Professor Bridget Wills

Research Area: Global Health
Scientific Themes: Tropical Medicine & Global Health
Keywords: Dengue, Emerging infectious diseases, Diagnosis and clinical management and International Child Health
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Dengue is the most important mosquito-borne viral infection of humans. It represents a major problem for public health services around the world, with recent estimates indicating that 390 million infections occur each year, of which almost 100 million are symptomatic. Infection can be caused by any one of four closely related dengue viral (DENV) serotypes, transmitted between people by Aedes mosquitoes. The disease is endemic across much of Asia and Latin America, and the global footprint is expanding rapidly in parallel with the dispersal of efficient mosquito vectors across tropical and subtropical regions of the world.

One of the most important aims of the research carried out is to improve diagnosis and clinical management of patients. The majority of people who get bitten by an infected mosquito can experience an unpleasant febrile illness that is a bit like flu, but in a very small proportion of cases there can be complications which can be very serious and occasionally result in death. Differentiating dengue from other common viral illnesses during the initial febrile phase, and identifying patients at risk of disease progression, are key questions for dengue case-management as the appropriate dengue therapeutic intervention can be chosen. There are two main approaches to dengue therapeutic interventions. The first is to target the virus through the use of an antiviral, and the second is to target the immune response to the virus since this is thought to be partly responsible for the development of severe disease.

There are no collaborations listed for this principal investigator.

Anh NT, Nhu LNT, Van HMT, Hong NTT, Thanh TT, Hang VTT, Ny NTH, Nguyet LA, Phuong TTL, Nhan LNT et al. 2018. Emerging Coxsackievirus A6 Causing Hand, Foot and Mouth Disease, Vietnam. Emerg Infect Dis, 24 (4), pp. 654-662. | Show Abstract | Read more

Hand, foot and mouth disease (HFMD) is a major public health issue in Asia and has global pandemic potential. Coxsackievirus A6 (CV-A6) was detected in 514/2,230 (23%) of HFMD patients admitted to 3 major hospitals in southern Vietnam during 2011-2015. Of these patients, 93 (18%) had severe HFMD. Phylogenetic analysis of 98 genome sequences revealed they belonged to cluster A and had been circulating in Vietnam for 2 years before emergence. CV-A6 movement among localities within Vietnam occurred frequently, whereas viral movement across international borders appeared rare. Skyline plots identified fluctuations in the relative genetic diversity of CV-A6 corresponding to large CV-A6-associated HFMD outbreaks worldwide. These data show that CV-A6 is an emerging pathogen and emphasize the necessity of active surveillance and understanding the mechanisms that shape the pathogen evolution and emergence, which is essential for development and implementation of intervention strategies.

Mai NTH, Phu NH, Nghia HDT, Phuong TM, Duc DT, Chau NVV, Wills B, Lim CCT, Thwaites G, Simmons CP, Yacoub S. 2018. Dengue-Associated Posterior Reversible Encephalopathy Syndrome, Vietnam. Emerg Infect Dis, 24 (2), pp. 402-404. | Show Abstract | Read more

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.

Carrington LB, Tran BCN, Le NTH, Luong TTH, Nguyen TT, Nguyen PT, Nguyen CVV, Nguyen HTC, Vu TT, Vo LT et al. 2018. Field- and clinically derived estimates ofWolbachia-mediated blocking of dengue virus transmission potential inAedes aegyptimosquitoes. Proc Natl Acad Sci U S A, 115 (2), pp. 361-366. | Show Abstract | Read more

ThewMel strain ofWolbachiacan reduce the permissiveness ofAedes aegyptimosquitoes to disseminated arboviral infections. Here, we report thatwMel-infectedAe. aegypti(Ho Chi Minh City background), when directly blood-fed on 141 viremic dengue patients, have lower dengue virus (DENV) transmission potential and have a longer extrinsic incubation period than their wild-type counterparts. ThewMel-infected mosquitoes that are field-reared have even greater relative resistance to DENV infection when fed on patient-derived viremic blood meals. This is explained by an increased susceptibility of field-reared wild-type mosquitoes to infection than laboratory-reared counterparts. Collectively, these field- and clinically relevant findings support the continued careful field-testing ofwMel introgression for the biocontrol ofAe. aegypti-born arboviruses.

Duyen HTL, Cerny D, Trung DT, Pang J, Velumani S, Toh YX, Qui PT, Hao NV, Simmons C, Haniffa M et al. 2017. Skin dendritic cell and T cell activation associated with dengue shock syndrome. Sci Rep, 7 (1), pp. 14224. | Show Abstract | Read more

The pathogenesis of severe dengue remains unclear, particularly the mechanisms underlying the plasma leakage that results in hypovolaemic shock in a small proportion of individuals. Maximal leakage occurs several days after peak viraemia implicating immunological pathways. Skin is a highly vascular organ and also an important site of immune reactions with a high density of dendritic cells (DCs), macrophages and T cells. We obtained skin biopsies and contemporaneous blood samples from patients within 24 hours of onset of dengue shock syndrome (DSS), and from healthy controls. We analyzed cell subsets by flow cytometry, and soluble mediators and antibodies by ELISA; the percentage of migratory CD1a+dermal DCs was significantly decreased in the DSS patients, and skin CD8+T cells were activated, but there was no accumulation of dengue-specific antibodies. Inflammatory monocytic cells were not observed infiltrating the skin of DSS cases on whole-mount histology, although CD14dimcells disappeared from blood.

Trieu HT, Anh NTK, Vuong HNT, Dao TTM, Hoa NTX, Tuong VNC, Dinh PT, Wills B, Qui PT, Van Tan L et al. 2017. Long-term outcome in survivors of neonatal tetanus following specialist intensive care in Vietnam. BMC Infect Dis, 17 (1), pp. 646. | Show Abstract | Read more

BACKGROUND: Neonatal tetanus continues to occur in many resource-limited settings but there are few data regarding long-term neurological outcome from the disease, especially in settings with critical care facilities. METHODS: We assessed long-term outcome following neonatal tetanus in infants treated in a pediatric intensive care unit in southern Vietnam. Neurological and neurodevelopmental testing was performed in 17 survivors of neonatal tetanus and 18 control children from the same communities using tools previously validated in Vietnamese children. RESULTS: The median age of children assessed was 36 months. Eight neonatal tetanus survivors and 9 community control cases aged < 42 months were tested using the Bayley III Scales of Infant and Toddler Development (Bayley III-VN) and 8 neonatal tetanus survivors and 9 community controls aged ≥42 months were tested using the Movement Assessment Battery for Children. No significant reductions in growth indices or neurodevelopmental scores were shown in survivors of neonatal tetanus compared to controls although there was a trend towards lower scores in neonatal tetanus survivors. Neurological examination was normal in all children except for two neonatal tetanus survivors with perceptive deafness and one child with mild gross motor abnormality. Neonatal tetanus survivors who had expienced severe disease (Ablett grade ≥ 3) had lower total Bayley III-VN scores than those with mild disease (15 (IQR 14-18) vs 24 (IQR 19-27), p = 0.05) with a significantly lower cognitive domain score (3 (IQR 2-6) severe disease vs 7 (IQR 7-8) mild disease, p = 0.02). CONCLUSIONS: Neonatal tetanus is associated with long-term sequelae in those with severe disease. In view of these findings, prevention of neonatal tetanus should remain a priority.

Rabaa MA, Girerd-Chambaz Y, Duong Thi Hue K, Vu Tuan T, Wills B, Bonaparte M, van der Vliet D, Langevin E, Cortes M, Zambrano B et al. 2017. Genetic epidemiology of dengue viruses in phase III trials of the CYD tetravalent dengue vaccine and implications for efficacy. Elife, 6 | Show Abstract | Read more

This study defined the genetic epidemiology of dengue viruses (DENV) in two pivotal phase III trials of the tetravalent dengue vaccine, CYD-TDV, and thereby enabled virus genotype-specific estimates of vaccine efficacy (VE). Envelope gene sequences (n = 661) from 11 DENV genotypes in 10 endemic countries provided a contemporaneous global snapshot of DENV population genetics and revealed high amino acid identity between the E genes of vaccine strains and wild-type viruses from trial participants, including at epitope sites targeted by virus neutralising human monoclonal antibodies.Post-hocanalysis of all CYD14/15 trial participants revealed a statistically significant genotype-level VE association within DENV-4, where efficacy was lowest against genotype I. In subgroup analysis of trial participants age 9-16 years, VE estimates appeared more balanced within each serotype, suggesting that genotype-level heterogeneity may be limited in older children. Post-licensure surveillance is needed to monitor vaccine performance against the backdrop of DENV sequence diversity and evolution.

Yacoub S, Trung TH, Lam PK, Thien VHN, Hai DHT, Phan TQ, Nguyet OPK, Quyen NTH, Simmons CP, Broyd C et al. 2017. Cardio-haemodynamic assessment and venous lactate in severe dengue: Relationship with recurrent shock and respiratory distress. PLoS Negl Trop Dis, 11 (7), pp. e0005740. | Show Abstract | Read more

BACKGROUND: Dengue can cause plasma leakage that may lead to dengue shock syndrome (DSS). In approximately 30% of DSS cases, recurrent episodes of shock occur. These patients have a higher risk of fluid overload, respiratory distress and poor outcomes. We investigated the association of echocardiographically-derived cardiac function and intravascular volume parameters plus lactate levels, with the outcomes of recurrent shock and respiratory distress in severe dengue. METHODS/PRINCIPLE FINDINGS: We performed a prospective observational study in Paediatric and adult ICU, at the Hospital for Tropical Diseases (HTD), Ho Chi Minh City, Vietnam. Patients with dengue were enrolled within 12 hours of admission to paediatric or adult ICU. A haemodynamic assessment and portable echocardiograms were carried out daily for 5 days from enrolment and all interventions recorded. 102 patients were enrolled; 22 patients did not develop DSS, 48 had a single episode of shock and 32 had recurrent shock. Patients with recurrent shock had a higher enrolment pulse than those with 1 episode or no shock (median: 114 vs. 100 vs. 100 b/min, P = 0.002), significantly lower Stroke Volume Index (SVI), (median: 21.6 vs. 22.8 vs. 26.8mls/m2, P<0.001) and higher lactate levels (4.2 vs. 2.9 vs. 2.2 mmol/l, P = 0.001). Higher SVI and worse left ventricular function (higher Left Myocardial Performance Index) on study days 3-5 was associated with the secondary endpoint of respiratory distress. There was an association between the total IV fluid administered during the ICU admission and respiratory distress (OR: 1.03, 95% CI 1.01-1.06, P = 0.001). Admission lactate levels predicted patients who subsequently developed recurrent shock (P = 0.004), and correlated positively with the total IV fluid volume received (rho: 0.323, P = 0.001) and also with admission ALT (rho: 0.764, P<0.001) and AST (rho: 0.773, P<0.001). CONCLUSIONS/SIGNIFICANCE: Echo-derived intravascular volume assessment and venous lactate levels can help identify dengue patients at high risk of recurrent shock and respiratory distress in ICU. These findings may serve to, not only assist in the management of DSS patients, but also these haemodynamic endpoints could be used in future dengue fluid intervention trials.

Quyen NTH, Kien DTH, Rabaa M, Tuan NM, Vi TT, Van Tan L, Hung NT, Tuan HM, Van Tram T, Le Da Ha N et al. 2017. Chikungunya and Zika Virus Cases Detected against a Backdrop of Endemic Dengue Transmission in Vietnam. Am J Trop Med Hyg, 97 (1), pp. 146-150. | Show Abstract | Read more

Between 2010 and 2014, four chikungunya and two Zika virus infections were identified among 8,105 febrile children in southern Vietnam. Zika viruses were linked to French Polynesian strains, chikungunya to Cambodian strains. Against a backdrop of endemic dengue transmission, chikungunya and Zika present an additional arboviral disease burden in Vietnam.

Yacoub S, Lam PK, Huynh TT, Nguyen Ho HH, Dong Thi HT, Van NT, Lien LT, Ha QNT, Le DHT, Mongkolspaya J et al. 2017. Endothelial Nitric Oxide Pathways in the Pathophysiology of Dengue: A Prospective Observational Study. Clin Infect Dis, 65 (9), pp. 1453-1461. | Show Abstract | Read more

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.

Whitehorn J, Kien DTH, Quyen NTH, Wills B, Van Vinh Chau N, Tam DTH, Tuan NM, Jaenisch T, Hibberd M, Khor CC, Simmons CP. 2017. Genetic variants of MICB and PLCE1 and associations with the laboratory features of dengue. BMC Infect Dis, 17 (1), pp. 412. | Show Abstract | Read more

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.

Lam PK, Ngoc TV, Thu Thuy TT, Hong Van NT, Nhu Thuy TT, Hoai Tam DT, Dung NM, Hanh Tien NT, Thanh Kieu NT, Simmons C et al. 2017. The value of daily platelet counts for predicting dengue shock syndrome: Results from a prospective observational study of 2301 Vietnamese children with dengue. PLoS Negl Trop Dis, 11 (4), pp. e0005498. | Show Abstract | Read more

BACKGROUND: Dengue is the most important mosquito-borne viral infection to affect humans. Although it usually manifests as a self-limited febrile illness, complications may occur as the fever subsides. A systemic vascular leak syndrome that sometimes progresses to life-threatening hypovolaemic shock is the most serious complication seen in children, typically accompanied by haemoconcentration and thrombocytopenia. Robust evidence on risk factors, especially features present early in the illness course, for progression to dengue shock syndrome (DSS) is lacking. Moreover, the potential value of incorporating serial haematocrit and platelet measurements in prediction models has never been assessed. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed data from a prospective observational study of Vietnamese children aged 5-15 years admitted with clinically suspected dengue to the Hospital for Tropical Diseases in Ho Chi Minh City between 2001 and 2009. The analysis population comprised all children with laboratory-confirmed dengue enrolled between days 1-4 of illness. Logistic regression was the main statistical model for all univariate and multivariable analyses. The prognostic value of daily haematocrit levels and platelet counts were assessed using graphs and separate regression models fitted on each day of illness. Among the 2301 children included in the analysis, 143 (6%) progressed to DSS. Significant baseline risk factors for DSS included a history of vomiting, higher temperature, a palpable liver, and a lower platelet count. Prediction models that included serial daily platelet counts demonstrated better ability to discriminate patients who developed DSS from others, than models based on enrolment information only. However inclusion of daily haematocrit values did not improve prediction of DSS. CONCLUSIONS/SIGNIFICANCE: Daily monitoring of platelet counts is important to help identify patients at high risk of DSS. Development of dynamic prediction models that incorporate signs, symptoms, and daily laboratory measurements, could improve DSS prediction and thereby reduce the burden on health services in endemic areas.

Vi TT, Kien DT, Wills B, Simmons CP. 2017. A29 Wolbachia for dengue control; will dengue viruses evolve resistance? Virus Evol, 3 (Suppl 1), | Read more

Nguyen MT, Ho TN, Nguyen VVC, Nguyen TH, Ha MT, Ta VT, Nguyen LDH, Phan L, Han KQ, Duong THK et al. 2017. An Evidence-Based Algorithm for Early Prognosis of Severe Dengue in the Outpatient Setting. Clin Infect Dis, 64 (5), pp. 656-663. | Show Abstract | Read more

Background: Early prediction of severe dengue could significantly assist patient triage and case management. Methods: We prospectively investigated 7563 children with ≤3 days of fever recruited in the outpatient departments of 6 hospitals in southern Vietnam between 2010 and 2013. The primary endpoint of interest was severe dengue (2009 World Health Organization Guidelines), and predefined risk variables were collected at the time of enrollment to enable prognostic model development. Results: The analysis population comprised 7544 patients, of whom 2060 (27.3%) had laboratory-confirmed dengue; nested among these were 117 (1.5%) severe cases. In the multivariate logistic model, a history of vomiting, lower platelet count, elevated aspartate aminotransferase (AST) level, positivity in the nonstructural protein 1 (NS1) rapid test, and viremia magnitude were all independently associated with severe dengue. The final prognostic model (Early Severe Dengue Identifier [ESDI]) included history of vomiting, platelet count, AST level. and NS1 rapid test status. Conclusions: The ESDI had acceptable performance features (area under the curve = 0.95, sensitivity 87% (95% confidence interval [CI], 80%-92%), specificity 88% (95% CI, 87%-89%), positive predictive value 10% (95% CI, 9%-12%), and negative predictive value of 99% (95% CI, 98%-100%) in the population of all 7563 enrolled children. A score chart, for routine clinical use, was derived from the prognostic model and could improve triage and management of children presenting with fever in dengue-endemic areas.

Yacoub S, Lam PK, Vu LHM, Le TL, Ha NT, Toan TT, Van NT, Quyen NTH, Le Duyen HT, Van Kinh N et al. 2016. Association of Microvascular Function and Endothelial Biomarkers With Clinical Outcome in Dengue: An Observational Study. J Infect Dis, 214 (5), pp. 697-706. | Show Abstract | Read more

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.

Cerny D, Thi Le DH, The TD, Zuest R, Kg S, Velumani S, Khor CC, Mori L, Simmons CP, Poidinger M et al. 2016. Complete human CD1a deficiency on Langerhans cells due to a rare point mutation in the coding sequence. J Allergy Clin Immunol, 138 (6), pp. 1709-1712.e11. | Read more

Nguyen NM, Whitehorn JS, Luong Thi Hue T, Nguyen Thanh T, Mai Xuan T, Vo Xuan H, Nguyen Thi Cam H, Nguyen Thi Hong L, Nguyen HL, Dong Thi Hoai T et al. 2016. Physicians, Primary Caregivers and Topical Repellent: All Under-Utilised Resources in Stopping Dengue Virus Transmission in Affected Households. PLoS Negl Trop Dis, 10 (5), pp. e0004667. | Show Abstract | Read more

BACKGROUND: Primary health care facilities frequently manage dengue cases on an ambulatory basis for the duration of the patient's illness. There is a great opportunity for specific messaging, aimed to reduce dengue virus (DENV) transmission in and around the home, to be directly targeted toward this high-risk ambulatory patient group, as part of an integrated approach to dengue management. The extent however, to which physicians understand, and can themselves effectively communicate strategies to stop focal DENV transmission around an ambulatory dengue case is unknown; the matter of patient comprehension and recollection then ensues. In addition, the effectiveness of N,N-diethyl-3-methylbenzamide (DEET)-based insect repellent in protecting dengue patients from Aedes aegypti mosquitoes' bites has not been investigated. METHODOLOGY: A knowledge, attitude and practice (KAP) survey, focusing on the mechanisms of DENV transmission and prevention, was performed using semi-structured questionnaires. This survey was targeted towards the patients and family members providing supportive care, and physicians routinely involved in dengue patient management in Southern Vietnam. An additional clinical observational study was conducted to measure the efficacy of a widely-used 13% DEET-based insect repellent to repel Ae. aegypti mosquitoes from the forearms of dengue cases and matched healthy controls. PRINCIPAL FINDINGS: Among both the physician (n = 50) and patient (n = 49) groups there were several respondents lacking a coherent understanding of DENV transmission, leading to some inappropriate attitudes and inadequate acute preventive practices in the household. The application of insect repellent to protect patients and their relatives from mosquito bites was frequently recommended by majority of physicians (78%) participating in the survey. Nevertheless, our tested topical application of 13% DEET conferred only ~1hr median protection time from Ae. aegypti landing. This is notably shorter than that advertised on the manufacturer's label. No differences in landing time between febrile dengue cases or matched healthy controls (n = 19 experiments) were observed. CONCLUSION/SIGNIFICANCE: Our study identifies missed opportunities for primary care physicians to improve public health through communication of strategies that could prevent focal dengue transmission in and around a case household. We advocate better access to more efficient communication methods for physicians and auxilliary health workers, supporting to educate those at high risk of DENV transmission. Our empirical testing of a widely-available 13% DEET-based repellent was limited in its protective efficacy against Ae. aegypti mosquito bites, and therefore DENV transmission, suggesting more frequent application is necessary to be beneficial.

Van Vinh Chau N, Buu Chau L, Desquesnes M, Herder S, Phu Huong Lan N, Campbell JI, Van Cuong N, Yimming B, Chalermwong P, Jittapalapong S et al. 2016. A Clinical and Epidemiological Investigation of the First Reported Human Infection With the Zoonotic Parasite Trypanosoma evansi in Southeast Asia. Clin Infect Dis, 62 (8), pp. 1002-1008. | Show Abstract | Read more

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.

Rosenberger KD, Lum L, Alexander N, Junghanss T, Wills B, Jaenisch T, DENCO Clinical Study Group. 2016. Vascular leakage in dengue--clinical spectrum and influence of parenteral fluid therapy. Trop Med Int Health, 21 (3), pp. 445-453. | Show Abstract | Read more

OBJECTIVE: Clinical management of dengue relies on careful monitoring of fluid balance combined with judicious intravenous (IV) fluid therapy. However, in patients with significant vascular leakage, IV fluids may aggravate serosal fluid accumulation and result in respiratory distress. METHODS: Trained physicians followed suspected dengue cases prospectively at seven hospitals across Asia and Latin America, using a comprehensive case report form that included daily clinical assessment and detailed documentation of parenteral fluid therapy. Applying Cox regression, we evaluated risk factors for the development of shock or respiratory distress with fluid accumulation. RESULTS: Most confirmed dengue patients (1524/1734, 88%) never experienced dengue shock syndrome (DSS). Among those with DSS, 176/210 (84%) had fluid accumulation, and in the majority (83%), this was detectable clinically. Among all cases with clinically detectable fluid accumulation, 179/447 (40%) were diagnosed with shock or respiratory distress. The risk for respiratory distress with fluid accumulation increased significantly as the infused volume over the preceding 24 h increased (hazard ratio 1.18 per 10 ml/kg increase; P < 0.001). Longer duration of IV therapy, use of a fluid bolus in the preceding 24 h, female gender and poor nutrition also constituted independent risk factors. CONCLUSIONS: Shock and respiratory distress are relatively rare manifestations of dengue, but some evidence of fluid accumulation is seen in around 50% of cases. IV fluids play a crucial role in management, but they must be administered with caution. Clinically and/or radiologically detectable fluid accumulations have potential as intermediate severity endpoints for therapeutic intervention trials and/or pathogenesis studies.

Jaenisch T, Tam DTH, Kieu NTT, Van Ngoc T, Nam NT, Van Kinh N, Yacoub S, Chanpheaktra N, Kumar V, See LLC et al. 2016. Clinical evaluation of dengue and identification of risk factors for severe disease: protocol for a multicentre study in 8 countries. BMC Infect Dis, 16 (1), pp. 120. | Show Abstract | Read more

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.

Qui PT, Khanh TH, Trieu HT, Giang PT, Bich NN, Thoa LPK, Nhan LNT, Sabanathan S, Van Doorn R, Toan ND et al. 2016. Intravenous magnesium sulfate for the management of severe hand, foot, and mouth disease with autonomic nervous system dysregulation in Vietnamese children: study protocol for a randomized controlled trial. Trials, 17 (1), pp. 98. | Show Abstract | Read more

BACKGROUND: Over the last 15 years, hand, foot, and mouth disease (HFMD) has emerged as a major public health burden across the Asia-Pacific region. A small proportion of HFMD patients, typically those infected with enterovirus 71 (EV71), develop brainstem encephalitis with autonomic nervous system (ANS) dysregulation and may progress rapidly to cardiopulmonary failure and death. Although milrinone has been reported to control hypertension and support myocardial function in two small studies, in practice, a number of children still deteriorate despite this treatment. Magnesium sulfate (MgSO4) is a cheap, safe, and readily available medication that is effective in managing tetanus-associated ANS dysregulation and has shown promise when used empirically in EV71-confirmed severe HFMD cases. METHODS/DESIGN: We describe the protocol for a randomized, placebo-controlled, double-blind trial of intravenous MgSO4 in Vietnamese children diagnosed clinically with HFMD plus ANS dysregulation with systemic hypertension. A loading dose of MgSO4 or identical placebo is given over 20 min followed by a maintenance infusion for 72 h according to response, aiming for Mg levels two to three times the normal level in the treatment arm. The primary endpoint is a composite of disease progression within 72 h defined as follows: development of pre-specified blood pressure criteria necessitating the addition of milrinone, the need for ventilation, shock, or death. Secondary endpoints comprise these parameters singly, plus other clinical endpoints including the following: requirement for other inotropic agents; duration of hospitalization; presence of neurological sequelae at discharge in survivors; and neurodevelopmental status assessed 6 months after discharge. The number and severity of adverse events observed in the two treatment arms will also be compared. Based on preliminary data from a case series, and allowing for some losses, 190 patients (95 in each arm) will allow detection of a 50 % reduction in disease progression with 90 % power at a two-sided 5 % significance level. DISCUSSION: Given the large numbers of HFMD cases currently being seen in hospitals in Asia, if MgSO4 is shown to be effective in controlling ANS dysregulation and preventing severe HFMD complications, this finding would be important to pediatric care throughout the region. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01940250 (Registered 22 August 2013).

Whitehorn J, Nguyen CVV, Khanh LP, Kien DTH, Quyen NTH, Tran NTT, Hang NT, Truong NT, Hue Tai LT, Cam Huong NT et al. 2016. Lovastatin for the Treatment of Adult Patients With Dengue: A Randomized, Double-Blind, Placebo-Controlled Trial. Clin Infect Dis, 62 (4), pp. 468-476. | Show Abstract | Read more

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.

Trieu HT, Lubis IN, Qui PT, Yen LM, Wills B, Thwaites CL, Sabanathan S. 2016. Neonatal Tetanus in Vietnam: Comprehensive Intensive Care Support Improves Mortality. J Pediatric Infect Dis Soc, 5 (2), pp. 227-230. | Show Abstract | Read more

We report a 66% reduction in neonatal tetanus mortality after introducing a new management bundle integrating antibiotic therapy, muscle relaxation and invasive monitoring. The latter allowed rapid detection of autonomic instability which was treated with magnesium sulphate. This is the first report of its use in neonatal tetanus.

Tuan NM, Nhan HT, Chau NVV, Hung NT, Tuan HM, Tram TV, Ha NLD, Loi P, Quang HK, Kien DTH et al. 2015. Sensitivity and specificity of a novel classifier for the early diagnosis of dengue. PLoS Negl Trop Dis, 9 (4), pp. e0003638. | Show Abstract | Read more

BACKGROUND: Dengue is the commonest arboviral disease of humans. An early and accurate diagnosis of dengue can support clinical management, surveillance and disease control and is central to achieving the World Health Organisation target of a 50% reduction in dengue case mortality by 2020. METHODS: 5729 children with fever of <72 hrs duration were enrolled into this multicenter prospective study in southern Vietnam between 2010-2012. A composite of gold standard diagnostic tests identified 1692 dengue cases. Using statistical methods, a novel Early Dengue Classifier (EDC) was developed that used patient age, white blood cell count and platelet count to discriminate dengue cases from non-dengue cases. RESULTS: The EDC had a sensitivity of 74.8% (95%CI: 73.0-76.8%) and specificity of 76.3% (95%CI: 75.2-77.6%) for the diagnosis of dengue. As an adjunctive test alongside NS1 rapid testing, sensitivity of the composite test was 91.6% (95%CI: 90.4-92.9%). CONCLUSIONS: We demonstrate that the early diagnosis of dengue can be enhanced beyond the current standard of care using a simple evidence-based algorithm. The results should support patient management and clinical trials of specific therapies.

Sabanathan S, Wills B, Gladstone M. 2015. Child development assessment tools in low-income and middle-income countries: how can we use them more appropriately? Arch Dis Child, 100 (5), pp. 482-488. | Show Abstract | Read more

Global emphasis has shifted beyond reducing child survival rates to improving health and developmental trajectories in childhood. Optimum early childhood experience is believed to allow children to benefit fully from educational opportunities resulting in improved human capital. Investment in early childhood initiatives in low-income and middle-income countries (LMICs) is increasing. These initiatives use early childhood developmental assessment tools (CDATs) as outcome measures. CDATs are also key measures in the evaluation of programmatic health initiatives in LMICs, influencing public health policy. Interpretation of CDAT outcomes requires understanding of their structure and psychometric properties. This article reviews the structure and main methods of CDAT development with specific considerations when applied in LMICs.

Ferguson NM, Kien DTH, Clapham H, Aguas R, Trung VT, Chau TNB, Popovici J, Ryan PA, O'Neill SL, McGraw EA et al. 2015. Modeling the impact on virus transmission of Wolbachia-mediated blocking of dengue virus infection of Aedes aegypti. Sci Transl Med, 7 (279), pp. 279ra37. | Show Abstract | Read more

Dengue is the most common arboviral infection of humans and is a public health burden in more than 100 countries. Aedes aegypti mosquitoes stably infected with strains of the intracellular bacterium Wolbachia are resistant to dengue virus (DENV) infection and are being tested in field trials. To mimic field conditions, we experimentally assessed the vector competence of A. aegypti carrying the Wolbachia strains wMel and wMelPop after challenge with viremic blood from dengue patients. We found that wMelPop conferred strong resistance to DENV infection of mosquito abdomen tissue and largely prevented disseminated infection. wMel conferred less resistance to infection of mosquito abdomen tissue, but it did reduce the prevalence of mosquitoes with infectious saliva. A mathematical model of DENV transmission incorporating the dynamics of viral infection in humans and mosquitoes was fitted to the data collected. Model predictions suggested that wMel would reduce the basic reproduction number, R0, of DENV transmission by 66 to 75%. Our results suggest that establishment of wMelPop-infected A. aegypti at a high frequency in a dengue-endemic setting would result in the complete abatement of DENV transmission. Establishment of wMel-infected A. aegypti is also predicted to have a substantial effect on transmission that would be sufficient to eliminate dengue in low or moderate transmission settings but may be insufficient to achieve complete control in settings where R0 is high. These findings develop a framework for selecting Wolbachia strains for field releases and for calculating their likely impact.

Whitehorn J, Kien DTH, Nguyen NM, Nguyen HL, Kyrylos PP, Carrington LB, Tran CNB, Quyen NTH, Thi LV, Le Thi D et al. 2015. Comparative Susceptibility of Aedes albopictus and Aedes aegypti to Dengue Virus Infection After Feeding on Blood of Viremic Humans: Implications for Public Health. J Infect Dis, 212 (8), pp. 1182-1190. | Show Abstract | Read more

Aedes albopictus is secondary to Aedes aegypti as a vector of dengue viruses (DENVs) in settings of endemicity, but it plays an important role in areas of dengue emergence. This study compared the susceptibility of these 2 species to DENV infection by performing 232 direct blood-feeding experiments on 118 viremic patients with dengue in Vietnam. Field-derived A. albopictus acquired DENV infections as readily as A. aegypti after blood feeding. Once infected, A. albopictus permitted higher concentrations of DENV RNA to accumulate in abdominal tissues, compared with A. aegypti. However, the odds of A. albopictus having infectious saliva were lower than the odds observed for A. aegypti (odds ratio, 0.70; 95% confidence interval, .52-.93). These results quantitate the susceptibility of A. albopictus to DENV infection and will assist parameterization of models for predicting disease risk in settings where A. albopictus is present.

Yacoub S, Wertheim H, Simmons CP, Screaton G, Wills B. 2015. Microvascular and endothelial function for risk prediction in dengue: an observational study. Lancet, 385 Suppl 1 pp. S102. | Show Abstract | Read more

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.

Anders KL, Nga LH, Thuy NTV, Ngoc TV, Tam CT, Tai LTH, Truong NT, Duyen HTL, Trung VT, Kien DTH et al. 2015. Households as foci for dengue transmission in highly urban Vietnam. PLoS Negl Trop Dis, 9 (2), pp. e0003528. | Show Abstract | Read more

BACKGROUND: Dengue control programs commonly employ reactive insecticide spraying around houses of reported cases, with the assumption that most dengue virus (DENV) transmission occurs in the home. Focal household transmission has been demonstrated in rural settings, but it is unclear whether this holds true in dense and mobile urban populations. We conducted a prospective study of dengue clustering around households in highly urban Ho Chi Minh City, Vietnam. METHODS: We enrolled 71 index cases with suspected dengue (subsequently classified as 52 dengue cases and 19 non-dengue controls); each initiated the enrollment of a cluster of 25-35 household members and neighbors who were followed up over 14 days. Incident DENV infections in cluster participants were identified by RT-PCR, NS1-ELISA, and/or DENV-IgM/-IgG seroconversion, and recent infections by DENV-IgM positivity at baseline. PRINCIPAL FINDINGS/CONCLUSIONS: There was no excess risk of DENV infection within dengue case clusters during the two-week follow-up, compared to control clusters, but the prevalence of recent DENV infection at baseline was two-fold higher in case clusters than controls (OR 2.3, 95%CI 1.0-5.1, p = 0.05). Prevalence of DENV infection in Aedes aegypti was similar in case and control houses, and low overall (1%). Our findings are broadly consistent with household clustering of dengue risk, but indicate that any clustering is at a short temporal scale rather than sustained chains of localized transmission. This suggests that reactive perifocal insecticide spraying may have a limited impact in this setting.

Yacoub S, Wills B. 2015. Dengue: an update for clinicians working in non-endemic areas. Clin Med (Lond), 15 (1), pp. 82-85. | Show Abstract | Read more

Dengue is one of the most rapidly emerging viral infections globally, with 2.5 billion people now thought to live in dengue-endemic areas. In addition, reports of travel-related and autochthonous infections are increasing in non-endemic areas. Most patients with dengue experience a self-limiting febrile illness, but a proportion develop potentially life threatening complications around the time of fever clearance, including plasma leakage occasionally leading to shock, bleeding, and organ impairment. As dengue can present with non-specific symptoms of fever, headache and myalgias, the potential for misdiagnosis and inappropriate management by medical staff inexperienced with the disease is a concern. This short review will outline the latest World Health Organisation disease classification, potential complications, clinical assessment and management for clinicians working in non-endemic areas.

Simmons CP, McPherson K, Van Vinh Chau N, Hoai Tam DT, Young P, Mackenzie J, Wills B. 2015. Recent advances in dengue pathogenesis and clinical management. Vaccine, 33 (50), pp. 7061-7068. | Show Abstract | Read more

This review describes and commentates on recent advances in the understanding of dengue pathogenesis and immunity, plus clinical research on vaccines and therapeutics. We expand specifically on the role of the dermis in dengue virus infection, the contribution of cellular and humoral immune responses to pathogenesis and immunity, NS1 and mechanisms of virus immune evasion. Additionally we review a series of therapeutic intervention trials for dengue, as well as recent clinical research aimed at improving clinical diagnosis, risk prediction and disease classification.

Lam PK, Tam DTH, Dung NM, Tien NTH, Kieu NTT, Simmons C, Farrar J, Wills B, Wolbers M. 2015. A Prognostic Model for Development of Profound Shock among Children Presenting with Dengue Shock Syndrome. PLoS One, 10 (5), pp. e0126134. | Show Abstract | Read more

PURPOSE: To identify risk factors and develop a prediction model for the development of profound and recurrent shock amongst children presenting with dengue shock syndrome (DSS). METHODS: We analyzed data from a prospective cohort of children with DSS recruited at the Paediatric Intensive Care Unit of the Hospital for Tropical Disease in Ho Chi Minh City, Vietnam. The primary endpoint was "profound DSS", defined as ≥2 recurrent shock episodes (for subjects presenting in compensated shock), or ≥1 recurrent shock episodes (for subjects presenting initially with decompensated/hypotensive shock), and/or requirement for inotropic support. Recurrent shock was evaluated as a secondary endpoint. Risk factors were pre-defined clinical and laboratory variables collected at the time of presentation with shock. Prognostic model development was based on logistic regression and compared to several alternative approaches. RESULTS: The analysis population included 1207 children of whom 222 (18%) progressed to "profound DSS" and 433 (36%) had recurrent shock. Independent risk factors for both endpoints included younger age, earlier presentation, higher pulse rate, higher temperature, higher haematocrit and, for females, worse hemodynamic status at presentation. The final prognostic model for "profound DSS" showed acceptable discrimination (AUC=0.69 for internal validation) and calibration and is presented as a simple score-chart. CONCLUSIONS: Several risk factors for development of profound or recurrent shock among children presenting with DSS were identified. The score-chart derived from the prognostic models should improve triage and management of children presenting with DSS in dengue-endemic areas.

Bich TD, Pham OKN, Hai DHT, Nguyen NM, Van HN, The TD, Wills B, Yacoub S. 2015. A pregnant woman with acute cardiorespiratory failure: dengue myocarditis. Lancet, 385 (9974), pp. 1260. | Read more

Lam PK, Trieu HT, Lubis IND, Loan HT, Thuy TTD, Wills B, Parry CM, Day NPJ, Qui PT, Yen LM, Thwaites CL. 2015. Prognosis of neonatal tetanus in the modern management era: an observational study in 107 Vietnamese infants. Int J Infect Dis, 33 pp. 7-11. | Show Abstract | Read more

OBJECTIVES: Most data regarding the prognosis in neonatal tetanus originate from regions where limited resources have historically impeded management. It is not known whether recent improvements in critical care facilities in many low- and middle-income countries have affected indicators of a poor prognosis in neonatal tetanus. We aimed to determine the factors associated with worse outcomes in a Vietnamese hospital with neonatal intensive care facilities. METHODS: Data were collected from 107 cases of neonatal tetanus. Clinical features on admission were analyzed against mortality and a combined endpoint of 'death or prolonged hospital stay'. RESULTS: Multivariable analysis showed that only younger age (odds ratio (OR) for mortality 0.69, 95% confidence interval (CI) 0.48-0.98) and lower weight (OR for mortality 0.06, 95% CI 0.01-0.54) were significantly associated with both the combined endpoint and death. A shorter period of onset (OR 0.94, 95% CI 0.88-0.99), raised white cell count (OR 1.17, 95% CI 1.02-1.35), and time between first symptom and admission (OR 3.77, 95% CI 1.14-12.51) were also indicators of mortality. CONCLUSIONS: Risk factors for a poor outcome in neonatal tetanus in a setting with critical care facilities include younger age, lower weight, delay in admission, and leukocytosis.

Jaenisch T, Junghanss T, Wills B, Brady OJ, Eckerle I, Farlow A, Hay SI, McCall PJ, Messina JP, Ofula V et al. 2014. Dengue expansion in Africa-not recognized or not happening? Emerg Infect Dis, 20 (10), | Show Abstract | Read more

An expert conference on Dengue in Africa was held in Accra, Ghana, in February 2013 to consider key questions regarding the possible expansion of dengue in Africa. Four key action points were highlighted to advance our understanding of the epidemiology of dengue in Africa. First, dengue diagnostic tools must be made more widely available in the healthcare setting in Africa. Second, representative data need to be collected across Africa to uncover the true burden of dengue. Third, established networks should collaborate to produce these types of data. Fourth, policy needs to be informed so the necessary steps can be taken to provide dengue vector control and health services.

Yacoub S, Wills B. 2014. Predicting outcome from dengue. BMC Med, 12 (1), pp. 147. | Show Abstract | Read more

Dengue is emerging as one of the most abundant vector-borne disease globally. Although the majority of infections are asymptomatic or result in only a brief systemic viral illness, a small proportion of patients develop potentially fatal complications. These severe manifestations, including a unique plasma leakage syndrome, a coagulopathy sometimes accompanied by bleeding, and organ impairment, occur relatively late in the disease course, presenting a window of opportunity to identify the group of patients likely to progress to these complications. However, as yet, differentiating this group from the thousands of milder cases seen each day during outbreaks remains challenging, and simple and inexpensive strategies are urgently needed in order to improve case management and to facilitate appropriate use of limited resources. This review will cover the current understanding of the risk factors associated with poor outcome in dengue. We focus particularly on the clinical features of the disease and on conventional investigations that are usually accessible in mid-level healthcare facilities in endemic areas, and then discuss a variety of viral, immunological and vascular biomarkers that have the potential to improve risk prediction. We conclude with a description of several novel methods of assessing vascular function and intravascular volume status non-invasively.

Whitehorn J, Yacoub S, Anders KL, Macareo LR, Cassetti MC, Nguyen Van VC, Shi P-Y, Wills B, Simmons CP. 2014. Dengue therapeutics, chemoprophylaxis, and allied tools: state of the art and future directions. PLoS Negl Trop Dis, 8 (8), pp. e3025. | Show Abstract | Read more

Dengue is the most common arboviral disease of humans. There is an unmet need for a therapeutic intervention that reduces the duration and severity of dengue symptoms and diminishes the likelihood of severe complications. To this end, there are active discovery efforts in industry and academia to develop interventions, with a focus on small molecule inhibitors of dengue virus replication that are suitable for therapy or chemoprophylaxis. Advancements in animal models of dengue virus infection together with the possibility of a dengue human infection model have further enhanced the platform for dengue drug discovery. Whilst drug discovery efforts gestate, there are ongoing clinical research designed to benefit today's patients, including trials of supportive care interventions, and descriptive studies that should improve the ability of clinicians to make an accurate diagnosis early in the illness course and to identify patients most at risk of progression to severe disease. This review provides a state of the art summary of dengue drug discovery, clinical trials, and supportive allied research and reflects discussions at the 2nd International Dengue Therapeutics Workshop held in Ho Chi Minh City, Vietnam, in December 2013.

Yacoub S, Wertheim H, Simmons CP, Screaton G, Wills B. 2014. Cardiovascular manifestations of the emerging dengue pandemic. Nat Rev Cardiol, 11 (6), pp. 335-345. | Show Abstract | Read more

Dengue is one of the most important emerging viral diseases globally. The majority of symptomatic infections result in a relatively benign disease course. However, a small proportion of patients develop severe clinical manifestations, including bleeding, organ impairment, and endothelial dysfunction with increased capillary permeability causing hypovolaemic shock that can lead to cardiovascular collapse. Evidence is increasing that dengue can also cause myocardial impairment, arrhythmias and, occasionally, fulminant myocarditis. No antiviral agents or vaccines are licensed for dengue, and treatment remains supportive with judicious fluid replacement for patients with severe disease. Defining the role of cardiac dysfunction in the haemodynamic compromise of severe dengue has potentially important management implications. In this Review, we will outline the current understanding of the cardiovascular manifestations of dengue, including myocardial and vascular involvement, and conclude with a discussion of the available therapeutic options and potential future research directions.

Lam PK, Wills B. 2014. Reply to Thomas et al. Clin Infect Dis, 58 (7), pp. 1039-1040. | Read more

Cited:

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Scopus

Sabanathan S, Van Tan L, Thwaites L, Wills B, Qui PT, Van Doorn HR. 2014. Enterovirus 71 related severe hand, foot and mouth disease outbreaks in South-East Asia: Current situation and ongoing challenges Journal of Epidemiology and Community Health, 68 (6), pp. 500-502. | Read more

Jaenisch T, Sakuntabhai A, Wilder-Smith A. 2013. Dengue Research Funded by the European Commission-Scientific Strategies of Three European Dengue Research Consortia PLoS Neglected Tropical Diseases, 7 (12), pp. e2320-e2320. | Read more

Lam PK, Tam DTH, Diet TV, Tam CT, Tien NTH, Kieu NTT, Simmons C, Farrar J, Nga NTN, Qui PT et al. 2013. Clinical characteristics of Dengue shock syndrome in Vietnamese children: a 10-year prospective study in a single hospital. Clin Infect Dis, 57 (11), pp. 1577-1586. | Show Abstract | Read more

BACKGROUND: Dengue shock syndrome (DSS) is a severe manifestation of dengue virus infection that particularly affects children and young adults. Despite its increasing global importance, there are no prospective studies describing the clinical characteristics, management, or outcomes of DSS. METHODS: We describe the findings at onset of shock and the clinical evolution until discharge or death, from a comprehensive prospective dataset of 1719 Vietnamese children with laboratory-confirmed DSS managed on a single intensive care unit between 1999 and 2009. RESULTS: The median age of patients was 10 years. Most cases had secondary immune responses, with only 6 clear primary infections, and all 4 dengue virus serotypes were represented during the 10-year study. Shock occurred commonly between days 4 and 6 of illness. Clinical signs and symptoms were generally consistent with empirical descriptions of DSS, although at presentation 153 (9%) were still febrile and almost one-third had no bleeding. Overall, 31 (2%) patients developed severe bleeding, primarily from the gastrointestinal tract, 26 of whom required blood transfusion. Only 8 patients died, although 123 of 1719 (7%) patients had unrecordable blood pressure at presentation and 417 of the remaining 1596 (26%) were hypotensive for age. The majority recovered well with standard crystalloid resuscitation or following a single colloid infusion. All cases were classified as severe dengue, while only 70% eventually fulfilled all 4 criteria for the 1997 World Health Organization classification of dengue hemorrhagic fever. CONCLUSIONS: With prompt intervention and assiduous clinical care by experienced staff, the outcome of this potentially fatal condition can be excellent.

Lam PK, Tam DTH, Diet TV, Tam CT, Tien NTH, Kieu NTT, Simmons C, Farrar J, Qui PT, Dung NM et al. 2013. Dengue shock syndrome in children: clinical features and a prognostic model for profound shock TROPICAL MEDICINE & INTERNATIONAL HEALTH, 18 pp. 53-54.

Farrar JJ, Hien TT, Horstick O, Hung NT, Jaenisch T, Junghanns T, Kroeger A, Laksono IS, Lum L, Martinez E et al. 2013. Dogma in classifying dengue disease. Am J Trop Med Hyg, 89 (2), pp. 198-201. | Read more

Nguyet MN, Duong THK, Trung VT, Nguyen THQ, Tran CNB, Long VT, Dui LT, Nguyen HL, Farrar JJ, Holmes EC et al. 2013. Host and viral features of human dengue cases shape the population of infected and infectious Aedes aegypti mosquitoes. Proc Natl Acad Sci U S A, 110 (22), pp. 9072-9077. | Show Abstract | Read more

Dengue is the most prevalent arboviral disease of humans. The host and virus variables associated with dengue virus (DENV) transmission from symptomatic dengue cases (n = 208) to Aedes aegypti mosquitoes during 407 independent exposure events was defined. The 50% mosquito infectious dose for each of DENV-1-4 ranged from 6.29 to 7.52 log10 RNA copies/mL of plasma. Increasing day of illness, declining viremia, and rising antibody titers were independently associated with reduced risk of DENV transmission. High early DENV plasma viremia levels in patients were a marker of the duration of human infectiousness, and blood meals containing high concentrations of DENV were positively associated with the prevalence of infectious mosquitoes 14 d after blood feeding. Ambulatory dengue cases had lower viremia levels compared with hospitalized dengue cases but nonetheless at levels predicted to be infectious to mosquitoes. These data define serotype-specific viremia levels that vaccines or drugs must inhibit to prevent DENV transmission.

Tam DTH, Simmons CP, Wills BA. 2013. Reply to Halstead and Sayce et al. Clin Infect Dis, 56 (6), pp. 903-904. | Read more

Huynh TT, Nguyen VC, Wills BA. 2013. Resuscitation fluids. N Engl J Med, 369 (25), pp. 2461. | Read more

Nguyen THT, Nguyen THQ, Vu TT, Farrar J, Hoang TL, Dong THT, Ngoc Tran V, Phung KL, Wolbers M, Whitehead SS et al. 2013. Corticosteroids for dengue - why don't they work? PLoS Negl Trop Dis, 7 (12), pp. e2592. | Show Abstract | Read more

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.

Tam DTH, Simmons CP, Wills BA. 2013. Reply to Halstead and Sayce et al Clinical Infectious Diseases, 56 (6), pp. 903-904. | Read more

Whitehorn J, Chau TNB, Nguyet NM, Kien DTH, Quyen NTH, Trung DT, Pang J, Wills B, Van Vinh Chau N, Farrar J et al. 2013. Genetic variants of MICB and PLCE1 and associations with non-severe dengue. PLoS One, 8 (3), pp. e59067. | Show Abstract | Read more

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.

Hanh Tien NT, Lam PK, Duyen HTL, Ngoc TV, Ha PTT, Kieu NTT, Simmons C, Wolbers M, Wills B. 2013. Assessment of microalbuminuria for early diagnosis and risk prediction in dengue infections. PLoS One, 8 (1), pp. e54538. | Show Abstract | Read more

BACKGROUND: Dengue is the most important arboviral infection of humans. Following an initial febrile period, a small proportion of infected patients develop a vasculopathy, with children at particular risk for severe vascular leakage and shock. Differentiation between dengue and other common childhood illnesses is difficult during the early febrile phase, and risk prediction for development of shock is poor. The presence of microalbuminuria is recognized as a useful early predictor for subsequent complications in a number of other disorders with vascular involvement. Significant proteinuria occurs in association with dengue shock syndrome and it is possible that early-phase microalbuminuria may be helpful both for diagnosis of dengue and for identification of patients likely to develop severe disease. METHODOLOGY/PRINCIPAL FINDINGS: We measured formal urine albumin to creatinine ratios (UACRs) in daily samples obtained from a large cohort of children with suspected dengue recruited at two outpatient clinics in Ho Chi Minh City, Vietnam. Although UACRs were increased in the 465 confirmed dengue patients, with a significant time trend showing peak values around the critical period for dengue-associated plasma leakage, urine albumin excretion was also increased in the comparison group of 391 patients with other febrile illnesses (OFI). The dengue patients generally had higher UACRs than the OFI patients, but microalbuminuria, using the conventional cutoff of 30 mg albumin/g creatinine discriminated poorly between the two diagnostic groups in the early febrile phase. Secondly UACRs did not prove useful in predicting either development of warning signs for severe dengue or need for hospitalization. CONCLUSION/SIGNIFICANCE: Low-level albuminuria is common, even in relatively mild dengue infections, but is also present in many OFIs. Simple point-of-care UACR tests are unlikely to be useful for early diagnosis or risk prediction in dengue endemic areas.

Simmons CP, Wolbers M, Nguyen MN, Whitehorn J, Shi PY, Young P, Petric R, Nguyen VVC, Farrar J, Wills B. 2012. Therapeutics for dengue: recommendations for design and conduct of early-phase clinical trials. PLoS Negl Trop Dis, 6 (9), pp. e1752. | Read more

Tam DTH, Ngoc TV, Tien NTH, Kieu NTT, Thuy TTT, Thanh LTC, Tam CT, Truong NT, Dung NT, Qui PT et al. 2012. Effects of short-course oral corticosteroid therapy in early dengue infection in Vietnamese patients: a randomized, placebo-controlled trial. Clin Infect Dis, 55 (9), pp. 1216-1224. | Show Abstract | Read more

BACKGROUND: Patients with dengue can experience a variety of serious complications including hypovolemic shock, thrombocytopenia, and bleeding. These problems occur as plasma viremia is resolving and are thought to be immunologically mediated. Early corticosteroid therapy may prevent the development of such complications but could also prolong viral clearance. METHODS: We performed a randomized, placebo-controlled, blinded trial of low-dose (0.5 mg/kg) or high-dose (2 mg/kg) oral prednisolone therapy for 3 days in Vietnamese patients aged 5-20 years admitted with dengue and fever for ≤72 hours, aiming to assess potential harms from steroid use during the viremic phase. Intention-to-treat analysis was performed using linear trend tests with a range of clinical and virological endpoints specified in advance. In addition to recognized complications of dengue, we focused on the are under the curve for serial plasma viremia measurements and the number of days after enrollment to negative viremia and dengue nonstructural protein 1 status. RESULTS: Between August 2009 and January 2011, 225 participants were randomized to 1 of the 3 treatment arms. Baseline characteristics were similar across the groups. All patients recovered fully and adverse events were infrequent. Aside from a trend toward hyperglycemia in the steroid recipients, we found no association between treatment allocation and any of the predefined clinical, hematological, or virological endpoints. CONCLUSIONS: Use of oral prednisolone during the early acute phase of dengue infection was not associated with prolongation of viremia or other adverse effects. Although not powered to assess efficacy, we found no reduction in the development of shock or other recognized complications of dengue virus infection in this study.

Chau NVV, Simmons CP, Wills B. 2012. Dengue REPLY NEW ENGLAND JOURNAL OF MEDICINE, 367 (2), pp. 181-181.

Anders KL, Nguyet NM, Quyen NTH, Ngoc TV, Tram TV, Gan TT, Tung NT, Dung NT, Chau NVV, Wills B, Simmons CP. 2012. An evaluation of dried blood spots and oral swabs as alternative specimens for the diagnosis of dengue and screening for past dengue virus exposure. Am J Trop Med Hyg, 87 (1), pp. 165-170. | Show Abstract | Read more

Non-invasive specimens for dengue diagnosis may be preferable where venous blood is difficult to collect and/or process, such as community-based or remote settings or when sampling from young children. We evaluated the performance of oral swabs and dried blood spots (DBS), compared with plasma, in diagnosing acute dengue and screening for past dengue virus (DENV) exposure. DENV-specific immunoglobulin (Ig) M, IgG, and NS1 antigen were detected both in oral swabs and DBS from acute patients. Oral swabs were less sensitive (IgM: 68.7%, IgG: 91.9%, NS1: 64.7%), but retained good specificity (100%, 92.3%, 95.8%, respectively) compared with plasma. DBS displayed high sensitivity (IgM: 100%, IgG: 96%, NS1: 100%) and specificity (IgM: 75%, IgG: 93%). DENV RNA was amplified from DBS (sensitivity 95.6%) but not from oral swabs. DENV-IgG (indicative of past flavivirus exposure) were detected with moderate sensitivity (61.1%) but poor specificity (50%) in oral swabs from healthy volunteers. Dried blood spots allow sensitive and specific diagnosis of acute dengue by serological, molecular, and antigen detection methods. Oral swabs may be an adequate alternative where blood cannot be collected.

Trung DT, Thao LTT, Dung NM, Ngoc TV, Hien TT, Chau NVV, Wolbers M, Tam DTH, Farrar J, Simmons C, Wills B. 2012. Clinical features of dengue in a large Vietnamese cohort: intrinsically lower platelet counts and greater risk for bleeding in adults than children. PLoS Negl Trop Dis, 6 (6), pp. e1679. | Show Abstract | Read more

BACKGROUND: As dengue spreads to new geographical regions and the force of infection changes in existing endemic areas, a greater breadth of clinical presentations is being recognised. Clinical experience suggests that adults manifest a pattern of complications different from those observed in children, but few reports have described the age-related spectrum of disease in contemporaneous groups of patients recruited at the same geographical location. METHODOLOGY/PRINCIPAL FINDINGS: Using detailed prospectively collected information from ongoing studies that encompass the full spectrum of hospitalised dengue cases admitted to a single hospital in southern Vietnam, we compared clinical and laboratory features, management, and outcome for 647 adults and 881 children with confirmed dengue. Signs of vascular leakage and shock were more frequent and more severe in children than adults, while bleeding manifestations and organ involvement were more common in adults. Additionally, adults experienced significantly more severe thrombocytopenia. Secondary infection but not serotype was independently associated with greater thrombocytopenia, although with a smaller effect than age-group. The effect of age-group on platelet count was also apparent in the values obtained several weeks after recovery, indicating that healthy adults have intrinsically lower counts compared to children. CONCLUSIONS/SIGNIFICANCE: There are clear distinctions between adults and children in the pattern of complications seen in association with dengue infection, and these depend partly on intrinsic age-dependent physiological differences. Knowledge of such differences is important to inform research on disease pathogenesis, as well as to encourage development of management guidelines that are appropriate to the age-groups at risk.

Whitehorn J, Rodriguez Roche R, Guzman MG, Martinez E, Gomez WV, Nainggolan L, Laksono IS, Mishra A, Lum L, Faiz A et al. 2012. Prophylactic platelets in dengue: survey responses highlight lack of an evidence base. PLoS Negl Trop Dis, 6 (6), pp. e1716. | Show Abstract | Read more

Dengue is the most important arboviral infection of humans. Thrombocytopenia is frequently observed in the course of infection and haemorrhage may occur in severe disease. The degree of thrombocytopenia correlates with the severity of infection, and may contribute to the risk of haemorrhage. As a result of this prophylactic platelet transfusions are sometimes advocated for the prevention of haemorrhage. There is currently no evidence to support this practice, and platelet transfusions are costly and sometimes harmful. We conducted a global survey to assess the different approaches to the use of platelets in dengue. Respondents were all physicians involved with the treatment of patients with dengue. Respondents were asked that their answers reflected what they would do if they were the treating physician. We received responses from 306 physicians from 20 different countries. The heterogeneity of the responses highlights the variation in clinical practice and lack of an evidence base in this area and underscores the importance of prospective clinical trials to address this key question in the clinical management of patients with dengue.

Yacoub S, Grifiths A, Chau TTH, Simmons C, Wills B, Hien TT, Henein M, Farrar J. 2012. Cardiac function and haemodynamics in Vietnemese patients with different dengue severity grades INTERNATIONAL JOURNAL OF INFECTIOUS DISEASES, 16 pp. E119-E119. | Read more

Akbar NA, Allende I, Balmaseda A, Coelho ICB, da Cunha RV, Datta B, Devi SS, Farrar J, Gaczkowski R, Guzman MG et al. 2012. Regarding "Dengue--how best to classify it". Clin Infect Dis, 54 (12), pp. 1820-1821. | Read more

Whitehorn J, Van Vinh Chau N, Truong NT, Tai LTH, Van Hao N, Hien TT, Wolbers M, Merson L, Dung NTP, Peeling R et al. 2012. Lovastatin for adult patients with dengue: protocol for a randomised controlled trial. Trials, 13 (1), pp. 203. | Show Abstract | Read more

BACKGROUND: Dengue is the most important vector-borne viral infection of man, with approximately 2 billion people living in areas at risk. Infection results in a range of manifestations from asymptomatic infection through to life-threatening shock and haemorrhage. One of the hallmarks of severe dengue is vascular endothelial disruption. There is currently no specific therapy and clinical management is limited to supportive care. Statins are a class of drug initially developed for lipid lowering. There has been considerable recent interest in their effects beyond lipid lowering. These include anti-inflammatory effects at the endothelium. In addition, it is possible that lovastatin may have an anti-viral effect against dengue. Observational data suggest that the use of statins may improve outcomes for such conditions as sepsis and pneumonia. This paper describes the protocol for a randomised controlled trial investigating a short course of lovastatin therapy in adult patients with dengue. METHODS/DESIGN: A randomised, double-blind, placebo-controlled trial will investigate the effects of lovastatin therapy in the treatment of dengue. The trial will be conducted in two phases with an escalation of dose between phases if an interim safety review is satisfactory. This is an exploratory study focusing on safety and there are no data on which to base a sample size calculation. A target sample size of 300 patients in the second phase, enrolled over two dengue seasons, was chosen based on clinical judgement and feasibility considerations. In a previous randomised trial in dengue, about 10% and 30% of patients experienced at least one serious adverse event or adverse event, respectively. With 300 patients, we will have 80% power to detect an increase of 12% (from 10% to 22%) or 16% (from 30% to 46%) in the frequency of adverse events. Furthermore, this sample size ensures some power to explore the efficacy of statins. DISCUSSION: The development of a dengue therapeutic that can attenuate disease would be an enormous advance in global health. The favourable effects of statins on the endothelium, their good safety profile and their low cost make lovastatin an attractive therapeutic candidate. TRIAL REGISTRATION: International Standard Randomised Controlled Trial Number ISRCTN03147572.

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Simmons CP, Farrar JJ, Chau NVV, Wills B. 2012. CURRENT CONCEPTS Dengue NEW ENGLAND JOURNAL OF MEDICINE, 366 (15), pp. 1423-1432. | Read more

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Yacoub S, Griffiths A, Hong Chau TT, Simmons CP, Wills B, Hien TT, Henein M, Farrar J. 2012. Cardiac function in Vietnamese patients with different dengue severity grades Critical Care Medicine, 40 (2), pp. 477-483. | Show Abstract | Read more

Objective: Dengue continues to cause significant global morbidity and mortality. Severe disease is characterized by cardiovascular compromise from capillary leakage. Cardiac involvement in dengue has also been reported but has not been adequately studied. Setting: H ospital for Tropical Diseases, Ho Chi Minh City, Vietnam. Subjects and Design: Seventy-nine patients aged 8-6 yrs with different dengue severity grades were studied using echocardiography including tissue Doppler imaging. The patients were split into severity grades: dengue, dengue with warning signs, and severe dengue. Changes in cardiac functional parameters and hemodynamic indices were monitored over the hospital stay. Intervention: None. Measurements and Main Results; Patients with severe dengue had worse cardiac function compared with dengue in the form of left ventricular systolic dysfunction with increased left myocardial performance index (0.58 [0.26-0.80] vs. 0.38 [0.22-0.70] , p = .006). Septal myocardial systolic velocities were reduced (6.4 [4.8-10] vs. 8.1 [6-13] cm/s, p = .01) as well as right ventricular systolic (11.4 [7.5-17] vs. 13.5 [10-17] cm/s, p = .016) and diastolic velocities (13 [8-23] vs. 17 [12-25] cm/s, p = .0026). In the severe group, these parameters improved from hospital admission to discharge; septal myocardial systolic velocities to 8.8 (7-11) cm/s (p = .002), right ventricular myocardial systolic velocities to 15.0 (11.8-23) cm/s, (p = .003), and diastolic velocity to 21 (11-25) cm/s (p = .002). Patients with cardiac impairment were more likely to have significant pleural effusions. Conclusions: Patients with severe dengue have evidence of systolic and diastolic cardiac impairment with septal and right ventricular wall being predominantly affected. Copyright © 2012 by the Society of Critical Care Medicine and Lippincott Williams & Wilkins.

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Midgley CM, Bajwa-Joseph M, Vasanawathana S, Limpitikul W, Wills B, Flanagan A, Waiyaiya E, Tran HB, Cowper AE, Chotiyarnwong P et al. 2011. An In-Depth Analysis of Original Antigenic Sin in Dengue Virus Infection (vol 85, pg 410, 2011) JOURNAL OF VIROLOGY, 85 (22), pp. 12100-12100. | Read more

Yacoub S, Griffiths A, Chau TTH, Simmons CP, Wills B, Hien TT, Henein M, Farrar J. 2012. Cardiac function in Vietnamese patients with different dengue severity grades. Crit Care Med, 40 (2), pp. 477-483. | Show Abstract | Read more

OBJECTIVE: Dengue continues to cause significant global morbidity and mortality. Severe disease is characterized by cardiovascular compromise from capillary leakage. Cardiac involvement in dengue has also been reported but has not been adequately studied. SETTING: Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam. SUBJECTS AND DESIGN: Seventy-nine patients aged 8-6 yrs with different dengue severity grades were studied using echocardiography including tissue Doppler imaging. The patients were split into severity grades: dengue, dengue with warning signs, and severe dengue. Changes in cardiac functional parameters and hemodynamic indices were monitored over the hospital stay. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Patients with severe dengue had worse cardiac function compared with dengue in the form of left ventricular systolic dysfunction with increased left myocardial performance index (0.58 [0.26-0.80] vs. 0.38 [0.22-0.70], p = .006). Septal myocardial systolic velocities were reduced (6.4 [4.8-10] vs. 8.1 [6-13] cm/s, p = .01) as well as right ventricular systolic (11.4 [7.5-17] vs. 13.5 [10-17] cm/s, p = .016) and diastolic velocities (13 [8-23] vs. 17 [12-25] cm/s, p = .0026). In the severe group, these parameters improved from hospital admission to discharge; septal myocardial systolic velocities to 8.8 (7-11) cm/s (p = .002), right ventricular myocardial systolic velocities to 15.0 (11.8-23) cm/s, (p = .003), and diastolic velocity to 21 (11-25) cm/s (p = .002). Patients with cardiac impairment were more likely to have significant pleural effusions. CONCLUSIONS: Patients with severe dengue have evidence of systolic and diastolic cardiac impairment with septal and right ventricular wall being predominantly affected.

Nguyen-Pouplin J, Pouplin T, Van TP, The TD, Thi DN, Farrar J, Tinh HT, Wills B. 2011. Dextran fractional clearance studies in acute dengue infection. PLoS Negl Trop Dis, 5 (8), pp. e1282. | Show Abstract | Read more

BACKGROUND: Although increased capillary permeability is the major clinical feature associated with severe dengue infections the mechanisms underlying this phenomenon remain unclear. Dextran clearance methodology has been used to investigate the molecular sieving properties of the microvasculature in clinical situations associated with altered permeability, including during pregnancy and in various renal disorders. In order to better understand the characteristics of the vascular leak associated with dengue we undertook formal dextran clearance studies in Vietnamese dengue patients and healthy volunteers. METHODOLOGY/PRINCIPAL FINDINGS: We carried out serial clearance studies in 15 young adult males with acute dengue and evidence of vascular leakage a) during the phase of maximal leakage and b) one and three months later, as well as in 16 healthy control subjects. Interestingly we found no difference in the clearance profiles of neutral dextran solutions among the dengue patients at any time-point or in comparison to the healthy volunteers. CONCLUSIONS/SIGNIFICANCE: The surface glycocalyx layer, a fibre-matrix of proteoglycans, glycosaminoglycans, and plasma proteins, forms a complex with the underlying endothelial cells to regulate plasma volume within circumscribed limits. It is likely that during dengue infections loss of plasma proteins from this layer alters the permeability characteristics of the complex; physical and/or electrostatic interactions between the dextran molecules and the glycocalyx structure may temporarily restore normal function, rendering the technique unsuitable for assessing permeability in these patients. The implications for resuscitation of patients with dengue shock syndrome (DSS) are potentially important. It is possible that continuous low-dose infusions of dextran may help to stabilize the permeability barrier in patients with profound or refractory shock, reducing the need for repeated boluses, limiting the total colloid volume required. Formal clinical studies should help to assess this strategy as an alternative to conventional fluid resuscitation for severe DSS.

Raghwani J, Rambaut A, Holmes EC, Hang VT, Hien TT, Farrar J, Wills B, Lennon NJ, Birren BW, Henn MR, Simmons CP. 2011. Endemic dengue associated with the co-circulation of multiple viral lineages and localized density-dependent transmission. PLoS Pathog, 7 (6), pp. e1002064. | Show Abstract | Read more

Dengue is one of the most important infectious diseases of humans and has spread throughout much of the tropical and subtropical world. Despite this widespread dispersal, the determinants of dengue transmission in endemic populations are not well understood, although essential for virus control. To address this issue we performed a phylogeographic analysis of 751 complete genome sequences of dengue 1 virus (DENV-1) sampled from both rural (Dong Thap) and urban (Ho Chi Minh City) populations in southern Viet Nam during the period 2003-2008. We show that DENV-1 in Viet Nam exhibits strong spatial clustering, with likely importation from Cambodia on multiple occasions. Notably, multiple lineages of DENV-1 co-circulated in Ho Chi Minh City. That these lineages emerged at approximately the same time and dispersed over similar spatial regions suggests that they are of broadly equivalent fitness. We also observed an important relationship between the density of the human host population and the dispersion rate of dengue, such that DENV-1 tends to move from urban to rural populations, and that densely populated regions within Ho Chi Minh City act as major transmission foci. Despite these fluid dynamics, the dispersion rates of DENV-1 are relatively low, particularly in Ho Chi Minh City where the virus moves less than an average of 20 km/year. These low rates suggest a major role for mosquito-mediated dispersal, such that DENV-1 does not need to move great distances to infect a new host when there are abundant susceptibles, and imply that control measures should be directed toward the most densely populated urban environments.

Duyen HTL, Ngoc TV, Ha DT, Hang VTT, Kieu NTT, Young PR, Farrar JJ, Simmons CP, Wolbers M, Wills BA. 2011. Kinetics of plasma viremia and soluble nonstructural protein 1 concentrations in dengue: differential effects according to serotype and immune status. J Infect Dis, 203 (9), pp. 1292-1300. | Show Abstract | Read more

We describe the magnitude and kinetics of plasma viremia and nonstructural protein 1 (sNS1) levels in sequential samples from 167 children with acute dengue, enrolled early in a community study in Vietnam. All children recovered fully, and only 5 required hospitalization. Among those with dengue virus type 1 (DENV-1), plasma viremia was significantly greater in primary (49) than secondary (44) infections and took longer to resolve. In primary DENV-2 and 3 infections, viremia was significantly lower than among primary DENV-1 infections. Concentrations of sNS1 were significantly higher for DENV-1 than for DENV-2 after adjusting for viremia, with marked differences in the kinetic profiles between primary and secondary infections. Secondary infection and higher viremia were independent predictors of more severe thrombocytopenia, and higher viremia was associated with a small increase in hemoconcentration. Our findings identify clear serotype and immune-status related effects on the dynamics of dengue viremia and sNS1 responses, together with associations with important clinical parameters.

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Khor CC, Chau TNB, Pang J, Davila S, Long HT, Ong RTH, Dunstan SJ, Wills B, Farrar J, Van Tram T et al. 2011. Genome-wide association study identifies susceptibility loci for dengue shock syndrome at MICB and PLCE1 Nature Genetics, 43 (11), pp. 1139-1141. | Show Abstract | Read more

Hypovolemic shock (dengue shock syndrome (DSS)) is the most common life-threatening complication of dengue. We conducted a genome-wide association study of 2,008 pediatric cases treated for DSS and 2,018 controls from Vietnam. Replication of the most significantly associated markers was carried out in an independent Vietnamese sample of 1,737 cases and 2,934 controls. SNPs at two loci showed genome-wide significant association with DSS. We identified a susceptibility locus at MICB (major histocompatibility complex (MHC) class I polypeptide-related sequence B), which was within the broad MHC region on chromosome 6 but outside the class I and class II HLA loci (rs3132468, P meta = 4.41 × 10 -11 , per-allele odds ratio (OR) = 1.34 (95% confidence interval: 1.23-1.46)). We identified associated variants within PLCE1 (phospholipase C, epsilon 1) on chromosome 10 (rs3765524, P meta = 3.08 × 10 -10 , per-allele OR = 0.80 (95% confidence interval: 0.75-0.86)). We identify two loci associated with susceptibility to DSS in people with dengue, suggesting possible mechanisms for this severe complication of dengue. © 2011 Nature America, Inc. All rights reserved.

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Hue KDT, Tuan TV, Thi HTN, Bich CTN, Anh HHL, Wills BA, Simmons CP. 2011. Validation of an internally controlled one-step real-time multiplex RT-PCR assay for the detection and quantitation of dengue virus RNA in plasma Journal of Virological Methods, 177 (2), pp. 168-173. | Show Abstract | Read more

Dengue is mosquito-borne virus infection that annually causes ~50 million clinically apparent cases worldwide. An internally controlled one-step real-time multiplex RT-PCR assay was developed for detection and quantitation of DENV RNA in plasma sample by using specific primers and fluorogenic TaqMan probes. All primers and probes targeted sequences near the 3' end of the NS5 gene. The method comprised two multiplex assays and was validated for sensitivity, specificity, linearity, reproducibility and precision. An internal control template was spiked into each clinical specimen to provide quality assurance for each experimental step. The assay allowed for detection of between 0.5 and 3 infectious particles per mL, is rapid and has been operationally characterized in 287 Vietnamese dengue patients from two therapeutic intervention trials at the Hospital for Tropical Diseases, Ho Chi Minh City, Viet Nam. © 2011 Elsevier B.V.

Do AHL, van Doorn HR, Nghiem MN, Bryant JE, Hoang THT, Do QH, Van TL, Tran TT, Wills B, Nguyen VCV et al. 2011. Viral etiologies of acute respiratory infections among hospitalized Vietnamese children in Ho Chi Minh City, 2004-2008. PLoS One, 6 (3), pp. e18176. | Show Abstract | Read more

BACKGROUND: The dominant viral etiologies responsible for acute respiratory infections (ARIs) are poorly understood, particularly among hospitalized children in resource-limited tropical countries where morbidity and mortality caused by ARIs are highest. Improved etiological insight is needed to improve clinical management and prevention. OBJECTIVES: We conducted a three-year prospective descriptive study of severe respiratory illness among children from 2 months to 13 years of age within the largest referral hospital for infectious diseases in southern Vietnam. METHODS: Molecular detection for 15 viral species and subtypes was performed on three types of respiratory specimens (nose, throat swabs and nasopharyngeal aspirates) using a multiplex RT-PCR kit (Seeplex™ RV detection, Seegene) and additional monoplex real-time RT-PCRs. RESULTS: A total of 309 children were enrolled from November 2004 to January 2008. Viruses were identified in 72% (222/309) of cases, including respiratory syncytial virus (24%), influenza virus A and B (17%), human bocavirus (16%), enterovirus (9%), human coronavirus (8%), human metapneumovirus (7%), parainfluenza virus 1-3 (6%), adenovirus (5%), and human rhinovirus A (4%). Co-infections with multiple viruses were detected in 20% (62/309) of patients. When combined, diagnostic yields in nose and throat swabs were similar to nasopharyngeal aspirates. CONCLUSION: Similar to other parts in the world, RSV and influenza were the predominant viral pathogens detected in Vietnamese hospitalized children. Combined nasal and throat swabs are the specimens of choice for sensitive molecular detection of a broad panel of viral agents. Further research is required to better understand the clinical significance of single versus multiple viral coinfections and to address the role of bacterial (co-)infections involved in severe respiratory illness.

Tran CBN, Nguyen HT, Phan HTT, Tran NV, Wills B, Farrar J, Santangelo JD, Simmons CP. 2011. The seroprevalence and seroincidence of enterovirus71 infection in infants and children in Ho Chi Minh City, Viet Nam. PLoS One, 6 (7), pp. e21116. | Show Abstract | Read more

Enterovirus 71 (EV71)-associated hand, foot and mouth disease has emerged as a serious public health problem in South East Asia over the last decade. To better understand the prevalence of EV71 infection, we determined EV71 seroprevalence and seroincidence amongst healthy infants and children in Ho Chi Minh City, Viet Nam. In a cohort of 200 newborns, 55% of cord blood samples contained EV71 neutralizing antibodies and these decayed to undetectable levels by 6 months of age in 98% of infants. The EV71 neutralizing antibody seroconversion rate was 5.6% in the first year and 14% in the second year of life. In children 5-15 yrs of age, seroprevalence of EV71 neutralizing antibodies was 84% and in cord blood it was 55%. Taken together, these data suggest EV71 force of infection is high and highlights the need for more research into its epidemiology and pathogenesis in high disease burden countries.

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Alexander N, Balmaseda A, Coelho ICB, Dimaano E, Hien TT, Hung NT, Jäenisch T, Kroeger A, Lum LCS, Martinez E et al. 2011. Multicentre prospective study on dengue classification in four South-east Asian and three Latin American countries Tropical Medicine and International Health, 16 (8), pp. 936-948. | Show Abstract | Read more

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. © 2011 Blackwell Publishing Ltd.

Trung DT, Thao LTT, Hien TT, Hung NT, Vinh NN, Hien PTD, Chinh NT, Simmons C, Wills B. 2010. Liver involvement associated with dengue infection in adults in Vietnam. Am J Trop Med Hyg, 83 (4), pp. 774-780. | Show Abstract | Read more

Globally, the number of adults hospitalized with dengue has increased markedly in recent years. It has been suggested that hepatic dysfunction is more significant in this group than among children. We describe the spectrum and evolution of disease manifestations among 644 adults with dengue who were prospectively recruited on admission to a major infectious disease hospital in southern Vietnam and compare them with a group of patients with similar illnesses not caused by dengue. Transaminase levels increased in virtually all dengue patients and correlated with other markers of disease severity. However, peak enzyme values usually occurred later than other complications. Clinically severe liver involvement was infrequent and idiosyncratic, but usually resulted in severe bleeding. Chronic co-infection with hepatitis B was associated with modestly but significantly increased levels of alanine aminotransferase, but did not otherwise impact the clinical picture.

Dung NTP, Duyen HTL, Thuy NTV, Ngoc TV, Chau NVV, Hien TT, Rowland-Jones SL, Dong T, Farrar J, Wills B, Simmons CP. 2010. Timing of CD8+ T cell responses in relation to commencement of capillary leakage in children with dengue. J Immunol, 184 (12), pp. 7281-7287. | Show Abstract | Read more

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.

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Hang VTT, Holmes EC, Veasna D, Quy NT, Hien TT, Quail M, Churcher C, Parkhill J, Cardosa J, Farrar J et al. 2010. Emergence of the Asian 1 genotype of dengue Virus serotype 2 in viet Nam: in vivo fitness advantage and lineage replacement in South-East Asia PLoS Neglected Tropical Diseases, 4 (7), | Show Abstract | Read more

A better description of the extent and structure of genetic diversity in dengue virus (DENV) in endemic settings is central to its eventual control. To this end we determined the complete coding region sequence of 187 DENV-2 genomes and 68 E genes from viruses sampled from Vietnamese patients between 1995 and 2009. Strikingly, an episode of genotype replacement was observed, with Asian 1 lineage viruses entirely displacing the previously dominant Asian/American lineage viruses. This genotype replacement event also seems to have occurred within DENV- 2 in Thailand and Cambodia, suggestive of a major difference in viral fitness. To determine the cause of this major evolutionary event we compared both the infectivity of the Asian 1 and Asian/American genotypes in mosquitoes and their viraemia levels in humans. Although there was little difference in infectivity in mosquitoes, we observed significantly higher plasma viraemia levels in paediatric patients infected with Asian 1 lineage viruses relative to Asian/ American viruses, a phenotype that is predicted to result in a higher probability of human-to-mosquito transmission. These results provide a mechanistic basis to a marked change in the genetic structure of DENV-2 and more broadly underscore that an understanding of DENV evolutionary dynamics can inform the development of vaccines and antiviral drugs. © 2010 Ty Hang et al.

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Scopus

Dondorp AM, Fanello CI, Hendriksen IC, Gomes E, Seni A, Chhaganlal KD, Bojang K, Olaosebikan R, Anunobi N, Maitland K et al. 2010. Artesunate versus quinine in the treatment of severe falciparum malaria in African children (AQUAMAT): An open-label, randomised trial The Lancet, 376 (9753), pp. 1647-1657. | Show Abstract | Read more

Severe malaria is a major cause of childhood death and often the main reason for paediatric hospital admission in sub-Saharan Africa. Quinine is still the established treatment of choice, although evidence from Asia suggests that artesunate is associated with a lower mortality. We compared parenteral treatment with either artesunate or quinine in African children with severe malaria. This open-label, randomised trial was undertaken in 11 centres in nine African countries. Children ( < 15 years) with severe falciparum malaria were randomly assigned to parenteral artesunate or parenteral quinine. Randomisation was in blocks of 20, with study numbers corresponding to treatment allocations kept inside opaque sealed paper envelopes. The trial was open label at each site, and none of the investigators or trialists, apart from for the trial statistician, had access to the summaries of treatment allocations. The primary outcome measure was in-hospital mortality, analysed by intention to treat. This trial is registered, number ISRCTN50258054. 5425 children were enrolled; 2712 were assigned to artesunate and 2713 to quinine. All patients were analysed for the primary outcome. 230 (8·5) patients assigned to artesunate treatment died compared with 297 (10·9) assigned to quinine treatment (odds ratio [OR] stratified for study site 0·75, 95 CI 0·63-0·90; relative reduction 22·5, 95 CI 8·1-36·9; p=0·0022). Incidence of neurological sequelae did not differ significantly between groups, but the development of coma (65/1832 [3·5] with artesunate vs 91/1768 [5·1] with quinine; OR 0·69 95 CI 0·49-0·95; p=0·0231), convulsions (224/2712 [8·3] vs 273/2713 [10·1] ; OR 0·80, 0·66-0·97; p=0·0199), and deterioration of the coma score (166/2712 [6·1] vs 208/2713 [7·7] ; OR 0·78, 0·64-0·97; p=0·0245) were all significantly less frequent in artesunate recipients than in quinine recipients. Post-treatment hypoglycaemia was also less frequent in patients assigned to artesunate than in those assigned to quinine (48/2712 [1·8] vs 75/2713 [2·8] ; OR 0·63, 0·43-0·91; p=0·0134). Artesunate was well tolerated, with no serious drug-related adverse effects. Artesunate substantially reduces mortality in African children with severe malaria. These data, together with a meta-analysis of all trials comparing artesunate and quinine, strongly suggest that parenteral artesunate should replace quinine as the treatment of choice for severe falciparum malaria worldwide. The Wellcome Trust. © 2010 Elsevier Ltd.

Trung DT, Wills B. 2010. Systemic vascular leakage associated with dengue infections - the clinical perspective. Curr Top Microbiol Immunol, 338 (1), pp. 57-66. | Show Abstract | Read more

Vascular leakage is the most serious complication of dengue infection. However, despite considerable progress in understanding the immunological derangements associated with dengue, the pathogenic mechanisms underlying the change in vascular permeability remain unclear. Lack of suitable model systems that manifest permeability characteristics similar to human vascular endothelium has seriously impeded research in this area. Similarly, limited knowledge of the factors regulating intrinsic microvascular permeability in health, together with limited understanding of the alterations seen in disease states in general, has also hampered progress. Fortunately considerable advances have been made in the field of endothelial biology in recent years, especially following appreciation of the crucial role played by the endothelial surface glycocalyx, acting in concert with underlying cellular structures, in regulating fluid flow across the microvasculature. We review what is known about vascular leakage during dengue infections, particularly in relation to current knowledge of vascular physiology, and discuss potential areas of research that may help to elucidate the complex nature of this singular phenomenon in the future.

Tricou V, Minh NN, Van TP, Lee SJ, Farrar J, Wills B, Tran HT, Simmons CP. 2010. A randomized controlled trial of chloroquine for the treatment of dengue in Vietnamese adults. PLoS Negl Trop Dis, 4 (8), pp. e785. | Show Abstract | Read more

BACKGROUND: There is currently no licensed antiviral drug for treatment of dengue. Chloroquine (CQ) inhibits the replication of dengue virus (DENV) in vitro. METHODS AND FINDINGS: A double-blind, randomized, placebo-controlled trial of CQ in 307 adults hospitalized for suspected DENV infection was conducted at the Hospital for Tropical Diseases (Ho Chi Minh City, Vietnam) between May 2007 and July 2008. Patients with illness histories of 72 hours or less were randomized to a 3-day course of CQ (n = 153) or placebo (n = 154). Laboratory-confirmation of DENV infection was made in 257 (84%) patients. The primary endpoints were time to resolution of DENV viraemia and time to resolution of DENV NS1 antigenaemia. In patients treated with CQ there was a trend toward a longer duration of DENV viraemia (hazard ratio (HR) = 0.80, 95% CI 0.62-1.05), but we did not find any difference for the time to resolution of NS1 antigenaemia (HR = 1.07, 95% CI 0.76-1.51). Interestingly, CQ was associated with a significant reduction in fever clearance time in the intention-to-treat population (HR = 1.37, 95% CI 1.08-1.74) but not in the per-protocol population. There was also a trend towards a lower incidence of dengue hemorrhagic fever (odds ratio = 0.60, PP 95% CI 0.34-1.04) in patients treated with CQ. Differences in levels of T cell activation or pro- or anti-inflammatory plasma cytokine concentrations between CQ- and placebo-treated patients did not explain the trend towards less dengue hemorrhagic fever in the CQ arm. CQ was associated with significantly more adverse events, primarily vomiting. CONCLUSIONS: CQ does not reduce the durations of viraemia and NS1 antigenaemia in dengue patients. Further trials, with appropriate endpoints, would be required to determine if CQ treatment has any clinical benefit in dengue. TRIAL REGISTRATION: Current Controlled Trials number ISRCTN38002730.

Tricou V, Vu HTT, Quynh NVN, Nguyen CVV, Tran HT, Farrar J, Wills B, Simmons CP. 2010. Comparison of two dengue NS1 rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses. BMC Infect Dis, 10 (1), pp. 142. | Show Abstract | Read more

BACKGROUND: Dengue is a major public health problem in tropical and subtropical countries. Rapid and easy diagnosis of dengue can assist patient triage and care-management. The detection of DENV NS1 on rapid lateral flow tests offers a fast route to a presumptive dengue diagnosis but careful evaluations are urgently needed as more and more people use them. METHODS: The sensitivity and specificity of the Bio-Rad NS1 Ag Strip and SD Dengue Duo (NS1/IgM/IgG) lateral flow rapid tests were evaluated in a panel of plasma samples from 245 Vietnamese patients with RT-PCR confirmed dengue and 47 with other febrile illnesses. RESULTS: The NS1 rapid tests had similar diagnostic sensitivities (respectively 61.6% and 62.4%) in confirmed dengue cases but were 100% specific. When IgM/IgG results from the SD Dengue Duo were included in the test interpretation, the sensitivity improved significantly from 62.4% with NS1 alone to 75.5% when NS1 and/or IgM was positive and 83.7% when NS1 and/or IgM and/or IgG was positive. Both NS1 assays were significantly more sensitive for primary than secondary dengue. NS1 positivity was associated with the underlying viraemia as NS1-positive samples had a significantly higher viraemia than NS1-negative samples. CONCLUSIONS: These data suggest that the NS1 test component of these assays are highly specific and have similar levels of sensitivity. The IgM parameter in the SD Duo test improved overall test sensitivity without compromising specificity. The SD Dengue Duo lateral flow rapid test deserves further prospective evaluation in dengue endemic settings.

Dung NM, Turtle L, Chong WK, Mai NT, Thao TT, Thuy TT, Kneen R, Phu NH, Wills B, Farrar J et al. 2009. An evaluation of the usefulness of neuroimaging for the diagnosis of Japanese encephalitis. J Neurol, 256 (12), pp. 2052-2060. | Show Abstract | Read more

Japanese encephalitis virus (JEV) is estimated to cause 30–50,000 cases of encephalitis every year. The disease occurs mainly in rural Asia and is transmitted to humans from birds and pigs by mosquitoes of the genus Culex. JE is diagnosed with antibody testing of the serum and CSF, but this is not available in many hospitals. Neuroimaging abnormalities, particularly thalamic hypodensity on computed tomography (CT) and hyperintensity on T2 weighted magnetic resonance imaging (MRI) have been described in case studies, but their usefulness for diagnosing JE is not known. We have therefore evaluated the usefulness of neuroimaging (CT and MRI) for the diagnosis of JE. The findings of thalamic lesions were compared with the final serological diagnosis in a cohort of 75 patients (children and adults) with suspected CNS infections in Southern Vietnam, a JEV endemic area. Thalamic lesions on CT and/or MRI combined had sensitivity 23% (95% confidence interval 12.9–33.1%), specificity 100%, positive predictive value 100% and negative predictive value 42.1% (95% confidence interval 30.2–53.8%) for a diagnosis of JE in this cohort. Over time, the thalamic lesions resolved in some patients. One patient showed disappearance of lesions on CT followed by reappearance of the lesions some time later, known as the fogging effect. In this setting, the presence of thalamic abnormalities suggested the diagnosis of JE, but their absence did not exclude it.

Wills B, Tran VN, Nguyen THV, Truong TTT, Tran TNT, Nguyen MD, Tran VD, Nguyen VVC, Dinh TT, Farrar J. 2009. Hemostatic changes in Vietnamese children with mild dengue correlate with the severity of vascular leakage rather than bleeding. Am J Trop Med Hyg, 81 (4), pp. 638-644. | Show Abstract | Read more

The mechanisms underlying the bleeding manifestations and coagulopathy associated with dengue remain unclear, in part because of the focus of much previous work on severe disease without an appropriate comparison group. We describe detailed clinical and laboratory profiles for a large group of children with dengue of all severities, and a group with similar non-dengue febrile illnesses, all followed prospectively from early presentation through to recovery. Among the dengue-infected patients but not the controls, thrombocytopenia, increased partial thromboplastin times and reduced fibrinogen concentrations were apparent from an early stage, and these abnormalities correlated strongly with the severity and timing of vascular leakage but not bleeding. There was little evidence of procoagulant activation. The findings do not support a primary diagnosis of disseminated intravascular coagulation to explain the intrinsic coagulopathy. An alternative biologically plausible hypothesis is discussed.

Wills B. 2009. Mechanisms of vascular leakage and bleeding during dengue infections TROPICAL MEDICINE & INTERNATIONAL HEALTH, 14 pp. 38-39.

Jaenisch T, Wills B, Alexander N, Arana B, Balmaseda A, Jr SJB, Castelobranco I, Dimaano E, Farrar J, Gaczkowski R et al. 2009. Evidence for a revised dengue classification: a multi-centre prospective study across Southeast Asia and Latin America TROPICAL MEDICINE & INTERNATIONAL HEALTH, 14 pp. 39-40.

Recker M, Blyuss KB, Simmons CP, Hien TT, Wills B, Farrar J, Gupta S. 2009. Immunological serotype interactions and their effect on the epidemiological pattern of dengue. Proc Biol Sci, 276 (1667), pp. 2541-2548. | Show Abstract | Read more

Long-term epidemiological data reveal multi-annual fluctuations in the incidence of dengue fever and dengue haemorrhagic fever, as well as complex cyclical behaviour in the dynamics of the four serotypes of the dengue virus. It has previously been proposed that these patterns are due to the phenomenon of the so-called antibody-dependent enhancement (ADE) among dengue serotypes, whereby viral replication is increased during secondary infection with a heterologous serotype; however, recent studies have implied that this positive reinforcement cannot account for the temporal patterns of dengue and that some form of cross-immunity or external forcing is necessary. Here, we show that ADE alone can produce the observed periodicities and desynchronized oscillations of individual serotypes if its effects are decomposed into its two possible manifestations: enhancement of susceptibility to secondary infections and increased transmissibility from individuals suffering from secondary infections. This decomposition not only lowers the level of enhancement necessary for realistic disease patterns but also reduces the risk of stochastic extinction. Furthermore, our analyses reveal a time-lagged correlation between serotype dynamics and disease incidence rates, which could have important implications for understanding the irregular pattern of dengue epidemics.

Long HT, Hibberd ML, Hien TT, Dung NM, Van Ngoc T, Farrar J, Wills B, Simmons CP. 2009. Patterns of gene transcript abundance in the blood of children with severe or uncomplicated dengue highlight differences in disease evolution and host response to dengue virus infection. J Infect Dis, 199 (4), pp. 537-546. | Show Abstract | Read more

DNA microarrays and specific reverse-transcription polymerase chain reaction assays were used to reveal transcriptional patterns in the blood of children presenting with dengue shock syndrome (DSS) and well-matched patients with uncomplicated dengue. The transcriptome of patients with acute uncomplicated dengue was characterized by a metabolically demanding "host-defense" profile; transcripts related to oxidative metabolism, interferon signaling, protein ubiquination, apoptosis, and cytokines were prominent. In contrast, the transcriptome of patients with DSS was surprisingly benign, particularly with regard to transcripts derived from apoptotic and type I interferon pathways. These data highlight significant heterogeneity in the type or timing of host transcriptional immune responses precipitated by dengue virus infection independent of the duration of illness. In particular, they suggest that, if transcriptional events in the blood compartment contribute to capillary leakage leading to hypovolemic shock, they occur before cardiovascular decompensation, a finding that has implications for rational adjuvant therapy in this syndrome.

Hang VT, Nguyet NM, Trung DT, Tricou V, Yoksan S, Dung NM, Van Ngoc T, Hien TT, Farrar J, Wills B, Simmons CP. 2009. Diagnostic accuracy of NS1 ELISA and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses. PLoS Negl Trop Dis, 3 (1), pp. e360. | Show Abstract | Read more

BACKGROUND: Dengue is a public health problem in many countries. Rapid diagnosis of dengue can assist patient triage and management. Detection of the dengue viral protein, NS1, represents a new approach to dengue diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity and specificity of the Platelia NS1 ELISA assay and an NS1 lateral flow rapid test (LFRT) were compared against a gold standard reference diagnostic algorithm in 138 Vietnamese children and adults. Overall, the Platelia NS1 ELISA was modestly more sensitive (82%) than the NS1 LFRT (72%) in confirmed dengue cases. Both ELISA and LFRT assays were more sensitive for primary than secondary dengue, and for specimens collected within 3 days of illness onset relative to later time points. The presence of measurable DENV-reactive IgG and to a lesser extent IgM in the test sample was associated with a significantly lower rate of NS1 detection in both assays. NS1 positivity was associated with the underlying viraemia, as NS1-positive samples had a significantly higher viraemia than NS1-negative samples matched for duration of illness. The Platelia and NS1 LFRT were 100% specific, being negative in all febrile patients without evidence of recent dengue, as well as in patients with enteric fever, malaria, Japanese encephalitis and leptospirosis. CONCLUSIONS/SIGNIFICANCE: Collectively, these data suggest NS1 assays deserve inclusion in the diagnostic evaluation of dengue patients, but with due consideration for the limitations in patients who present late in their illness or have a concomitant humoral immune response.

Moran E, Simmons C, Chau NV, Luhn K, Wills B, Phuong Dung N, Thao LTT, Hien TT, Farrar J, Rowland-Jones S, Dong T. 2008. Preservation of a critical epitope core region is associated with the high degree of flaviviral cross-reactivity exhibited by a dengue-specific CD4+ T cell clone. Eur J Immunol, 38 (4), pp. 1050-1057. | Show Abstract | Read more

Dengue is a member of the Flaviviridae, a large group of related viruses some of which co-circulate in certain regions (e.g. dengue and Yellow fever in South America). Immune responses cross-reactive between different dengue serotypes are important in the pathogenesis of dengue disease but it is not known whether previous infection with one flavivirus might affect the clinical course of subsequent infections with other members of the family. CD4+ T cells have been shown to be important in the production of cytokines in response to dengue infection and can demonstrate significant epitope cross-reactivity. Here, we describe the generation and characterisation of CD4+ T cell clones from a patient experiencing acute dengue infection. These clones were DRB1*15+ and recognised epitope variants not only within other dengue viruses but certain other flaviviruses. This cross-reactivity was dependent upon the presence of a five-amino acid core region, consistent with structural observations of class II MHC binding to TCR demonstrating that only a subset of residues within an epitope bound to a class II molecule are "read out" by the TCR. This capacity of CD4+ T cell clones to recognise a given epitope despite considerable variation between viruses may be of pathological significance, particularly in regions where related viruses co-circulate.

Moxon C, Wills B. 2008. Management of severe dengue in children. Adv Exp Med Biol, 609 pp. 131-144. | Show Abstract | Read more

Dengue is a major global disease which, in its severe form, affects up to 500,000 people worldwide each year, most of whom are children. The development of a safe and effective vaccine is a clear priority, together with public health measures to prevent the spread of infection. However, while major epidemics continue to occur, clinicians must also focus on optimising management. Although no specific treatment is available at present, with good supportive care, mortality for children with DHF can be reduced to well below 1%. In patients without signs of shock, fluid replacement can be attempted orally, but in children with DSS parenteral treatment is essential. Very careful titration of fluid therapy is necessary combined with frequent reassessment for signs of worsening shock or the development of fluid overload. In most DSS cases isotonic crystalloid solutions are as effective as colloid solutions, but the question whether early intervention with colloid solutions improves outcome in more advanced shock requires further investigation. The outcome of studies to address this question, together with further research to examine the pathophysiological mechanisms underlying the plasma leakage, will hopefully result in better management of children with severe dengue but may also provide useful insights into other diseases that affect endothelial function.

Lühn K, Simmons CP, Moran E, Dung NTP, Chau TNB, Quyen NTH, Thao LTT, Van Ngoc T, Dung NM, Wills B et al. 2007. Increased frequencies of CD4+ CD25(high) regulatory T cells in acute dengue infection. J Exp Med, 204 (5), pp. 979-985. | Show Abstract | Read more

Dengue virus infection is an increasingly important tropical disease, causing 100 million cases each year. Symptoms range from mild febrile illness to severe hemorrhagic fever. The pathogenesis is incompletely understood, but immunopathology is thought to play a part, with antibody-dependent enhancement and massive immune activation of T cells and monocytes/macrophages leading to a disproportionate production of proinflammatory cytokines. We sought to investigate whether a defective population of regulatory T cells (T reg cells) could be contributing to immunopathology in severe dengue disease. CD4(+)CD25(high)FoxP3(+) T reg cells of patients with acute dengue infection of different severities showed a conventional phenotype. Unexpectedly, their capacity to suppress T cell proliferation and to secrete interleukin-10 was not altered. Moreover, T reg cells suppressed the production of vasoactive cytokines after dengue-specific stimulation. Furthermore, T reg cell frequencies and also T reg cell/effector T cell ratios were increased in patients with acute infection. A strong indication that a relative rise of T reg cell/effector T cell ratios is beneficial for disease outcome comes from patients with mild disease in which this ratio is significantly increased (P < 0.0001) in contrast to severe cases (P = 0.2145). We conclude that although T reg cells expand and function normally in acute dengue infection, their relative frequencies are insufficient to control the immunopathology of severe disease.

Dong T, Moran E, Vinh Chau N, Simmons C, Luhn K, Peng Y, Wills B, Phuong Dung N, Thi Thu Thao L, Hien TT et al. 2007. High pro-inflammatory cytokine secretion and loss of high avidity cross-reactive cytotoxic T-cells during the course of secondary dengue virus infection. PLoS One, 2 (12), pp. e1192. | Show Abstract | Read more

BACKGROUND: Dengue is one of the most important human diseases transmitted by an arthropod vector and the incidence of dengue virus infection has been increasing - over half the world's population now live in areas at risk of infection. Most infections are asymptomatic, but a subset of patients experience a potentially fatal shock syndrome characterised by plasma leakage. Severe forms of dengue are epidemiologically associated with repeated infection by more than one of the four dengue virus serotypes. Generally attributed to the phenomenon of antibody-dependent enhancement, recent observations indicate that T-cells may also influence disease phenotype. METHODS AND FINDINGS: Virus-specific cytotoxic T lymphocytes (CTL) showing high level cross reactivity between dengue serotypes could be expanded from blood samples taken during the acute phase of secondary dengue infection. These could not be detected in convalescence when only CTL populations demonstrating significant serotype specificity were identified. Dengue cross-reactive CTL clones derived from these patients were of higher avidity than serotype-specific clones and produced much higher levels of both type 1 and certain type 2 cytokines, many previously implicated in dengue pathogenesis. CONCLUSION: Dengue serotype cross-reactive CTL clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. That such cells cannot be expanded from convalescent samples suggests that they may be depleted, perhaps as a consequence of activation-induced cell death. Such high avidity cross-reactive memory CTL may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. These cells appear to be subsequently deleted leaving a more serotype-specific memory CTL pool. Further studies are needed to relate these cellular observations to disease phenotype in a large group of patients. If confirmed they have significant implications for understanding the role of virus-specific CTL in pathogenesis of dengue disease.

Nguyen D, Dong T, Nguyen VVC, Nguyen MD, Wills B, Rowland-Jones S, Farrar J, Simmons C. 2006. Spectrum and kinetic of T cell responses to dengue virus epitopes and the influence of hla polymorphisms in dengue disease pathogenesis AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 75 (5), pp. 289-290.

Deen JL, Harris E, Wills B, Balmaseda A, Hammond SN, Rocha C, Dung NM, Hung NT, Hien TT, Farrar JJ. 2006. The WHO dengue classification and case definitions: time for a reassessment. Lancet, 368 (9530), pp. 170-173. | Read more

Thuong NTT, Dunstan SJ, Dung NM, Charlieu JP, Loan HT, Wills B, Solomon T, Farrar JJ. 2006. Polymorphisms of the gene coding for copper/zinc superoxide dismutase (SOD1) in patients with Japanese encephalitis Annals of Tropical Medicine and Parasitology, 100 (7), pp. 631-636. | Show Abstract | Read more

Japanese encephalitis is the commonest form of encephalitis globally. Most cases develop characteristic encephalitis but some also present with flaccid paralysis. The paralysis is secondary to damage at the alpha motor neurone, the site that is also damaged in amyotrophic lateral sclerosis (ALS). The gene coding for superoxide dismutase 1 (SOD1) is thought to be involved in ALS and may also be linked to susceptibility to Japanese encephalitis. To investigate this possibility, polymorphisms in the SOD1 gene were investigated, in 61 cases of Japanese encephalitis, 61 matched controls and 171 population controls, in Vietnam. Novel polymorphisms, found only in three of the cases and one of the population controls, may be involved with susceptibility to Japanese encephalitis and potentially to other flavivirus infections that lead to damage to the cells of the anterior horn. Further research on this possible association is required. © 2006 The Liverpool School of Tropical Medicine.

Wills BA, Dung NM, Farrar JJ. 2005. Fluid solutions in dengue shock syndrome - Reply NEW ENGLAND JOURNAL OF MEDICINE, 353 (23), pp. 2511-2511.

Wills B, Dung NM, Loan HT, Tam DT, Thu TT, Minh LT, Diet TV, Hao NT, Chau NV, Stepniewska K et al. 2005. Fluid resuscitation for dengue shock syndrome - The evidence from formal randomised and blinded intervention trials in Vietnam AMERICAN JOURNAL OF TROPICAL MEDICINE AND HYGIENE, 73 (6), pp. 361-361.

Wills BA, Nguyen MD, Ha TL, Dong THT, Tran TNT, Le TTM, Tran VD, Nguyen TH, Nguyen VC, Stepniewska K et al. 2005. Comparison of three fluid solutions for resuscitation in dengue shock syndrome. N Engl J Med, 353 (9), pp. 877-889. | Show Abstract | Read more

BACKGROUND: Dengue shock syndrome is characterized by severe vascular leakage and disordered hemostasis and progresses to death in 1 to 5 percent of cases. Although volume replacement is recognized as the critical therapeutic intervention, World Health Organization management guidelines remain empirical rather than evidence-based. METHODS: We performed a double-blind, randomized comparison of three fluids for initial resuscitation of Vietnamese children with dengue shock syndrome. We randomly assigned 383 children with moderately severe shock to receive Ringer's lactate, 6 percent dextran 70 (a colloid), or 6 percent hydroxyethyl starch (a colloid) and 129 children with severe shock to receive one of the colloids. The primary outcome measure was requirement for rescue colloid at any time after administration of the study fluid. RESULTS: Only one patient died (<0.2 percent mortality). The primary outcome measure--requirement for rescue colloid--was similar for the different fluids in the two severity groups. The relative risk of requirement for rescue colloid was 1.08 (95 percent confidence interval, 0.78 to 1.47; P=0.65) among children with moderate shock who received Ringer's lactate as compared with either of the colloid solutions, 1.13 (95 percent confidence interval, 0.74 to 1.74; P=0.59) among children who received dextran as compared with starch in the group with severe shock, and 0.88 (95 percent confidence interval, 0.66 to 1.17; P=0.38) among children who received dextran as compared with starch in the combined analysis. Although treatment with Ringer's lactate resulted in less rapid improvement in the hematocrit and a marginally longer time to initial recovery than did treatment with either of the colloid solutions, there were no differences in all other measures of treatment response. Only minor differences in efficacy were detected between the two colloids, but significantly more recipients of dextran than of starch had adverse reactions. Bleeding manifestations, coagulation derangements, and severity of fluid overload were similar for all fluid-treatment groups. CONCLUSIONS: Initial resuscitation with Ringer's lactate is indicated for children with moderately severe dengue shock syndrome. Dextran 70 and 6 percent hydroxyethyl starch perform similarly in children with severe shock, but given the adverse reactions associated with the use of dextran, starch may be preferable for this group.

Zinderman CE, Wise R, Landow L. 2005. Fluid solutions in dengue shock syndrome. N Engl J Med, 353 (23), pp. 2510-2511. | Read more

Winter PM, Dung NM, Loan HT, Kneen R, Wills B, Thu LT, House D, White NJ, Farrar JJ, Hart CA, Solomon T. 2004. Proinflammatory cytokines and chemokines in humans with Japanese encephalitis. J Infect Dis, 190 (9), pp. 1618-1626. | Show Abstract | Read more

BACKGROUND: Japanese encephalitis virus (JEV), the mosquito-borne flavivirus, annually causes an estimated 35,000-50,000 encephalitis cases and 10,000-15,000 deaths in Asia, and there is no antiviral treatment. The role played by the immune response in determining the outcome of human infection with JEV is poorly understood, although, in animal models of flavivirus encephalitis, unregulated proinflammatory cytokine responses can be detrimental. METHODS: We studied the innate, cellular, and humoral immune responses in 118 patients infected with JEV, of whom 13 (11%) died. RESULTS: Levels of interferon (IFN)- alpha , the proinflammatory cytokine interleukin (IL)-6, and the chemokine IL-8 were all higher in the cerebrospinal fluid (CSF) of the nonsurvivors than of the survivors (P=.04, P=.006, and P=.04, respectively), as were both the IL-6 : IL-4 ratio in CSF (a marker of the balance of pro- and anti-inflammatory cytokines) and the level of the chemokine RANTES (regulated on activation, normally T cell expressed and secreted) in plasma (P=.03). In contrast, levels of immunoglobulin (Ig) M and IgG in CSF and of IgM in plasma were higher in the survivors (P=.035, P=.003, and P=.009, respectively). Levels of IFN- gamma and nitric oxide did not vary with outcome. CONCLUSIONS: During JEV infection, elevated levels of proinflammatory cytokines and chemokines are associated with a poor outcome, but whether they are simply a correlate of severe disease or contribute to pathogenesis remains to be determined.

Wills BA, Oragui EE, Dung NM, Loan HT, Chau NV, Farrar JJ, Levin M. 2004. Size and charge characteristics of the protein leak in dengue shock syndrome. J Infect Dis, 190 (4), pp. 810-818. | Show Abstract | Read more

BACKGROUND: he mechanism underlying the transient vascular leak syndrome of dengue hemorrhagic fever (DHF) is unknown. We aimed to determine whether molecular size and charge selectivity, which help restrict plasma proteins within the intravascular space, are altered in patients with DHF and whether a disturbance of the anionic glycosaminoglycan (GAG) layer on the luminal endothelial surface contributes to disease pathogenesis. METHODS: We measured serial plasma levels and fractional clearances of proteins with different size and charge characteristics in 48 children with dengue shock syndrome (DSS) and urinary excretion profiles of heparan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate in affected children and healthy control subjects. RESULTS: Compared with convalescent values, acute plasma concentrations of all proteins were reduced, with increased fractional clearances. Smaller proteins were more affected than larger molecules. Albumin, which is normally protected from leakage by its strong negative charge, demonstrated a clearance pattern similar to that of transferrin, a neutral molecule of similar size. Urinary heparan sulfate excretion was significantly increased in children with DSS. CONCLUSIONS: The endothelial size-dependent sieving mechanism for plasma proteins is at least partially retained, whereas selective restriction based on negative charge is impaired. The increased heparan sulfate excretion suggests a role for GAGs in the pathogenesis of the vascular leak.

Solomon T, Dung NM, Wills B, Winter PM, Loan HT, Kneen R, Diet TV, Thuy TTN, Khanh VC, Farrar JJ, Hart CA. 2004. Interferon alpha in the pathogenesis and treatment Japanese encephalitis JOURNAL OF NEUROVIROLOGY, 10 pp. 28-28.

Phuong CXT, Nhan NT, Kneen R, Thuy PTT, van Thien C, Nga NTT, Thuy TT, Solomon T, Stepniewska K, Wills B, Dong Nai Study Group. 2004. Clinical diagnosis and assessment of severity of confirmed dengue infections in Vietnamese children: is the world health organization classification system helpful? Am J Trop Med Hyg, 70 (2), pp. 172-179. | Show Abstract

Classification of dengue using the current World Health Organization (WHO) system is not straightforward. In a large prospective study of pediatric dengue, no clinical or basic laboratory parameters clearly differentiated between children with and without dengue, although petechiae and hepatomegaly were independently associated with the diagnosis. Among the 712 dengue-infected children there was considerable overlap in the major clinical features. Mucosal bleeding was observed with equal frequency in those with dengue fever and dengue hemorrhagic fever (DHF), and petechiae, thrombocytopenia, and the tourniquet test differentiated poorly between the two diagnostic categories. Fifty-seven (18%) of 310 with shock did not fulfill all four criteria considered necessary for a diagnosis of DHF by the WHO, but use of the WHO provisional classification scheme resulted in considerable over-inflation of the DHF figures. If two separate entities truly exist rather than a continuous spectrum of disease, it is essential that some measure of capillary leak is included in any classification system, with less emphasis on bleeding and a specific platelet count.

Phuong CXT, Nhan NT, Kneen R, Bethell D, Dep LT, Nga NTT, Thuy PTT, Luat TD, Solomon T, Wills B, Parry CM. 2004. Evaluation of an algorithm for integrated management of childhood illness in an area of Vietnam with dengue transmission Tropical Medicine and International Health, 9 (5), pp. 573-581. | Show Abstract | Read more

OBJECTIVES: To determine whether nurses, using the WHO/UNICEF algorithm for integrated management of childhood illness (IMCI), modified to include dengue infection, satisfactorily classified children in an area endemic for dengue haemorrhagic fever (DHF). METHODS: Nurses assessed and classified, using the modified IMCI algorithm, a systematic sample of 1250 children aged 2 months to 10 years (n = 1250) presenting to a paediatric hospital in Dong Nai Province, Vietnam. Their classification was compared with that of a paediatrician, blind to the result of the nurses' assessment, which could be modified in the light of simple investigations, e.g. dengue serology. RESULTS: In children aged 2-59 months (n = 859), the nurses were able to classify, using the modified chart, the presenting illness in > 99% of children and found more than one classification in 70%. For the children with pneumonia, diarrhoea, dengue shock syndrome, severe DHF and severe disease requiring urgent admission, the nurse's classification was > 60% sensitive and > 85% specific compared with that of the paediatrician. For the nurse's classification of DHF the specificity was 50-55% for the children < 5 years and in children with definitive dengue serology. Alterations in the DHF algorithm improved specificity at the expense of sensitivity. CONCLUSION: Using the IMCI chart, nurses classified appropriately many of the major clinical problems in sick children < 5 years in southern Vietnam. However, further modifications will be required in the fever section, particularly for dengue. The impact of using the IMCI chart in peripheral health stations remains to be evaluated.

Solomon T, Dung NM, Wills B, Kneen R, Gainsborough M, Diet TV, Thuy TTN, Loan HT, Khanh VC, Vaughn DW et al. 2003. Interferon alfa-2a in Japanese encephalitis: a randomised double-blind placebo-controlled trial. Lancet, 361 (9360), pp. 821-826. | Show Abstract | Read more

BACKGROUND: Japanese encephalitis virus (JEV), although confined to Asia, causes about 35000-50000 cases and 10000 deaths every year, and is the most important cause of encephalitis worldwide. There is no known antiviral treatment for any flavivirus. Results from in-vitro studies and work in animals have shown inteferon alfa has antiviral activity on Japanese encephalitis and other flaviviruses; therefore, we aimed to assess the efficacy of inteferon alfa-2a in Japanese encephalitis. METHODS: We did a randomised double-blind placebo-controlled trial of interferon alfa-2a (10 million units/m2, daily for 7 days) in 112 Vietnamese children with suspected Japanese encephalitis, 87 of whom had serologically confirmed infections. Our primary endpoints were hospital death or severe sequelae at discharge. Analysis was by intention to treat. FINDINGS: Overall, 21 children (19%) died, and 17 (15%) had severe sequelae. Outcome at discharge and 3 months did not differ between the two treatment groups; 20 children in the interferon group had a poor outcome (death or severe sequelae), compared with 18 in the placebo group (p=0.85, difference 0.1%, 95% CI -17.5 to 17.6%), there were no long-term side effects of interferon. INTERPRETATION: The doses of interferon alfa-2a given in this regimen did not improve the outcome of patients with Japanese encephalitis.

Wills BA, Oragui EE, Stephens AC, Daramola OA, Dung NM, Loan HT, Chau NV, Chambers M, Stepniewska K, Farrar JJ, Levin M. 2002. Coagulation abnormalities in dengue hemorrhagic Fever: serial investigations in 167 Vietnamese children with Dengue shock syndrome. Clin Infect Dis, 35 (3), pp. 277-285. | Show Abstract | Read more

The pathophysiological basis of hemorrhage in dengue infections remains poorly understood, despite the increasing global importance of these infections. A large prospective study of 167 Vietnamese children with dengue shock syndrome documented only minor prolongations of prothrombin and partial thromboplastin times but moderate to severe depression of plasma fibrinogen concentrations. A detailed study of 48 children revealed low plasma concentrations of the anticoagulant proteins C, S, and antithrombin III, which decreased with increasing severity of shock, probably because of capillary leakage. Concurrent increases in the levels of thrombomodulin, tissue factor, and plasminogen activator inhibitor type 1 (PAI-1) indicated increased production of these proteins. Thrombomodulin levels suggestive of endothelial activation correlated with increasing shock severity, whereas PAI-1 levels correlated with bleeding severity. Dengue virus can directly activate plasminogen in vitro. Rather than causing true disseminated intravascular coagulation, dengue infection may activate fibrinolysis primarily, degrading fibrinogen directly and prompting secondary activation of procoagulant homeostatic mechanisms.

Cao XTP, Ngo TN, Wills B, Kneen R, Nguyen TTH, Ta TTM, Tran TTH, Doan TKL, Solomon T, Simpson JA et al. 2002. Evaluation of the World Health Organization standard tourniquet test and a modified tourniquet test in the diagnosis of dengue infection in Viet Nam. Trop Med Int Health, 7 (2), pp. 125-132. | Show Abstract | Read more

OBJECTIVES: A positive tourniquet test is one of several clinical parameters considered by the World Health Organization to be important in the diagnosis of dengue haemorrhagic fever, but no formal evaluation of the test has been undertaken. As many doctors remain unconvinced of its usefulness, this study was designed to assess the diagnostic utility of both the standard test and a commonly employed modified test. METHODS: A prospective evaluation of the standard sphygmomanometer cuff tourniquet test, compared with a simple elastic cuff tourniquet test, was carried out in 1136 children with suspected dengue infection admitted to a provincial paediatric hospital in southern Viet Nam. RESULTS: There was good agreement between independent observers for both techniques, but the sphygmomanometer method resulted in consistently greater numbers of petechiae. This standard method had a sensitivity of 41.6% for dengue infection, with a specificity of 94.4%, positive predictive value of 98.3% and negative predictive value of 17.3%. The test differentiated poorly between dengue haemorrhagic fever (45% positive) and dengue fever (38% positive). The simple elastic tourniquet was less sensitive than the sphygmomanometer cuff, but at a threshold of 10 petechiae (compared with the WHO recommendation of 20) per 2.5 cm2 the sensitivity for the elastic tourniquet rose to 45% (specificity 85%). Other evidence of bleeding was frequently present and the tourniquet test provided additional information to aid diagnosis in only 5% of cases. CONCLUSION: The conventional tourniquet test adds little to the diagnosis of dengue in hospitalized children. The simple, cheap elastic tourniquet may be useful in diagnosing dengue infection in busy rural health stations in dengue endemic areas of the tropics. A positive test should prompt close observation or early hospital referral, but a negative test does not exclude dengue infection.

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Twiddy SS, Farrar JJ, Vinh Chau N, Wills B, Gould EA, Gritsun T, Lloyd G, Holmes EC. 2002. Phylogenetic relationships and differential selection pressures among genotypes of dengue-2 virus Virology, 298 (1), pp. 63-72. | Show Abstract | Read more

To elucidate the processes controlling the emergence and spread of dengue-2 virus (DEN-2) we examined the evolution of viral isolates sampled from both local (Viet Nam) and global populations. Our phylogenetic analysis, incorporating envelope (E) glycoprotein sequences from 147 isolates of DEN-2, provided a more complete picture of viral diversity, with a newly defined "Cosmopolitan" genotype having a near global distribution and two other genotypes restricted to Asia. By analyzing rates of synonymous and nonsynonymous substitution we determined that genotypes have experienced different selection pressures, with some evidence of positive selection in the Cosmopolitan genotype and one of the two Asian genotypes, but that the transition from sylvatic to human transmission was not accompanied by adaptive evolution of the E gene. Although there was no association between selection pressures acting on the E gene and proposed virulence differences among genotypes, some putatively selected amino acid sites have previously been implicated in changing viral pathogenicity, most notably E-390, and may also affect transmittability. These findings have implications for the future spread of DEN-2. © 2002 Elsevier Science (USA).

Ngo NT, Cao XT, Kneen R, Wills B, Nguyen VM, Nguyen TQ, Chu VT, Nguyen TT, Simpson JA, Solomon T et al. 2001. Acute management of dengue shock syndrome: a randomized double-blind comparison of 4 intravenous fluid regimens in the first hour. Clin Infect Dis, 32 (2), pp. 204-213. | Show Abstract | Read more

Dengue hemorrhagic fever is an important cause of morbidity among Asian children, and the more severe dengue shock syndrome (DSS) causes a significant number of childhood deaths. DSS is characterized by a massive increase in systemic capillary permeability with consequent hypovolemia. Fluid resuscitation is critical, but as yet there have been no large trials to determine the optimal fluid regimen. We undertook a randomized blinded comparison of 4 fluids (dextran, gelatin, lactated Ringer's, and "normal" saline) for initial resuscitation of 230 Vietnamese children with DSS. All the children survived, and there was no clear advantage to using any of the 4 fluids, but the longest recovery times occurred in the lactated Ringer's group. The most significant factor determining clinical response was the pulse pressure at presentation. A comparison of the colloid and crystalloid groups suggested benefits in children presenting with lower pulse pressures who received one of the colloids. Further large-scale studies, stratified for admission pulse pressure, are indicated.

Wills B. 2001. Volume replacement in dengue shock syndrome Dengue Bulletin, 25 pp. 50-55. | Show Abstract

The treatment of dengue shock syndrome (DSS) is a medical emergency. Prompt and vigorous volume replacement therapy is required, with extreme care to avoid fluid overload. Recognition of the importance of increased vascular permeability in the pathophysiology of DSS and of the critical need for parenteral fluids in resuscitation has had a dramatic effect on mortality from the disease over the last 40 years. However, until recently there has been little research to determine the optimal fluid regimen, and the choice of fluid has remained largely empirical. Colloid and crystalloid fluid have different physicochemical properties which influence the patterns of distribution and elimination, as well as the secondary osmotic effects. In two recent double-blind randomized trials in Viet Nam, initial resuscitation with colloids fluids (dextran 70 or 3% gelatin) restored cardiac index and pulse pressure and normalized haemotocrit more quickly than either of the crystalloid fluids (physiological saline or Ringer's lactate). There was no difference, however, in the overall recovery time or the subsequent requirement for fluids. From the larger study it was apparent that the major determinant of clinical response was the width of the pulse pressure at presentation with shock, the small number of children with pulse pressures of ≤ 10 mm Hg requiring significantly more resuscitation than those with higher pulse pressures. Within this more compromised group there was a trend to earlier, sustained recovery among those who received one of the colloids. It appears that the majority of children with DSS recover with infusion of crystallois fluids alone, but that an important minority may benefit initial resuscitation with a colloid. Large trials will be necessary to confirm this effect and to clearly characterize the subgroup of children who might benefit from initial colloid therapy. Given the huge burden of dengue disease in South-East Asia, if a true benefit is established in a subgroup of patients, this may have a significant influence on mortality in the region.

Wills B, Farrar J. 2000. Central nervous system infections in the tropics: diagnosis, treatment and prevention. Curr Opin Infect Dis, 13 (3), pp. 259-264. | Show Abstract | Read more

The present review is a brief discussion of some of the important new developments that have been reported relating to bacterial and viral infections of the central nervous system. For many of these conditions, certain issues remain unresolved, including what is the best diagnostic approach, what is the optimum treatment and how best to prevent such conditions. The development of improved surveillance, more effective vaccines and a greater understanding of the pathophysiology of these diseases all offer great potential benefits. The hope is that these advances, when they come, will be affordable to those who need them most.

Parry CM, Wuthiekanun V, Hoa NT, Diep TS, Thao LT, Loc PV, Wills BA, Wain J, Hien TT, White NJ, Farrar JJ. 1999. Melioidosis in Southern Vietnam: clinical surveillance and environmental sampling. Clin Infect Dis, 29 (5), pp. 1323-1326. | Show Abstract | Read more

From 1992-1998, Burkholderia pseudomallei was isolated from only 9 (0.25%) of 3653 cultures of blood from febrile patients admitted to the Centre for Tropical Diseases in Ho Chi Minh City, an infectious disease referral center for southern Vietnam. Soil was sampled from 407 sites in 147 rice fields along the 5 major roads radiating from Ho Chi Minh City. B. pseudomallei was isolated from 73 sites (18%) in 39 rice fields (27%), but only 15 (21%) of the 71 isolates from 9 (6%) of 147 fields were the virulent l-arabinose (ara)-negative biotype. All except 1 of the fields with the ara-negative biotype were close to the homes of the patients with melioidosis. The low incidence of melioidosis in the provinces around Ho Chi Minh City may be explained by the restricted distribution of ara-negative B. pseudomallei in the soil in this area.

Lewallen S, Taylor TE, Molyneux ME, Semba RD, Wills BA, Courtright P. 1998. Association between measures of vitamin A and the ocular fundus findings in cerebral malaria. Arch Ophthalmol, 116 (3), pp. 293-296. | Show Abstract | Read more

OBJECTIVE: To investigate the relationship between serum vitamin A levels and conjunctival impression cytology and retinal whitening present in Malawian children with cerebral malaria. METHODS: Standard retinal examination and conjunctival impression cytology were performed at hospital admission on 101 consecutively admitted children with cerebral malaria. Blood samples were drawn from 56 children at 24 hours, frozen at -20 degrees C, and transported for assessment of vitamin A levels by high-performance liquid chromatography. Associations among fundus findings and vitamin A measurements were sought. RESULTS: The whitening of the retina that we have previously described in children with cerebral malaria was found to be associated with a mean+/-SD serum vitamin A level of 0.29+/-0.1 micromol/L, compared with a mean vitamin A level of 0.41+/-0.2 micromol/L in children without retinal whitening. Children with retinal whitening were 2.77 (95% CI, 1.06-7.3) times more likely to have abnormal conjunctival impression cytology results than those without whitening. No child had any clinical or ophthalmologic evidence of chronic vitamin A deficiency. CONCLUSIONS: The retinal whitening described in children with cerebral malaria is associated with low serum vitamin A levels and with abnormal conjunctival impression cytology results and may be due to acute vitamin A deficiency at the tissue level.

Taylor TE, Wills BA, Courval JM, Molyneux ME. 1998. Intramuscular artemether vs intravenous quinine: an open, randomized trial in Malawian children with cerebral malaria. Trop Med Int Health, 3 (1), pp. 3-8. | Show Abstract | Read more

OBJECTIVES: To compare artemether (by intramuscular injection) and quinine (by intravenous infusion) as treatments for cerebral malaria in African children. METHODS: An open, randomized trial conducted at the Queen Elizabeth Central Hospital in Blantyre, Malawi. This trial was part of a multicentre study designed to determine if treatment with artemether would significantly lower mortality rates compared with quinine. Data from 83 artemether recipients and 81 quinine recipients are reported here. RESULTS: Overall mortality rates and coma resolution times were not significantly different in the two treatment groups. Parasite and fever clearance times were significantly more rapid in the artemether recipients. Analyses which took into account the possible confounding variables did not significantly alter the findings of these unadjusted analyses. CONCLUSION: These results do not suggest that treatment with artemether would confer a survival advantage in children with life-threatening malaria. The power and precision of the estimated treatment effects of artemether would be enhanced by a meta-analysis of all relevant clinical trials.

Lewallen S, Bakker H, Taylor TE, Wills BA, Courtright P, Molyneux ME. 1996. Retinal findings predictive of outcome in cerebral malaria. Trans R Soc Trop Med Hyg, 90 (2), pp. 144-146. | Show Abstract | Read more

The pathogenesis of cerebral malaria is poorly understood. Direct and indirect ophthalmoscope examinations of 141 Malawian children with strictly defined cerebral malaria revealed 2 distinct and prognostically significant findings: papilloedema and extramacular retinal oedema. The relative risk of death in patients with papilloedema was 6.7 times that in patients without papilloedema. Extramacular retinal oedema was associated with a 2.9 fold increase in the relative risk of dying. The mortality rate in patients with neither of these signs was only 1.3% compared to an overall mortality rate of 9.2%. The clinical and laboratory features associated with each of these ophthalmological findings were different, suggesting that there may be at least 2 different pathogenetic processes in patients with cerebral malaria.

Taylor T, Wills B, Wirima J, Molyneux M. 1993. Artemether in cerebral malaria. Lancet, 341 (8860), pp. 1604. | Read more

Lewallen S, Taylor TE, Molyneux ME, Wills BA, Courtright P. 1993. Ocular fundus findings in Malawian children with cerebral malaria. Ophthalmology, 100 (6), pp. 857-861. | Show Abstract | Read more

BACKGROUND: Cerebral malaria is a major cause of mortality and morbidity in children in tropical regions. The pathogenesis of this important complication of Plasmodium falciparum infection is not well understood. A number of observers have commented on the presence of retinal pathology in various types of malaria. Previous reports have not demonstrated that fundus findings are significantly associated with outcome. METHODS: The authors examined the ocular fundi, by direct and indirect ophthalmoscopy, of 56 children admitted consecutively with cerebral malaria. RESULTS: Every child with a normal fundus on admission recovered fully, but two conditions were found to be associated with a poor outcome. Patients with papilledema had a relative risk of poor outcome 5.2 times greater than those without this finding (P < 0.01). Patients with retinal edema outside the posterior vascular arcades had a relative risk of poor outcome 3.9 times greater than those without this finding (P < 0.01). These two fundus findings were independently predictive of a poor outcome. CONCLUSION: Fundus findings are useful as predictors of outcome in children with cerebral malaria. The authors' findings suggest that there may be two distinct mechanisms associated with poor outcome in these children.

Taylor TE, Wills BA, Kazembe P, Chisale M, Wirima JJ, Ratsma EY, Molyneux ME. 1993. Rapid coma resolution with artemether in Malawian children with cerebral malaria. Lancet, 341 (8846), pp. 661-662. | Show Abstract | Read more

Artemisinin compounds clear parasitaemia more rapidly than other drugs do in both mild and severe malaria, but no advantage in clinical efficacy has been shown. We have compared artemether treatment with standard quinine treatment in Malawian children with cerebral malaria. 65 unconscious children were randomly allocated to intravenous quinine (n = 37) or intramuscular artemether (n = 28) treatment. The two groups were well matched for various prognostic features. Median parasite clearance times were shorter in the artemether group (28 [interquartile range 18-34] vs 40 [36-44] h in the quinine group, p = 0.0002). Coma resolution times were also shorter with artemether than with quinine (8 [4-15] vs 14 [10-36] h, p = 0.01).

Lewallen S, Wills BA. 1993. Retinal haemorrhage in children with malaria. Lancet, 341 (8842), pp. 442. | Read more

Jaenisch T, Junghanss T, Wills B, Brady OJ, Eckerle I, Farlow A, Hay SI, McCall PJ, Messina JP, Ofula V et al. 2014. Dengue expansion in Africa-not recognized or not happening? Emerg Infect Dis, 20 (10), | Show Abstract | Read more

An expert conference on Dengue in Africa was held in Accra, Ghana, in February 2013 to consider key questions regarding the possible expansion of dengue in Africa. Four key action points were highlighted to advance our understanding of the epidemiology of dengue in Africa. First, dengue diagnostic tools must be made more widely available in the healthcare setting in Africa. Second, representative data need to be collected across Africa to uncover the true burden of dengue. Third, established networks should collaborate to produce these types of data. Fourth, policy needs to be informed so the necessary steps can be taken to provide dengue vector control and health services.

Yacoub S, Wills B. 2014. Predicting outcome from dengue. BMC Med, 12 (1), pp. 147. | Show Abstract | Read more

Dengue is emerging as one of the most abundant vector-borne disease globally. Although the majority of infections are asymptomatic or result in only a brief systemic viral illness, a small proportion of patients develop potentially fatal complications. These severe manifestations, including a unique plasma leakage syndrome, a coagulopathy sometimes accompanied by bleeding, and organ impairment, occur relatively late in the disease course, presenting a window of opportunity to identify the group of patients likely to progress to these complications. However, as yet, differentiating this group from the thousands of milder cases seen each day during outbreaks remains challenging, and simple and inexpensive strategies are urgently needed in order to improve case management and to facilitate appropriate use of limited resources. This review will cover the current understanding of the risk factors associated with poor outcome in dengue. We focus particularly on the clinical features of the disease and on conventional investigations that are usually accessible in mid-level healthcare facilities in endemic areas, and then discuss a variety of viral, immunological and vascular biomarkers that have the potential to improve risk prediction. We conclude with a description of several novel methods of assessing vascular function and intravascular volume status non-invasively.

Whitehorn J, Yacoub S, Anders KL, Macareo LR, Cassetti MC, Nguyen Van VC, Shi P-Y, Wills B, Simmons CP. 2014. Dengue therapeutics, chemoprophylaxis, and allied tools: state of the art and future directions. PLoS Negl Trop Dis, 8 (8), pp. e3025. | Show Abstract | Read more

Dengue is the most common arboviral disease of humans. There is an unmet need for a therapeutic intervention that reduces the duration and severity of dengue symptoms and diminishes the likelihood of severe complications. To this end, there are active discovery efforts in industry and academia to develop interventions, with a focus on small molecule inhibitors of dengue virus replication that are suitable for therapy or chemoprophylaxis. Advancements in animal models of dengue virus infection together with the possibility of a dengue human infection model have further enhanced the platform for dengue drug discovery. Whilst drug discovery efforts gestate, there are ongoing clinical research designed to benefit today's patients, including trials of supportive care interventions, and descriptive studies that should improve the ability of clinicians to make an accurate diagnosis early in the illness course and to identify patients most at risk of progression to severe disease. This review provides a state of the art summary of dengue drug discovery, clinical trials, and supportive allied research and reflects discussions at the 2nd International Dengue Therapeutics Workshop held in Ho Chi Minh City, Vietnam, in December 2013.

Yacoub S, Wertheim H, Simmons CP, Screaton G, Wills B. 2014. Cardiovascular manifestations of the emerging dengue pandemic. Nat Rev Cardiol, 11 (6), pp. 335-345. | Show Abstract | Read more

Dengue is one of the most important emerging viral diseases globally. The majority of symptomatic infections result in a relatively benign disease course. However, a small proportion of patients develop severe clinical manifestations, including bleeding, organ impairment, and endothelial dysfunction with increased capillary permeability causing hypovolaemic shock that can lead to cardiovascular collapse. Evidence is increasing that dengue can also cause myocardial impairment, arrhythmias and, occasionally, fulminant myocarditis. No antiviral agents or vaccines are licensed for dengue, and treatment remains supportive with judicious fluid replacement for patients with severe disease. Defining the role of cardiac dysfunction in the haemodynamic compromise of severe dengue has potentially important management implications. In this Review, we will outline the current understanding of the cardiovascular manifestations of dengue, including myocardial and vascular involvement, and conclude with a discussion of the available therapeutic options and potential future research directions.

Lam PK, Tam DTH, Diet TV, Tam CT, Tien NTH, Kieu NTT, Simmons C, Farrar J, Nga NTN, Qui PT et al. 2013. Clinical characteristics of Dengue shock syndrome in Vietnamese children: a 10-year prospective study in a single hospital. Clin Infect Dis, 57 (11), pp. 1577-1586. | Show Abstract | Read more

BACKGROUND: Dengue shock syndrome (DSS) is a severe manifestation of dengue virus infection that particularly affects children and young adults. Despite its increasing global importance, there are no prospective studies describing the clinical characteristics, management, or outcomes of DSS. METHODS: We describe the findings at onset of shock and the clinical evolution until discharge or death, from a comprehensive prospective dataset of 1719 Vietnamese children with laboratory-confirmed DSS managed on a single intensive care unit between 1999 and 2009. RESULTS: The median age of patients was 10 years. Most cases had secondary immune responses, with only 6 clear primary infections, and all 4 dengue virus serotypes were represented during the 10-year study. Shock occurred commonly between days 4 and 6 of illness. Clinical signs and symptoms were generally consistent with empirical descriptions of DSS, although at presentation 153 (9%) were still febrile and almost one-third had no bleeding. Overall, 31 (2%) patients developed severe bleeding, primarily from the gastrointestinal tract, 26 of whom required blood transfusion. Only 8 patients died, although 123 of 1719 (7%) patients had unrecordable blood pressure at presentation and 417 of the remaining 1596 (26%) were hypotensive for age. The majority recovered well with standard crystalloid resuscitation or following a single colloid infusion. All cases were classified as severe dengue, while only 70% eventually fulfilled all 4 criteria for the 1997 World Health Organization classification of dengue hemorrhagic fever. CONCLUSIONS: With prompt intervention and assiduous clinical care by experienced staff, the outcome of this potentially fatal condition can be excellent.

Hanh Tien NT, Lam PK, Duyen HTL, Ngoc TV, Ha PTT, Kieu NTT, Simmons C, Wolbers M, Wills B. 2013. Assessment of microalbuminuria for early diagnosis and risk prediction in dengue infections. PLoS One, 8 (1), pp. e54538. | Show Abstract | Read more

BACKGROUND: Dengue is the most important arboviral infection of humans. Following an initial febrile period, a small proportion of infected patients develop a vasculopathy, with children at particular risk for severe vascular leakage and shock. Differentiation between dengue and other common childhood illnesses is difficult during the early febrile phase, and risk prediction for development of shock is poor. The presence of microalbuminuria is recognized as a useful early predictor for subsequent complications in a number of other disorders with vascular involvement. Significant proteinuria occurs in association with dengue shock syndrome and it is possible that early-phase microalbuminuria may be helpful both for diagnosis of dengue and for identification of patients likely to develop severe disease. METHODOLOGY/PRINCIPAL FINDINGS: We measured formal urine albumin to creatinine ratios (UACRs) in daily samples obtained from a large cohort of children with suspected dengue recruited at two outpatient clinics in Ho Chi Minh City, Vietnam. Although UACRs were increased in the 465 confirmed dengue patients, with a significant time trend showing peak values around the critical period for dengue-associated plasma leakage, urine albumin excretion was also increased in the comparison group of 391 patients with other febrile illnesses (OFI). The dengue patients generally had higher UACRs than the OFI patients, but microalbuminuria, using the conventional cutoff of 30 mg albumin/g creatinine discriminated poorly between the two diagnostic groups in the early febrile phase. Secondly UACRs did not prove useful in predicting either development of warning signs for severe dengue or need for hospitalization. CONCLUSION/SIGNIFICANCE: Low-level albuminuria is common, even in relatively mild dengue infections, but is also present in many OFIs. Simple point-of-care UACR tests are unlikely to be useful for early diagnosis or risk prediction in dengue endemic areas.

Tam DTH, Ngoc TV, Tien NTH, Kieu NTT, Thuy TTT, Thanh LTC, Tam CT, Truong NT, Dung NT, Qui PT et al. 2012. Effects of short-course oral corticosteroid therapy in early dengue infection in Vietnamese patients: a randomized, placebo-controlled trial. Clin Infect Dis, 55 (9), pp. 1216-1224. | Show Abstract | Read more

BACKGROUND: Patients with dengue can experience a variety of serious complications including hypovolemic shock, thrombocytopenia, and bleeding. These problems occur as plasma viremia is resolving and are thought to be immunologically mediated. Early corticosteroid therapy may prevent the development of such complications but could also prolong viral clearance. METHODS: We performed a randomized, placebo-controlled, blinded trial of low-dose (0.5 mg/kg) or high-dose (2 mg/kg) oral prednisolone therapy for 3 days in Vietnamese patients aged 5-20 years admitted with dengue and fever for ≤72 hours, aiming to assess potential harms from steroid use during the viremic phase. Intention-to-treat analysis was performed using linear trend tests with a range of clinical and virological endpoints specified in advance. In addition to recognized complications of dengue, we focused on the are under the curve for serial plasma viremia measurements and the number of days after enrollment to negative viremia and dengue nonstructural protein 1 status. RESULTS: Between August 2009 and January 2011, 225 participants were randomized to 1 of the 3 treatment arms. Baseline characteristics were similar across the groups. All patients recovered fully and adverse events were infrequent. Aside from a trend toward hyperglycemia in the steroid recipients, we found no association between treatment allocation and any of the predefined clinical, hematological, or virological endpoints. CONCLUSIONS: Use of oral prednisolone during the early acute phase of dengue infection was not associated with prolongation of viremia or other adverse effects. Although not powered to assess efficacy, we found no reduction in the development of shock or other recognized complications of dengue virus infection in this study.

Trung DT, Thao LTT, Dung NM, Ngoc TV, Hien TT, Chau NVV, Wolbers M, Tam DTH, Farrar J, Simmons C, Wills B. 2012. Clinical features of dengue in a large Vietnamese cohort: intrinsically lower platelet counts and greater risk for bleeding in adults than children. PLoS Negl Trop Dis, 6 (6), pp. e1679. | Show Abstract | Read more

BACKGROUND: As dengue spreads to new geographical regions and the force of infection changes in existing endemic areas, a greater breadth of clinical presentations is being recognised. Clinical experience suggests that adults manifest a pattern of complications different from those observed in children, but few reports have described the age-related spectrum of disease in contemporaneous groups of patients recruited at the same geographical location. METHODOLOGY/PRINCIPAL FINDINGS: Using detailed prospectively collected information from ongoing studies that encompass the full spectrum of hospitalised dengue cases admitted to a single hospital in southern Vietnam, we compared clinical and laboratory features, management, and outcome for 647 adults and 881 children with confirmed dengue. Signs of vascular leakage and shock were more frequent and more severe in children than adults, while bleeding manifestations and organ involvement were more common in adults. Additionally, adults experienced significantly more severe thrombocytopenia. Secondary infection but not serotype was independently associated with greater thrombocytopenia, although with a smaller effect than age-group. The effect of age-group on platelet count was also apparent in the values obtained several weeks after recovery, indicating that healthy adults have intrinsically lower counts compared to children. CONCLUSIONS/SIGNIFICANCE: There are clear distinctions between adults and children in the pattern of complications seen in association with dengue infection, and these depend partly on intrinsic age-dependent physiological differences. Knowledge of such differences is important to inform research on disease pathogenesis, as well as to encourage development of management guidelines that are appropriate to the age-groups at risk.

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Simmons CP, Farrar JJ, Chau NVV, Wills B. 2012. CURRENT CONCEPTS Dengue NEW ENGLAND JOURNAL OF MEDICINE, 366 (15), pp. 1423-1432. | Read more

Duyen HTL, Ngoc TV, Ha DT, Hang VTT, Kieu NTT, Young PR, Farrar JJ, Simmons CP, Wolbers M, Wills BA. 2011. Kinetics of plasma viremia and soluble nonstructural protein 1 concentrations in dengue: differential effects according to serotype and immune status. J Infect Dis, 203 (9), pp. 1292-1300. | Show Abstract | Read more

We describe the magnitude and kinetics of plasma viremia and nonstructural protein 1 (sNS1) levels in sequential samples from 167 children with acute dengue, enrolled early in a community study in Vietnam. All children recovered fully, and only 5 required hospitalization. Among those with dengue virus type 1 (DENV-1), plasma viremia was significantly greater in primary (49) than secondary (44) infections and took longer to resolve. In primary DENV-2 and 3 infections, viremia was significantly lower than among primary DENV-1 infections. Concentrations of sNS1 were significantly higher for DENV-1 than for DENV-2 after adjusting for viremia, with marked differences in the kinetic profiles between primary and secondary infections. Secondary infection and higher viremia were independent predictors of more severe thrombocytopenia, and higher viremia was associated with a small increase in hemoconcentration. Our findings identify clear serotype and immune-status related effects on the dynamics of dengue viremia and sNS1 responses, together with associations with important clinical parameters.

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Khor CC, Chau TNB, Pang J, Davila S, Long HT, Ong RTH, Dunstan SJ, Wills B, Farrar J, Van Tram T et al. 2011. Genome-wide association study identifies susceptibility loci for dengue shock syndrome at MICB and PLCE1 Nature Genetics, 43 (11), pp. 1139-1141. | Show Abstract | Read more

Hypovolemic shock (dengue shock syndrome (DSS)) is the most common life-threatening complication of dengue. We conducted a genome-wide association study of 2,008 pediatric cases treated for DSS and 2,018 controls from Vietnam. Replication of the most significantly associated markers was carried out in an independent Vietnamese sample of 1,737 cases and 2,934 controls. SNPs at two loci showed genome-wide significant association with DSS. We identified a susceptibility locus at MICB (major histocompatibility complex (MHC) class I polypeptide-related sequence B), which was within the broad MHC region on chromosome 6 but outside the class I and class II HLA loci (rs3132468, P meta = 4.41 × 10 -11 , per-allele odds ratio (OR) = 1.34 (95% confidence interval: 1.23-1.46)). We identified associated variants within PLCE1 (phospholipase C, epsilon 1) on chromosome 10 (rs3765524, P meta = 3.08 × 10 -10 , per-allele OR = 0.80 (95% confidence interval: 0.75-0.86)). We identify two loci associated with susceptibility to DSS in people with dengue, suggesting possible mechanisms for this severe complication of dengue. © 2011 Nature America, Inc. All rights reserved.

Trung DT, Thao LTT, Hien TT, Hung NT, Vinh NN, Hien PTD, Chinh NT, Simmons C, Wills B. 2010. Liver involvement associated with dengue infection in adults in Vietnam. Am J Trop Med Hyg, 83 (4), pp. 774-780. | Show Abstract | Read more

Globally, the number of adults hospitalized with dengue has increased markedly in recent years. It has been suggested that hepatic dysfunction is more significant in this group than among children. We describe the spectrum and evolution of disease manifestations among 644 adults with dengue who were prospectively recruited on admission to a major infectious disease hospital in southern Vietnam and compare them with a group of patients with similar illnesses not caused by dengue. Transaminase levels increased in virtually all dengue patients and correlated with other markers of disease severity. However, peak enzyme values usually occurred later than other complications. Clinically severe liver involvement was infrequent and idiosyncratic, but usually resulted in severe bleeding. Chronic co-infection with hepatitis B was associated with modestly but significantly increased levels of alanine aminotransferase, but did not otherwise impact the clinical picture.

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