve dengue disease management. Working closely with local staff at the Hospital for Tropical Diseases, Ho Chi Minh City, we developed and tested a prototype of the wearable device. We obtained perspectives on design and use of the sensor from patients. To develop the assessment tool, we used existing research data sets, mapped workflows and clinical priorities, interviewed stakeholders and held workshops with hospital staff. Local setting: In Viet Nam, a lower middle-income country, the health-care system is in the nascent stage of implementing digital health technologies. Relevant changes: Based on patient feedback, we are altering the design of the wearable sensor to increase comfort. We built the user interface of the assessment tool based on the core functionalities selected by workshop attendees. The interface was subsequently tested for usability in an iterative manner by the clinical staff members. Lessons learnt: The development and implementation of digital health technologies need an interoperable and appropriate plan for data management including collection, sharing and integration. Engagements and implementation studies should be conceptualized and conducted alongside the digital health technology development. The priorities of end-users, and understanding context and regulatory landscape are crucial for success.

Clinical benefit of AI-assisted lung ultrasound in a resource-limited intensive care unit.

BACKGROUND: Interpreting point-of-care lung ultrasound (LUS) images from intensive care unit (ICU) patients can be challenging, especially in low- and middle- income countries (LMICs) where there is limited training available. Despite recent advances in the use of Artificial Intelligence (AI) to automate many ultrasound imaging analysis tasks, no AI-enabled LUS solutions have been proven to be clinically useful in ICUs, and specifically in LMICs. Therefore, we developed an AI solution that assists LUS practitioners and assessed its usefulness in  a low resource ICU. METHODS: This was a three-phase prospective study. In the first phase, the performance of four different clinical user groups in interpreting LUS clips was assessed. In the second phase, the performance of 57 non-expert clinicians with and without the aid of a bespoke AI tool for LUS interpretation was assessed in retrospective offline clips. In the third phase, we conducted a prospective study in the ICU where 14 clinicians were asked to carry out LUS examinations in 7 patients with and without our AI tool and we interviewed the clinicians regarding the usability of the AI tool. RESULTS: The average accuracy of beginners' LUS interpretation was 68.7% [95% CI 66.8-70.7%] compared to 72.2% [95% CI 70.0-75.6%] in intermediate, and 73.4% [95% CI 62.2-87.8%] in advanced users. Experts had an average accuracy of 95.0% [95% CI 88.2-100.0%], which was significantly better than beginners, intermediate and advanced users (p < 0.001). When supported by our AI tool for interpreting retrospectively acquired clips, the non-expert clinicians improved their performance from an average of 68.9% [95% CI 65.6-73.9%] to 82.9% [95% CI 79.1-86.7%], (p < 0.001). In prospective real-time testing, non-expert clinicians improved their baseline performance from 68.1% [95% CI 57.9-78.2%] to 93.4% [95% CI 89.0-97.8%], (p < 0.001) when using our AI tool. The time-to-interpret clips improved from a median of 12.1 s (IQR 8.5-20.6) to 5.0 s (IQR 3.5-8.8), (p < 0.001) and clinicians' median confidence level improved from 3 out of 4 to 4 out of 4 when using our AI tool. CONCLUSIONS: AI-assisted LUS can help non-expert clinicians in an LMIC ICU improve their performance in interpreting LUS features more accurately, more quickly and more confidently.

Mapping patient pathways and understanding clinical decision-making in dengue management to inform the development of digital health tools.

BACKGROUND: Dengue is a common viral illness and severe disease results in life-threatening complications. Healthcare services in low- and middle-income countries treat the majority of dengue cases worldwide. However, the clinical decision-making processes which result in effective treatment are poorly characterised within this setting. In order to improve clinical care through interventions relating to digital clinical decision-support systems (CDSS), we set out to establish a framework for clinical decision-making in dengue management to inform implementation. METHODS: We utilised process mapping and task analysis methods to characterise existing dengue management at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. This is a tertiary referral hospital which manages approximately 30,000 patients with dengue each year, accepting referrals from Ho Chi Minh city and the surrounding catchment area. Initial findings were expanded through semi-structured interviews with clinicians in order to understand clinical reasoning and cognitive factors in detail. A grounded theory was used for coding and emergent themes were developed through iterative discussions with clinician-researchers. RESULTS: Key clinical decision-making points were identified: (i) at the initial patient evaluation for dengue diagnosis to decide on hospital admission and the provision of fluid/blood product therapy, (ii) in those patients who develop severe disease or other complications, (iii) at the point of recurrent shock in balancing the need for fluid therapy with complications of volume overload. From interviews the following themes were identified: prioritising clinical diagnosis and evaluation over existing diagnostics, the role of dengue guidelines published by the Ministry of Health, the impact of seasonality and caseload on decision-making strategies, and the potential role of digital decision-support and disease scoring tools. CONCLUSIONS: The study highlights the contemporary priorities in delivering clinical care to patients with dengue in an endemic setting. Key decision-making processes and the sources of information that were of the greatest utility were identified. These findings serve as a foundation for future clinical interventions and improvements in healthcare. Understanding the decision-making process in greater detail also allows for development and implementation of CDSS which are suited to the local context.

Hyperinflammatory Syndrome, Natural Killer Cell Function, and Genetic Polymorphisms in the Pathogenesis of Severe Dengue.

BACKGROUND: Severe dengue, characterized by shock and organ dysfunction, is driven by an excessive host immune response. We investigated the role of hyperinflammation in dengue pathogenesis. METHODS: Patients recruited into an observational study were divided into 3 plasma leak severity grades. Hyperinflammatory biomarkers were measured at 4 time points. Frequencies, activation, and cytotoxic potential of natural killer (NK) cells were analyzed by flow cytometry. RNA was extracted from sorted CD56+ NK cells and libraries were prepared using SMART-Seq and sequenced using HiSeq3000 (Illumina). RESULTS: Sixty-nine patients were included (grade 0, 42 patients; grade 1, 19 patients; grade 2, 8 patients). Patients with grade 2 leakage had higher biomarkers than grade 0, including higher peak ferritin levels (83.3% vs 45.2%) and H-scores (median, 148.5 vs 105.5). NK cells from grade 2 patients exhibited decreased expression of perforin and granzyme B and activation markers. RNA sequencing revealed 3 single-nucleotide polymorphisms in NK cell functional genes associated with more severe leakage-NK cell lectin-like receptor K1 gene (KLRK1) and perforin 1 (PRF1). CONCLUSIONS: Features of hyperinflammation are associated with dengue severity, including higher biomarkers, impaired NK cell function, and polymorphisms in NK cell cytolytic function genes (KLRK1 and PRF1). Trials of immunomodulatory therapy in these patients is now warranted.

Novel Clinical Monitoring Approaches for Reemergence of Diphtheria Myocarditis, Vietnam.

Diphtheria is a life-threatening, vaccine-preventable disease caused by toxigenic Corynebacterium bacterial species that continues to cause substantial disease and death worldwide, particularly in vulnerable populations. Further outbreaks of vaccine-preventable diseases are forecast because of health service disruptions caused by the coronavirus disease pandemic. Diphtheria causes a spectrum of clinical disease, ranging from cutaneous forms to severe respiratory infections with systemic complications, including cardiac and neurologic. In this synopsis, we describe a case of oropharyngeal diphtheria in a 7-year-old boy in Vietnam who experienced severe myocarditis complications. We also review the cardiac complications of diphtheria and discuss how noninvasive bedside imaging technologies to monitor myocardial function and hemodynamic parameters can help improve the management of this neglected infectious disease.

The compensatory reserve index predicts recurrent shock in patients with severe dengue.

BACKGROUND: Dengue shock syndrome (DSS) is one of the major clinical phenotypes of severe dengue. It is defined by significant plasma leak, leading to intravascular volume depletion and eventually cardiovascular collapse. The compensatory reserve Index (CRI) is a new physiological parameter, derived from feature analysis of the pulse arterial waveform that tracks real-time changes in central volume. We investigated the utility of CRI to predict recurrent shock in severe dengue patients admitted to the ICU. METHODS: We performed a prospective observational study in the pediatric and adult intensive care units at the Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. Patients were monitored with hourly clinical parameters and vital signs, in addition to continuous recording of the arterial waveform using pulse oximetry. The waveform data was wirelessly transmitted to a laptop where it was synchronized with the patient's clinical data. RESULTS: One hundred three patients with suspected severe dengue were recruited to this study. Sixty-three patients had the minimum required dataset for analysis. Median age was 11 years (IQR 8-14 years). CRI had a negative correlation with heart rate and moderate negative association with blood pressure. CRI was found to predict recurrent shock within 12 h of being measured (OR 2.24, 95% CI 1.54-3.26), P < 0.001). The median duration from CRI measurement to the first recurrent shock was 5.4 h (IQR 2.9-6.8). A CRI cutoff of 0.4 provided the best combination of sensitivity and specificity for predicting recurrent shock (0.66 [95% CI 0.47-0.85] and 0.86 [95% CI 0.80-0.92] respectively). CONCLUSION: CRI is a useful non-invasive method for monitoring intravascular volume status in patients with severe dengue.

A modified Sequential Organ Failure Assessment score for dengue: development, evaluation and proposal for use in clinical trials.

BACKGROUND: Dengue is a neglected tropical disease, for which no therapeutic agents have shown clinical efficacy to date. Clinical trials have used strikingly variable clinical endpoints, which hampers reproducibility and comparability of findings. We investigated a delta modified Sequential Organ Failure Assessment (delta mSOFA) score as a uniform composite clinical endpoint for use in clinical trials investigating therapeutics for moderate and severe dengue. METHODS: We developed a modified SOFA score for dengue, measured and evaluated its performance at baseline and 48 h after enrolment in a prospective observational cohort of 124 adults admitted to a tertiary referral hospital in Vietnam with dengue shock. The modified SOFA score included pulse pressure in the cardiovascular component. Binary logistic regression, cox proportional hazard and linear regression models were used to estimate association between mSOFA, delta mSOFA and clinical outcomes. RESULTS: The analysis included 124 adults with dengue shock. 29 (23.4%) patients required ICU admission for organ support or due to persistent haemodynamic instability: 9/124 (7.3%) required mechanical ventilation, 8/124 (6.5%) required vasopressors, 6/124 (4.8%) required haemofiltration and 5/124 (4.0%) patients died. In univariate analyses, higher baseline and delta (48 h) mSOFA score for dengue were associated with admission to ICU, requirement for organ support and mortality, duration of ICU and hospital admission and IV fluid use. CONCLUSIONS: The baseline and delta mSOFA scores for dengue performed well to discriminate patients with dengue shock by clinical outcomes, including duration of ICU and hospital admission, requirement for organ support and death. We plan to use delta mSOFA as the primary endpoint in an upcoming host-directed therapeutic trial and investigate the performance of this score in other phenotypes of severe dengue in adults and children.

Higher Plasma Viremia in the Febrile Phase Is Associated With Adverse Dengue Outcomes Irrespective of Infecting Serotype or Host Immune Status: An Analysis of 5642 Vietnamese Cases.

BACKGROUND: One of the generally accepted constructs of dengue pathogenesis is that clinical disease severity is at least partially dependent upon plasma viremia, yet data on plasma viremia in primary versus secondary infections and in relation to clinically relevant endpoints remain limited and contradictory. METHODS: Using a large database comprising detailed clinical and laboratory characterization of Vietnamese participants enrolled in a series of research studies executed over a 15-year period, we explored relationships between plasma viremia measured by reverse transcription-polymerase chain reaction and 3 clinically relevant endpoints-severe dengue, plasma leakage, and hospitalization-in the dengue-confirmed cases. All 4 dengue serotypes and both primary and secondary infections were well represented. In our logistic regression models we allowed for a nonlinear effect of viremia and for associations between viremia and outcome to differ by age, serotype, host immune status, and illness day at study enrollment. RESULTS: Among 5642 dengue-confirmed cases we identified 259 (4.6%) severe dengue cases, 701 (12.4%) patients with plasma leakage, and 1441 of 4008 (40.0%) patients recruited in outpatient settings who were subsequently hospitalized. From the early febrile phase onwards, higher viremia increased the risk of developing all 3 endpoints, but effect sizes were modest (ORs ranging from 1.12-1.27 per 1-log increase) compared with the effects of a secondary immune response (ORs, 1.67-7.76). The associations were consistent across age, serotype, and immune status groups, and in the various sensitivity and subgroup analyses we undertook. CONCLUSIONS: Higher plasma viremia is associated with increased dengue severity, regardless of serotype or immune status.

Dengue: Status of current and under-development vaccines.

Dengue is an emerging mosquito-borne viral infection with increasing reports of outbreaks. The clinical picture ranges from a benign febrile illness through to severe and potentially fatal manifestations. No specific anti-viral treatment exists, and therapy only consists of supportive care. During the last three decades, several attempts to develop an effective vaccine have been made. The first dengue vaccine to obtain licensure was Dengvaxia, which was authorized in 2015 and is currently available in over 20 countries. Its use has been approved with strict limitations regarding age and serostatus of the recipients, highlighting the necessity for a more safe and efficacious vaccine. At present several vaccine, candidates are undergoing clinical and pre-clinical trials. The most advanced candidates are TDV and TDV 003/005, two live-attenuated vaccines, but another 15 vaccines are under development, introducing novel immunization strategies to the traditional dengue vaccine scenario. This work reviews the current research status on dengue vaccines.

C-reactive protein as a potential biomarker for disease progression in dengue: a multi-country observational study.

BACKGROUND: Dengue infection can cause a wide spectrum of clinical outcomes. The severe clinical manifestations occur sufficiently late in the disease course, during day 4-6 of illness, to allow a window of opportunity for risk stratification. Markers of inflammation may be useful biomarkers. We investigated the value of C-reactive protein (CRP) measured early on illness days 1-3 to predict dengue disease outcome and the difference in CRP levels between dengue and other febrile illnesses (OFI). METHOD: We performed a nested case-control study using the clinical data and samples collected from the IDAMS-consortium multi-country study. This was a prospective multi-center observational study that enrolled almost 8000 participants presenting with a dengue-like illness to outpatient facilities in 8 countries across Asia and Latin America. Predefined severity definitions of severe and intermediate dengue were used as the primary outcomes. A total of 281 cases with severe/intermediate dengue were compared to 836 uncomplicated dengue patients as controls (ratio 1:3), and also 394 patients with OFI. RESULTS: In patients with confirmed dengue, median (interquartile range) of CRP level within the first 3 days was 30.2 mg/L (12.4-61.2 mg/L) (uncomplicated dengue, 28.6 (10.5-58.9); severe or intermediate dengue, 34.0 (17.4-71.8)). Higher CRP levels in the first 3 days of illness were associated with a higher risk of severe or intermediate outcome (OR 1.17, 95% CI 1.07-1.29), especially in children. Higher CRP levels, exceeding 30 mg/L, also associated with hospitalization (OR 1.37, 95% CI 1.14-1.64) and longer fever clearance time (HR 0.84, 95% CI 0.76-0.93), especially in adults. CRP levels in patients with dengue were higher than patients with potential viral infection but lower than patients with potential bacterial infection, resulting in a quadratic association between dengue diagnosis and CRP, with levels of approximately 30 mg/L associated with the highest risk of having dengue. CRP had a positive correlation with total white cell count and neutrophils and negative correlation with lymphocytes, but did not correlate with liver transaminases, albumin, or platelet nadir. CONCLUSIONS: In summary, CRP measured in the first 3 days of illness could be a useful biomarker for early dengue risk prediction and may assist differentiating dengue from other febrile illnesses.

Definitions for warning signs and signs of severe dengue according to the WHO 2009 classification: Systematic review of literature.

Since warning signs and signs of severe dengue are defined differently between studies, we conducted a systematic review on how researchers defined these signs. We conducted an electronic search in Scopus to identify relevant articles, using key words including dengue, "warning signs," "severe dengue," and "classification." A total of 491 articles were identified through this search strategy and were subsequently screened by 2 independent reviewers for definitions of any of the warning or severe signs in the 2009 WHO dengue classification. We included all original articles published in English after 2009, classifying dengue by the 2009 WHO classification or providing the additional definition or criterion of warning signs and severity (besides the information of 2009 WHO). Analysis of the extracted data from 44 articles showed wide variations among definitions and cutoff values used by physicians to classify patients diagnosed with dengue infection. The establishment of clear definitions for warning signs and severity is essential to prevent unnecessary hospitalization and harmonizing the interpretation and comparability of epidemiological studies dedicated to dengue infection.

Dengue-Associated Posterior Reversible Encephalopathy Syndrome, Vietnam.

Dengue can cause neurologic complications in addition to the more common manifestations of plasma leakage and coagulopathy. Posterior reversible encephalopathy syndrome has rarely been described in dengue, although the pathophysiology of endothelial dysfunction likely underlies both. We describe a case of dengue-associated posterior reversible encephalopathy syndrome and discuss diagnosis and management.

Endothelial Nitric Oxide Pathways in the Pathophysiology of Dengue: A Prospective Observational Study.

BACKGROUND: Dengue can cause increased vascular permeability that may lead to hypovolemic shock. Endothelial dysfunction may underlie this; however, the association of endothelial nitric oxide (NO) pathways with disease severity is unknown. METHODS: We performed a prospective observational study in 2 Vietnamese hospitals, assessing patients presenting early (<72 hours of fever) and patients hospitalized with warning signs or severe dengue. The reactive hyperemic index (RHI), which measures endothelium-dependent vasodilation and is a surrogate marker of endothelial function and NO bioavailability, was evaluated using peripheral artery tonometry (EndoPAT), and plasma levels of l-arginine, arginase-1, and asymmetric dimethylarginine were measured at serial time-points. The main outcome of interest was plasma leakage severity. RESULTS: Three hundred fourteen patients were enrolled; median age of the participants was 21(interquartile range, 13-30) years. No difference was found in the endothelial parameters between dengue and other febrile illness. Considering dengue patients, the RHI was significantly lower for patients with severe plasma leakage compared to those with no leakage (1.46 vs 2.00; P < .001), over acute time-points, apparent already in the early febrile phase (1.29 vs 1.75; P = .012). RHI correlated negatively with arginase-1 and positively with l-arginine (P = .001). CONCLUSIONS: Endothelial dysfunction/NO bioavailability is associated with worse plasma leakage, occurs early in dengue illness and correlates with hypoargininemia and high arginase-1 levels.

Association of Microvascular Function and Endothelial Biomarkers With Clinical Outcome in Dengue: An Observational Study.

BACKGROUND: The hallmark of severe dengue is increased microvascular permeability, but alterations in the microcirculation and their evolution over the course of dengue are unknown. METHODS: We conducted a prospective observational study to evaluate the sublingual microcirculation using side-stream dark-field imaging in patients presenting early (<72 hours after fever onset) and patients hospitalized with warning signs or severe dengue in Vietnam. Clinical findings, microvascular function, global hemodynamics assessed with echocardiography, and serological markers of endothelial activation were determined at 4 time points. RESULTS: A total of 165 patients were enrolled. No difference was found between the microcirculatory parameters comparing dengue with other febrile illnesses. The proportion of perfused vessels (PPV) and the mean flow index (MFI) were lower in patients with dengue with plasma than those without leakage (PPV, 88.1% vs 90.6% [P = .01]; MFI, 2.1 vs 2.4 [P = .007]), most markedly during the critical phase. PPV and MFI were correlated with the endothelial activation markers vascular cell adhesion molecule 1 (P < .001 for both) and angiopoietin 2 (P < .001 for both), negatively correlated. CONCLUSIONS: Modest microcirculatory alterations occur in dengue, are associated with plasma leakage, and are correlate with molecules of endothelial activation, angiopoietin 2 and vascular cell adhesion molecule 1.

A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia.

BACKGROUND: Trypanosomais a genus of unicellular parasitic flagellate protozoa.Trypanosoma bruceispecies and Trypanosoma cruziare the major agents of human trypanosomiasis; other Trypanosomaspecies can cause human disease, but are rare. In March 2015, a 38-year-old woman presented to a healthcare facility in southern Vietnam with fever, headache, and arthralgia. Microscopic examination of blood revealed infection with Trypanosoma METHODS: Microscopic observation, polymerase chain reaction (PCR) amplification of blood samples, and serological testing were performed to identify the infecting species. The patient's blood was screened for the trypanocidal protein apolipoprotein L1 (APOL1), and a field investigation was performed to identify the zoonotic source. RESULTS: PCR amplification and serological testing identified the infecting species as Trypanosoma evansi.Despite relapsing 6 weeks after completing amphotericin B therapy, the patient made a complete recovery after 5 weeks of suramin. The patient was found to have 2 wild-type APOL1 alleles and a normal serum APOL1 concentration. After responsive animal sampling in the presumed location of exposure, cattle and/or buffalo were determined to be the most likely source of the infection, with 14 of 30 (47%) animal blood samples testing PCR positive forT. evansi. CONCLUSIONS: We report the first laboratory-confirmed case ofT. evansiin a previously healthy individual without APOL1 deficiency, potentially contracted via a wound while butchering raw beef, and successfully treated with suramin. A linked epidemiological investigation revealed widespread and previously unidentified burden ofT. evansiin local cattle, highlighting the need for surveillance of this infection in animals and the possibility of further human cases.

Microvascular and endothelial function for risk prediction in dengue: an observational study.

BACKGROUND: Dengue infection can result in a wide spectrum of disease. The defining feature of severe disease is increased capillary permeability, which can lead to hypovolaemic shock. Microvascular and endothelial dysfunction might underlie hypovolaemic shock, but they have not been assessed clinically. We aimed to investigate the use of microvascular assessment as a prognostic method in dengue. METHODS: This is an ongoing prospective observational study that aims to recruit 300 participants: children over the age of 3 years and adults presenting to two outpatient departments in Vietnam with fever of less than 72 h duration and suspected dengue, and patients admitted to hospital with warning signs or severe disease. Participants are being clinically assessed daily for 6 days, and 2 weeks later. Microvascular imaging using sublingual sidestream darkfield imaging (SDF) and endothelial function testing using peripheral artery tonometry are being performed at enrolment, defervescence, and follow-up FINDINGS: To date, 167 patients have been recruited (92 outpatient arm, 75 inpatient arm, median age 27 years [IQR 21-33], 78 male [47%]). Dengue has been confirmed in 67 individuals in the outpatient arm, of whom 29 (43%) developed warning signs, three (4%) developed severe disease, and 35 had uncomplicated dengue; the other 25 outpatients (27%) were diagnosed as other febrile illness. At enrolment, the reactive hyperaemic index, a marker of endothelial function, was lowest in the patients who went on to develop severe dengue (median 1·54, IQR 1·36-1·77) followed by those who developed warning signs (1·78, 1·43-2·36) and then uncomplicated dengue (2·18, 1·65-2·24). Initial SDF results showed a lower proportion of perfused vessels and mean flow index during the febrile phase of dengue compared with follow-up, and were worst in the severe group at defervescence. INTERPRETATION: This study of vascular function at serial timepoints in dengue is, to our knowledge, the first and most comprehensive. Our preliminary results suggest that microvascular and endothelial dysfunction are associated with severity of dengue, and occur before the appearance of severe clinical manifestations. These techniques might be useful in risk prediction in dengue. A limitation is that a formal sample size could not be calculated because no previous microvascular data in dengue exist. FUNDING: Wellcome Trust.

Clinical evaluation of dengue and identification of risk factors for severe disease: protocol for a multicentre study in 8 countries.

BACKGROUND: The burden of dengue continues to increase globally, with an estimated 100 million clinically apparent infections occurring each year. Although most dengue infections are asymptomatic, patients can present with a wide spectrum of clinical symptoms ranging from mild febrile illness through to severe manifestations of bleeding, organ impairment, and hypovolaemic shock due to a systemic vascular leak syndrome. Clinical diagnosis of dengue and identification of which patients are likely to develop severe disease remain challenging. This study aims to improve diagnosis and clinical management through approaches designed a) to differentiate between dengue and other common febrile illness within 72 h of fever onset, and b) among patients with dengue to identify markers that are predictive of the likelihood of evolving to a more severe disease course. METHOD/DESIGN: This is a prospective multi-centre observational study aiming to enrol 7-8000 participants aged ≥ 5 years presenting with a febrile illness consistent with dengue to outpatient health facilities in 8 countries across Asia and Latin America. Patients presenting within 72 h of fever onset who do not exhibit signs of severe disease are eligible for the study. A broad range of clinical and laboratory parameters are assessed daily for up to 6 days during the acute illness, and also at a follow up visit 1 week later. DISCUSSION: Data from this large cohort of patients, enrolled early with undifferentiated fever, will be used to develop a practical diagnostic algorithm and a robust clinical case definition for dengue. Additionally, among patients with confirmed dengue we aim to identify simple clinical and laboratory parameters associated with progression to a more severe disease course. We will also investigate early virological and serological correlates of severe disease, and examine genetic associations in this large heterogeneous cohort. In addition the results will be used to assess the new World Health Organization classification scheme for dengue in practice, and to update the guidelines for "Integrated Management of Childhood Illness" used in dengue-endemic countries. TRIAL REGISTRATION: NCT01550016. Registration Date: March 7, 2012.