Epidemiological, virological and clinical characterization of a Dengue/Zika outbreak in the Caribbean region of Costa Rica 2017-2018.

The increase in incidence and geographical expansion of viruses transmitted by the Aedes mosquitoes, such as dengue (DENV) and zika (ZIKV) in the Americas, represents a burden for healthcare systems in tropical and subtropical regions. These and other under-detected arboviruses co-circulate in Costa Rica, adding additional complexity to their management due to their shared epidemiological behavior and similarity of symptoms in early stages. Since diagnostics of febrile illness is mostly based on clinical symptoms alone, we gathered acute-phase serum and urine from 399 samples of acute dengue-like cases from two healthcare facilities of Costa Rica, during an outbreak of arboviruses from July 2017 to May 2018, and tested them using molecular and serological methods. The analyses showed that of the clinically presumptive arbovirus cases that were reported, only 39.4% (n=153) of the samples were confirmed positive by RT-PCR to be DENV (DENV (10.3%), CHIKV (0.2%), ZIKV (27.3%), or mixed infections (1.5%). RT-PCR for other alphaviruses and flaviviruses, and PCR for Leptospira sp were negative. Furthermore, to assess flavivirus positivity in post-acute patients, the negative sera were tested against Dengue-IgM. 20% of sera were found positive, confounding even more the definitive number of cases, and emphasizing the need of several distinct diagnostic tools for accurate diagnostics. Molecular characterization of the prM and E genes from isolated viruses revealed that the American/Asian genotype of DENV-2 and the Asian lineage of ZIKV were circulating during this outbreak. Two different clades of DENV-2 American/Asian genotype were identified to co-circulate in the same region and a difference in the platelet and leukocyte count was noted between people infected with each clade, suggesting a putative distinct virulence. Our study sheds light on the necessity for healthcare strategies in managing arbovirus outbreaks, emphasizing the importance of comprehensive molecular and serological diagnostic approaches, as well as molecular characterization. This approach aids in enhancing our understanding of the clinical and epidemiological aspects of arboviral diseases during outbreaks. Our research highlights the need to strengthen training programs for health professionals and the need to increase research-based on laboratory evidence for diagnostic accuracy, guidance, development and implementation of public health interventions and epidemiological surveillance.

Serological immune biomarker for disease severity in dengue-infected pediatric hospitalized patients.

Clinical manifestation of dengue disease ranges from asymptomatic, febrile fever without warning sign (DOS) to serious outcome dengue with warning sign (DWS) and severe disease (SD) leading to shock syndrome and death. The role of antibody response in natural dengue infection is complex and not completely understood. Here, we aimed to assess serological marker for disease severity. Antibody response of dengue-confirmed pediatric patients with acute secondary infection were evaluated against infecting virus, immature virus, and recombinant envelop protein. Immature virus antibody titers were significantly higher in DWS as compared to DOS (p = 0.0006). However, antibody titers against recombinant envelop protein were higher in DOS as compared to DWS, and antibody avidity was significantly higher against infecting virus in DOS. Serum samples of DOS patients displayed higher in vitro neutralization potential in plaque assay as compared to DWS, whereas DWS serum samples showed higher antibody-dependent enhancement in the in vitro enhancement assays. Thus, antibodies targeting immature virus can predict disease severity and could be used in early forecast of disease outcome using an enzyme-linked immunoassay assay system which is less laborious and cheaper than plaque assay system for correlates of protection and could help optimize medical care and resources.

Field-deployable viral diagnostic tools for dengue virus based on Cas13a and Cas12a.

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.

[Takotsubo syndrome in dengue patient]. / Síndrome de Takotsubo en paciente con dengue.

Takotsubo syndrome, was described in Japan in 1990, it is a stress cardiomyopathy, predominantly in women, usually postmenopausal. Cardiac hypokinesia occurs, with involvement of multiple coronary territories. In intensive care unit (ICU), it is considered underdiagnosed. Manifestations of severe dengue fever include cardiovascular involvement, mainly arrhythmias and systolic dysfunction. A case of a 72-year-old man is presented, who was hospitalized in ICU for dengue fever, with plateletopenia (15000 cells/mm3) and dehydration. After fluid management the patient reported respiratory discomfort, auscultating crackling rales. A pulmonary ultrasound was made where bilateral B lines were found with B7 pattern compatible with interstitial syndrome and pulmonary edema. Basal hyperkinesia, medial and apical hypokinesia with an image consistent with apical ballooning were observed in the transthoracic echocardiogram. The electrocardiogram showed complete right bundle branch block. Chagas serology was negative and quantitative troponin I was increased. In the context of severe dengue, a Takotsubo syndrome was diagnosed. The patient evolved favorably. After discharge, a normalization of the cardiac function was stated in ultrasound images. The case is of clinical importance due to the low association of these two diseases and the need to screen for cardiac involvement in severe dengue.

El síndrome de Takotsubo, fue descripto en Japón en 1990, se trata de una miocardiopatía por estrés, predominante en mujeres, generalmente postmenopáusicas. Se produce una hipoquinesia cardiaca, con compromiso de múltiples territorios coronarios. En las unidades de terapia intensiva (UTI), se considera subdiagnosticada. En las manifestaciones del dengue grave, se encuentra el compromiso cardiovascular, principalmente arritmias y disfunción sistólica. Se presenta el caso de un hombre de 72 años, internado en UTI por dengue, con plaquetopenia (15000 células/mm3) y deshidratación. Luego de la administración de fluidos refirió disconfort respiratorio, auscultándose estertores pulmonares. Se realizó ecografía pulmonar donde se apreció líneas B bilaterales con patrón B7 compatible con síndrome intersticial y edema pulmonar. En el ecocardiograma transtorácico se objetivó hiperquinesia basal, hipoquinesia medial y apical con imagen compatible con balonamiento apical. En el electrocardiograma se evidenció bloqueo completo de rama derecha. La serología para Chagas fue negativa y la troponina I cuantitativa se detectó aumentada. Se diagnosticó síndrome de Takotsubo en el contexto de dengue grave. El paciente evolucionó favorablemente. Posterior al alta, se constató normalización de la motilidad cardíaca, en las imágenes ecográficas. El caso es de importancia clínica por la baja asociación de las dos enfermedades y la necesidad de pesquisar el compromiso cardíaco en el dengue grave.

Utilization of machine learning for dengue case screening.

Dengue causes approximately 10.000 deaths and 100 million symptomatic infections annually worldwide, making it a significant public health concern. To address this, artificial intelligence tools like machine learning can play a crucial role in developing more effective strategies for control, diagnosis, and treatment. This study identifies relevant variables for the screening of dengue cases through machine learning models and evaluates the accuracy of the models. Data from reported dengue cases in the states of Rio de Janeiro and Minas Gerais for the years 2016 and 2019 were obtained through the National Notifiable Diseases Surveillance System (SINAN). The mutual information technique was used to assess which variables were most related to laboratory-confirmed dengue cases. Next, a random selection of 10,000 confirmed cases and 10,000 discarded cases was performed, and the dataset was divided into training (70%) and testing (30%). Machine learning models were then tested to classify the cases. It was found that the logistic regression model with 10 variables (gender, age, fever, myalgia, headache, vomiting, nausea, back pain, rash, retro-orbital pain) and the Decision Tree and Multilayer Perceptron (MLP) models achieved the best results in decision metrics, with an accuracy of 98%. Therefore, a tree-based model would be suitable for building an application and implementing it on smartphones. This resource would be available to healthcare professionals such as doctors and nurses.

Is there an overestimation of dengue compared with that of other acute febrile syndromes in childhood?

A group of children with clinical suspicion of dengue were assessed to determine if there was an overestimation of dengue compared with that of leptospirosis and leishmaniasis. This descriptive and analytical cross-sectional study, based on the active search of participants with acute febrile illness, was conducted at two pediatric hospitals. The collection of clinical and epidemiological data was performed using questionnaires, and laboratory tests specific for dengue were performed using immunochromatographic, serological, and molecular methods. Dengue-negative samples were assessed for Leptospira and Leishmania spp. using molecular tests. Data were assessed using analysis of variance (ANOVA), the chi-square test, and Fisher's exact test. In total, 86 participants were evaluated, of whom 39 (45%) were positive for dengue fever, 4 (5%) for leptospirosis, and 1 (1%) for leishmaniasis. Forty-two participants (49%) presented dengue-like symptoms. The predominant age range for the virus was 3-10 years. Most clinical manifestations were nonspecific, with frequent concomitant gastrointestinal and respiratory symptoms. Furthermore, we found that the acute febrile syndrome in childhood persists as a challenge for health professionals, especially in the early days of the disease, due to a plurality of diagnostic hypotheses, associated with the difficulty of establishing well-defined symptoms in children, especially in infants. Dengue fever continues to be a frequent pathology with acute febrile infections in childhood; however, there is an overestimation of the disease, especially in endemic regions, when one considers only the clinical epidemiological diagnosis.

Advances in Nucleic Acid Assays for Infectious Disease: The Role of Microfluidic Technology.

Within the fields of infectious disease diagnostics, microfluidic-based integrated technology systems have become a vital technology in enhancing the rapidity, accuracy, and portability of pathogen detection. These systems synergize microfluidic techniques with advanced molecular biology methods, including reverse transcription polymerase chain reaction (RT-PCR), loop-mediated isothermal amplification (LAMP), and clustered regularly interspaced short palindromic repeats (CRISPR), have been successfully used to identify a diverse array of pathogens, including COVID-19, Ebola, Zika, and dengue fever. This review outlines the advances in pathogen detection, attributing them to the integration of microfluidic technology with traditional molecular biology methods and smartphone- and paper-based diagnostic assays. The cutting-edge diagnostic technologies are of critical importance for disease prevention and epidemic surveillance. Looking ahead, research is expected to focus on increasing detection sensitivity, streamlining testing processes, reducing costs, and enhancing the capability for remote data sharing. These improvements aim to achieve broader coverage and quicker response mechanisms, thereby constructing a more robust defense for global public health security.

Knowledge, attitudes, and practices of health care professionals regarding dengue fever: need for training and provision of diagnostic equipment in Togo in 2022, a cross-sectional study.

Background: Health statistics on dengue are virtually non-existent, despite the fact that the virus is circulating in Togo. This study aimed to assess the knowledge, attitudes, and practices (KAP) of health professionals in the Kara health region. Methods: A cross-sectional study was conducted from March to June 2022 among healthcare professionals who had worked in the Kara region of northern Togo were selected using an exhaustive recruitment method. Data were collected by trained resident doctors with a face-to-face interview using a standardized, pretested questionnaire based on the WHO 2009 dengue guide. Three multivariate regression models were utilized to investigate factors associated with knowledge, attitudes and, and practices. Results: A total of 464 respondents (37.1% female), median age 35 years, interquartile range (29-43 years) were included. Only (3.0%) of the participants had received training on dengue fever diagnosis, treatment and prevention in the last 3 years, and 10.3% had dengue rapid diagnostic tests available at their hospital. Half of the respondents (49.1%) had good knowledge of dengue fever, compared with 30.0% who had positive attitudes. Of a total of 256 professionals who had encountered a case of dengue fever in their practice, only 24 (9.4%) had appropriate practices for diagnosing and treating dengue fever. In multivariate analysis, the healthcare professionals who had taken part in ongoing training on dengue fever were more likely to have adequate dengue diagnosis and treatment practice aOR = 8.1; CI 95% = [1.7-36.0]. Conclusion: Strengthening healthcare professionals' dengue-related skills through ongoing training and the provision of dengue diagnostic tests could help improve early detection practices and management of dengue fever in Togo.

Liver involvement in dengue: A systematic review.

Liver involvement is an unusual yet frequently overlooked dengue complication. Pivotal for an efficient clinical management, the early diagnosis of dengue-associated liver involvement relies on an accurate description of its clinical and biological characteristics, its prognosis factors, its association with severe dengue and its clinical management. We conducted a systematic review by searching PubMed and Web of Science databases for original case reports, cohort and cross-sectional studies reporting the clinical and/or biological features of dengue-associated liver involvement. The study was registered in PROSPERO (CRD42021262657). Of the 2552 articles identified, 167 were included. Dengue-associated liver involvement was characterised by clinical features including abdominal pain, hepatomegaly, jaundice, nausea/vomiting, and an echogenic liver exhibiting hepatocellular necrosis and minimal inflammation. Elevated Aspartate Aminotransferase and Alanine Aminotransferase but also elevated bilirubin, Alkaline Phosphatase, gamma-glutamyl transferase, increased International Normalised Ratio, creatinine and creatine kinase, lower albumin and prolonged prothrombin and activated partial thromboplastin time were prevalent in dengue-associated liver involvement. Cardiovascular and haematological systems were frequently affected, translating in a strong association with severe dengue. Liver involvement was more common in males and older adults. It was associated with dengue virus serotype-2 and secondary infections. Early paracetamol intake increased the risk of liver involvement, which clinical management was mostly conservative. In conclusion, this systematic review demonstrates that early monitoring of transaminases, clinical assessment, and ultrasound examination allow an efficient diagnosis of dengue-associated liver involvement, enabling the early identification and management of severe dengue.

Postdengue Acute Disseminated Encephalomyelitis.

A 38-year-old gentleman, following an uncomplicated dengue fever 2 weeks back, developed acute onset bilateral lower limb weakness and numbness for 5 days, associated with bladder and bowel incontinence and a band-like sensation in T4 dermatome. On examination, he had paraparesis with normal cranial nerves except for left upper motor neuron-type 7th cranial nerve palsy and normal higher mental function. Magnetic resonance imaging (MRI) of the brain and spine detected multiple demyelinating lesions. A diagnosis of postdengue acute disseminated encephalomyelitis (ADEM) was made as part of postinfective inflammatory process after the fever had subsided. Cerebrospinal fluid study ruled out active infection. He was treated with intravenous steroids and is currently recovering. An interesting point in our case was that the patient had significant imaging findings in MRI of the brain with no symptoms or signs suggestive of intracranial involvement-ADEM without evidence of encephalitis.

DengueSeq: a pan-serotype whole genome amplicon sequencing protocol for dengue virus.

BACKGROUND: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes. RESULTS: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/µL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments. CONCLUSIONS: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance.

Advancing arbovirus diagnosis in Brazil: strengthening diagnostic strategies and public health data collection.

BACKGROUND: The last five decades have seen a surge in viral outbreaks, particularly in tropical and subtropical regions like Brazil, where endemic arboviruses such as Dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) pose significant threats. However, current diagnostic strategies exhibit limitations, leading to gaps in infection screening, arbovirus differential diagnoses, DENV serotyping, and life-long infection tracking. This deficiency impedes critical information availability regarding an individual's current infection and past infection history, disease risk assessment, vaccination needs, and policy formulation. Additionally, the availability of point-of-care diagnostics and knowledge regarding immune profiles at the time of infection are crucial considerations. OBJECTIVES: This review underscores the urgent need to strengthen diagnostic methods for arboviruses in Brazil and emphasizes the importance of data collection to inform public health policies for improved diagnostics, surveillance, and policy formulation. METHODS: We evaluated the diagnostic landscape for arboviral infections in Brazil, focusing on tailored, validated methods. We assessed diagnostic methods available for sensitivity and specificity metrics in the context of Brazil. RESULTS: Our review identifies high-sensitivity, high-specificity diagnostic methods for arboviruses and co-infections. Grifols transcription-mediated amplification assays are recommended for DENV, CHIKV, and ZIKV screening, while IgG/IgM ELISA assays outperform Rapid Diagnostic Tests (RDTs). The Triplex real-time RT-PCR assay is recommended for molecular screening due to its sensitivity and specificity. CONCLUSION: Enhanced diagnostic methods, on-going screening, and tracking are urgently needed in Brazil to capture the complex landscape of arboviral infections in the country. Recommendations include nationwide arbovirus differential diagnosis for DENV, ZIKV, and CHIKV, along with increased DENV serotyping, and lifelong infection tracking to combat enduring viral threats and reduce severe presentations.

Development and assessment of a multiepitope synthetic antigen for the diagnosis of Dengue virus infection.

Immunodiagnostic tests for detecting dengue virus infections encounter challenges related to cross-reactivity with other related flaviviruses. Our research focuses on the development of a synthetic multiepitope antigen tailored for dengue immunodiagnostics. Selected dengue epitopes involved structural linearity and dissimilarity from the proteomes of Zika and Yellow fever viruses which served for computationally modeling the three-dimensional protein structure, resulting in the design of two proteins: rDME-C and rDME-BR. Both proteins consist of seven epitopes, separated by the GPGPG linker, and a carboxy-terminal 6 × -histidine tag. The molecular weights of the final proteins rDME-C and rDME-BR are 16.83 kDa and 16.80 kDa, respectively, both with an isoelectric point of 6.35. The distinguishing factor between the two proteins lies in the origin of their epitope sequences, where rDME-C is based on the reference dengue proteome, while rDME-BR utilizes sequences from prevalent Dengue genotypes in Brazil from 2008 to 2019. PyMol analysis revealed exposure of epitopes in the secondary structure. Successful expression of the antigens was achieved in soluble form and fluorescence experiments indicated a disordered structure. In subsequent testing, rDME-BR and rDME-C antigens were assessed using an indirect Elisa protocol against Dengue infected serum, previously examined with a commercial diagnostic test. Optimal concentrations for antigens were determined at 10 µg/mL for rDME-BR and 30 µg/mL for rDME-C, with serum dilutions ranging from 1:50 to 1:100. Both antigens effectively detected IgM and IgG antibodies in Dengue fever patients, with rDME-BR exhibiting higher sensitivity. Our in-house test showed a sensitivity of 77.3 % and 82.6 % and a specificity of 89.4 % and 71.4 % for rDME-C and rDEM-BR antigens. No cross-reactivity was observed with serum from Zika-infected mice but with COVID-19 serum samples. Our findings underscore the utility of synthetic biology in crafting Dengue-specific multiepitope proteins and hold promise for precise clinical diagnosis and monitoring responses to emerging Dengue vaccines.

Dengue: A review of laboratory diagnostics in the vaccine age.

Background. Dengue is an important arboviral infection of considerable public health significance. It occurs in a wide global belt within a variety of tropical regions. The timely laboratory diagnosis of Dengue infection is critical to inform both clinical management and an appropriate public health response. Vaccination against Dengue virus is being introduced in some areas.Discussion. Appropriate diagnostic strategies will vary between laboratories depending on the available resources and skills. Diagnostic methods available include viral culture, the serological detection of Dengue-specific antibodies in using enzyme immunoassays (EIAs), microsphere immunoassays, haemagglutination inhibition or in lateral flow point of care tests. The results of antibody tests may be influenced by prior vaccination and exposure to other flaviviruses. The detection of non-structural protein 1 in serum (NS1) has improved the early diagnosis of Dengue and is available in point-of-care assays in addition to EIAs. Direct detection of viral RNA from blood by PCR is more sensitive than NS1 antigen detection but requires molecular skills and resources. An increasing variety of isothermal nucleic acid detection methods are in development. Timing of specimen collection and choice of test is critical to optimize diagnostic accuracy. Metagenomics and the direct detection by sequencing of viral RNA from blood offers the ability to rapidly type isolates for epidemiologic purposes.Conclusion. The impact of vaccination on immune response must be recognized as it will impact test interpretation and diagnostic algorithms.

Towards a machine-learning assisted non-invasive classification of dengue severity using wearable PPG data: a prospective clinical study.

BACKGROUND: Dengue epidemics impose considerable strain on healthcare resources. Real-time continuous and non-invasive monitoring of patients admitted to the hospital could lead to improved care and outcomes. We evaluated the performance of a commercially available wearable (SmartCare) utilising photoplethysmography (PPG) to stratify clinical risk for a cohort of hospitalised patients with dengue in Vietnam. METHODS: We performed a prospective observational study for adult and paediatric patients with a clinical diagnosis of dengue at the Hospital for Tropical Disease, Ho Chi Minh City, Vietnam. Patients underwent PPG monitoring early during admission alongside standard clinical care. PPG waveforms were analysed using machine learning models. Adult patients were classified between 3 severity classes: i) uncomplicated (ward-based), ii) moderate-severe (emergency department-based), and iii) severe (ICU-based). Data from paediatric patients were split into 2 classes: i) severe (during ICU stay) and ii) follow-up (14-21 days after the illness onset). Model performances were evaluated using standard classification metrics and 5-fold stratified cross-validation. FINDINGS: We included PPG and clinical data from 132 adults and 15 paediatric patients with a median age of 28 (IQR, 21-35) and 12 (IQR, 9-13) years respectively. 1781 h of PPG data were available for analysis. The best performing convolutional neural network models (CNN) achieved a precision of 0.785 and recall of 0.771 in classifying adult patients according to severity class and a precision of 0.891 and recall of 0.891 in classifying between disease and post-disease state in paediatric patients. INTERPRETATION: We demonstrate that the use of a low-cost wearable provided clinically actionable data to differentiate between patients with dengue of varying severity. Continuous monitoring and connectivity to early warning systems could significantly benefit clinical care in dengue, particularly within an endemic setting. Work is currently underway to implement these models for dynamic risk predictions and assist in individualised patient care. FUNDING: EPSRC Centre for Doctoral Training in High-Performance Embedded and Distributed Systems (HiPEDS) (Grant: EP/L016796/1) and the Wellcome Trust (Grants: 215010/Z/18/Z and 215688/Z/19/Z).

A serotype-specific and tiled amplicon multiplex PCR method for whole genome sequencing of dengue virus.

Dengue fever, a mosquito-borne viral disease of significant public health concern in tropical and subtropical regions, is caused by any of the four serotypes of the dengue virus (DENV1-4). Cutting-edge technologies like next-generation sequencing (NGS) are revolutionizing virology, enabling in-depth exploration of DENV's genetic diversity. Here, we present an optimized workflow for full-genome sequencing of DENV 1-4 utilizing tiled amplicon multiplex PCR and Illumina sequencing. Our assay, sequenced on the Illumina MiSeq platform, demonstrates its ability to recover the full-length dengue genome across various viral abundances in clinical specimens with high-quality base coverage. This high quality underscores its suitability for precise examination of intra-host diversity, enriching our understanding of viral evolution and holding potential for improved diagnostic and intervention strategies in regions facing dengue outbreaks.