Final Report Summary - DENGUETOOLS (Innovative tools and strategies for surveillance and control of dengue)
Executive Summary:
Research area 1: Develop a comprehensive early warning surveillance system that has predictive capability for epidemic dengue using novel tools for laboratory diagnosis and vector monitoring
We successfully set up a laboratory enhanced sentinel surveillance system in Colombo District, Sri Lanka, recruited 5436 febrile patients, documented the high dengue disease burden, and achieved effective technology transfer for PCR and serotyping. We integrated climate variables, socioeconomic factors, and mapping of breeding sites to assess the predictive value. Rainfall and increasing mean temperatures were highly predictive for dengue activity, but the results showed considerable heterogeneity. We found that schools were associated with more breeding sites, and these results were replicated by cluster analyses in Thailand that confirmed clustering of dengue infections around schools and within classrooms. Thus, schools should be targeted for intensified vector control strategies. We documented the economic burden of dengue in Sri Lanka: the annual total cost of dengue control and reported hospitalizations to be USD 3.45 million (US$1.50 per capita) in Colombo district.
We developed primers and optimized RT-Recombinase Polymerase Amplification technology (RPA); our results suggest that RPA is superior over Loop-mediated Amplification Isothermal Amplification. RPA’s diagnostic sensitivity is comparable to qRT-PCR assays, however, it has several advantages over PCR, namely user-friendliness, speed of test performance, affordability, and temperature independence.
Research area 2: Develop novel strategies to prevent dengue infections in children
Effective scalable control strategies to protect children are lacking. Given the day-biting nature of Aedes mosquitoes, we hypothesize that insecticide treated school uniforms may be a target for school based intervention to reduce the incidence of dengue in school children.
We conducted a cluster-randomized controlled trial with 1,811 pupils over a school-term in 10 schools in Thailand. Laboratory confirmed dengue incidence was 3.7 versus 3.3% for intervention versus control groups (no significant difference). WHOPES cone tests showed a 100% knock down and mortality of Aedes aegypti mosquitoes exposed to impregnated clothing at baseline, but this efficacy rapidly declined to below 20% after 20 washes. Entomological assessments showed that the mean number of Aedes aegypti mosquitoes caught inside the classrooms of the intervention schools was significantly reduced in the month following the introduction of the impregnated uniforms (p =0.04). We conclude that if the rapid washing-out of permethrin can be overcome by novel technological approaches, insecticide-treated clothes would deserve to be re-evaluated as a potentially cost-effective and scalable intervention.
Research area 3: Geographic spread and risk of introduction into new areas including Europe
We found that urbanization with increased population densities is the main driver for dengue in endemic countries. Our findings have significant implications for predicting future trends; policy-makers and the scientific community should heed more attention to the negative impact of urbanization on diseases such as dengue. We documented a convergence of various factors that put Europe increasingly at risk of dengue introduction and establishment. By calculating temperature-dependent vectorial capacity for Europe, we highlighted seasonal dengue epidemic potential in ten European cities and predicted. Our field trials showed that thermal fogging may be the control method of choice in Europe despite being highly labor-intensive. De-novo mathematical models were developed to quantify the risk of dengue introduction (and Zika for that matter) into Europe.
Project Context and Objectives:
Dengue is a major international public health concern and one of the most important arthropod-borne diseases. Approximately 2.5 billion people – over 40% of world’s population in over 100 countries - are at risk of dengue virus (DENV) infection. Every year, an estimated 390 million people in more than 100 countries are infected with dengue. Infection with any of its four serotypes of dengue virus may result in a mild nonspecific febrile illness, classic dengue fever, or severe dengue manifested by plasma leakage, hemorrhagic tendencies, shock and possibly death. Dengue often requires hospitalization, thereby challenging already fragile health care systems. The often unpredictable nature of dengue epidemics further compounds the public health impact.
Dengue has increased more than 30 fold in the past 40 years and is being introduced to non-endemic areas such as the United States, Southern Europe and Australia with increasing frequency. Current surveillance systems and control efforts are clearly insufficient to combat dengue in endemic countries and to prevent spread to previously uninfected areas (including Europe). We lack understanding of individual or combined roles of viral, entomological, ecological, and environmental and climate factors that influence dengue transmission dynamics and their respective outbreak predictive capability and the most cost-effective approach for surveillance and early warning systems. For surveillance to effectively provide early warning for epidemic transmission, it must be active, laboratory-based, and comprehensive in its coverage of the spectrum of clinical illness and the factors that influence transmission dynamics. Early diagnostic assays at point-of-care that are affordable and can be used in the field are also missing. Vector surveillance in particular has many shortcomings, in particular the lack of sensitive, reliable and simple field methods for vector surveillance.
Furthermore, children are the most vulnerable group for dengue. Effective control strategies to protect children are lacking, in particular simple, cost-effective and scalable strategies. Given that Aedes mosquitoes mainly bite during the day, at a time when children spend most of their time at schools, we set out to (1) determine whether schools are target sites for infections (WP 1 and WP 4 and 10) and (2) to test our hypothesis that insecticide treated school uniforms may be a target for school-based interventions to reduce the incidence of dengue in school children (WP 4).
Lastly, gaps in understanding the risk of introduction of dengue to non-infected areas, including Europe, hampers effective preventive strategies. We currently have insufficient data on the magnitude and trends of importation and virus evolution over time and by geographic origin. We also only have a poor understanding of the vectorial capacity of Aedes in temperate climates that are needed for predictive models under changing climate conditions. It is important to develop a theoretical framework for dengue emergence in naive areas with a particular focus on Europe.
To address these knowledge gaps, we developed three research areas around surveillance (including diagnostics and predictive models) in endemic countries (research area 1), novel strategies for the prevention of dengue in children (research area 2) and dedicated the third research area to the risk of dengue in Europe (importation via returning travellers, control of Aedes albopictus in Europe, and predictive climate dependent models for the risk of introduction and establishment of dengue in Europe).
The objectives were the following, and these were also published in our “DengueTools Design paper”:
Objectives:
Research area 1: Develop a comprehensive, early warning, surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring
Research area 2: Develop novel strategies to prevent dengue in children
Research area 3: Describe and predict the risk of global spread of dengue in particular the risk of introduction and establishment in Europe in the context of parameters of vectorial capacity, global mobility and climate change.
Specific scientific and technical objectives
The Consortium collaborated on the following specific objectives:
Research area 1: Develop a comprehensive, early warning, surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring
1. Develop and validate novel diagnostic assays for point-of-care use
2. Field-test novel diagnostic assays
3. Develop improved field devices and attractants for vector monitoring
4. Develop novel assays for virus detection and characterisation in Aedes mosquitoes
5. Study entomological and environmental indicators that may influence dengue transmission dynamics
6. Develop geo-spatial modelling and risk maps
7. Evaluate the outbreak predictive capability of climate variables (weather variability) and their operational utility for surveillance
8. Develop a comprehensive, early warning, laboratory-based sentinel disease surveillance system
9. Study viral genomic sequence data and the potential role in causing outbreaks of more severe disease
10. Determine the usefulness of the revised 2009 WHO/TDR case definition for surveillance purposes
11. Describe the costs of novel strategies for dengue surveillance and prevention to their effect on the burden of the disease.
Research area 2: Develop novel strategies to prevent dengue in children
1. Conduct a literature review on the protective efficacy of impregnated clothing against vector-borne diseases
2. Determine the efficacy of impregnated school uniforms on the reduction of dengue incidence in school aged children
3. Determine the protection efficacy of school uniforms impregnated with repellent under different laboratory scenarios
4. Develop a cost-effectiveness framework and model for the school-based preventive intervention of insecticide treated school uniforms
5. Produce policy briefs and engage with policy makers to ensure the scalability of such school intervention programs
Research area 3: Describe and predict the risk of global spread of dengue in particular the risk of introduction and establishment in Europe in the context of parameters of vectorial capacity, global mobility and climate change.
1. Describe the trends of dengue importation into Europe
2. Conduct dengue sequencing and produce phylogenetic trees on imported dengue viruses to Europe
3. Compare clinical manifestations and the usefulness of the 2009 WHO dengue case of dengue in travellers (non-endemic populations) versus endemic populations (Sri Lanka)
4. Explore reasons for the resurgence and geographic spread of dengue
5. Estimate and model dengue virus introductions via travellers
6. Estimate the risk of dengue in travellers to the World Cup in 2014 and to the Summer Olympics in Rio de Janeiro in 2016
7. Model vectorial capacity for Aedes mosquitoes in temperate climate zones and globally under current and future climate scenarios
8. Describe typical breeding sites for Ae. albopictus in Southern France
9. Evaluate the impact of ultra-low volume (ULV) aerosols on Ae. albopictus in Sothern France
10. Determine the susceptibility of Ae. albopictus populations to DENV under different temperature regimes
Project Results:
In the following, we will describe the main findings and foreground by research area, whereby the contributions of all work packages will be addressed within the appropriate research areas, as outlined in our design papers.1,2 The findings are ordered by overarching objectives, and not by work packages. Only publications are cited that are a direct result of our DengueTools collaborative work.
Research area 1: Develop a comprehensive, early warning, surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring
Obj 1: Develop and validate novel diagnostic assays for point-of-care use
The main objective was to develop a rapid (15 minute) nucleic acid test for dengue virus that can be undertaken in a low resource setting using Recombinase Polymerase Amplification (RPA) in comparison with LAMP methods.
A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes.3 The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses. The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods. We then compared this with a new technique:
A prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed.4 The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. The only down-side is that it still needs the Twistdx equipment, which means also it is simple, affordable, user-friendly, independent of outside temperature, for field-use it is only of use if one has purchased the Twistdx diagnostic equipment.
In another study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n =375). 5The NS1 Ag Strip and qRT-PCR showed a fair concordance. While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2–94.8%) than in those from primary dengue (21.7–64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.
Obj 2: Field-test novel diagnostic assays
The diagnostic utility of the RT-RPA assay was assessed surveillance laboratory of the WHO Collaborating Centre for Arbovirus Reference & Research (Dengue/Severe Dengue) at the University of Malaya (UM). The easiness of performing the RT-RPA assay was evaluated among new users using the user usability test. 84.2% (16/19) of the participants performed the assay with only 1 to 5 deviations from the provided written protocol. 88.2% of the test results obtained by the participants were similar to the reference results. The user satisfaction towards the RT-RPA protocol and interpretation of results were 100% and 90%, respectively. The improved RT-RPA is now routinely used for detection of dengue at the WHO Collaborating Center for Arbovirus Research and Reference (Dengue/Severe dengue) at UM. In an analysis of
429 dengue virus IgM negative serum samples, 196 samples tested positive for dengue virus NS1 antigen using the rapid strip test (RDT). By using the RT-RPA assays, 238 samples were tested positive. All the in-house RT-PCR-positive serum samples were also positive by the RT-RPA assay. By using the RT-RPA assay, detection of positive samples among the dengue IgM negative samples increased from 39 (8.7%) to 238 (53%).
Obj 3: Develop improved field devices and attractants for vector monitoring
Novel traps that were developed for Panama were used again in the field in Sri Lanka with less success than in Panama. However, we caught sufficient numbers to feed obj 4
Obj 4: Develop novel assays for virus detection and characterisation in Aedes mosquitoes
We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.6
Obj 5: Study entomological and environmental indicators that may influence dengue transmission dynamics
We did an extensive survey in a sub-district in Sri Lanka to to assess Aedes mosquito breeding sites and the prevention practices of community members in a heavily urbanized part of Colombo. Hence, we did a cross-sectional entomological survey from April to June 2013 in 1469 premises located in a subdistrict of the City of Colombo.8 Types of breeding sites and their infestation with larvae or pupae were recorded. Furthermore, a questionnaire was administered to the occupants of these premises to record current practices of dengue vector control. The surveyed premises consisted of 1341 residential premises and 110 non-residential premises (11 schools, 99 work or public sites), 5 open lands, and 13 non-specified. In these 1469 premises, 15447 potential breeding sites suitable to host larvae of pupae were found; of these sites18.0% contained water. Among the 2775 potential breeding sites that contained water, 452 (16.3%) were positive for larvae and/or pupae. Schools were associated with the proportionally highest number of breeding sites; 85 out of 133 (63.9%) breeding sites were positive for larvae and/or pupae in schools compared with 338 out of 2288 (14.8%) in residential premises. The odds ratio (OR) for schools and work or public sites for being infested with larvae and/or pupae was 2.77 (95% CI 1.58 4.86) when compared to residential premises. Occupants of 80.8% of the residential premises, 54.5% of the schools and 67.7% of the work or public sites reported using preventive measures. The main prevention practices were coverage of containers and elimination of mosquito breeding places. Occupants of residential premises were much more likely to practice preventive measures than were those of non-residential premises (OR 2.23; 1.49 3.36). In conclusion, schools and working sites were associated with the highest numbers of breeding sites and lacked preventive measures for vector control. In addition to pursuing vector control measures at residential level, public health strategies should be expanded in schools and work places.
As we identified schools to be the sites with the highest number of Aedes breeding sites in Sri Lanka, we also investigated whether schools or even classrooms would be at higher risk for dengue incidence. To answer this question, we did a study in schools in Thailand. This manuscript is now under minor revision before final publication: Ratanawong P, Kittayapong P, Olanratmanee, P, Wilder-Smith A, Byass P, Tozan Y, Dambach P, Montenegro Quiñonez CA, Louis VR. Are schools important sites for dengue transmission in semi-rural and semi-urban Thailand? 2016 PLOS One. A cohort of 1,811 students from 10 schools in semi-rural and semi-urban Thailand participated in this study (WP 4). Seroconversion data and location of participants’ residences and schools were recorded to determine spatial patterns of dengue cases. Blood samples were taken to confirm dengue infections in participants. Entomological surveys recorded adult mosquito density and breeding sites of Aedes vectors in schools. Clustering analyses were performed to detect spatial aggregation of dengue cases among participants.
Results: A total of 57 dengue seroconversions were detected among the 1,655 participants who provided paired blood samples. Of the 57 confirmed dengue infections, 23 (40.0%) occurred in students from 6 (6.8%) of the 88 classrooms in 10 schools. Dengue cases did not show significant clustering by residential location in the study area. A total of 66 Aedes aegypti mosquitoes were identified from 278 mosquitoes caught using a portable vacuum aspirator in 88 classrooms. In a follow-up survey of breeding sites one year after the study, 484 out of 2,399 water containers surveyed (20.2%) were identified as active mosquito breeding sites.
Our findings suggest that dengue infections were clustered among schools and among classrooms within schools. The schools studied were found to contain a large number of different types of breeding sites. Aedes vector densities in schools were correlated with dengue infections and breeding sites in those schools. Given that only a small proportion of breeding sites in the schools were subjected to vector control measures (11%), this study emphasizes the urgent need to implement vector control strategies at schools, while maintaining efforts at the household level.
Obj 6 Develop geo-spatial modelling and risk maps
A systematic search was undertaken, using the PubMed, Web of Science, WHOLIS, Centers for Disease Control and Prevention (CDC) and OvidSP databases for published citations.7 A manual search of the titles and abstracts was carried out using predefined criteria, notably the inclusion of dengue cases. Data were extracted for pre-identified variables, including the type of predictors and the type of modeling approach used for risk mapping.
Our review revelealed that a wide variety of both predictors and modeling approaches was used to create dengue risk maps. No specific patterns could be identified in the combination of predictors or models across studies. The most important and commonly used predictors for the category of demographic and socio-economic variables were age, gender, education, housing conditions and level of income. Among environmental variables, precipitation and air temperature were often significant predictors. Remote sensing provided a source of varied land cover data that could act as a proxy for other predictor categories. Descriptive maps showing dengue case hotspots were useful for identifying high-risk areas. Predictive maps based on more complex methodology facilitated advanced data analysis and visualization, but their applicability in public health contexts remains to be established.
Availability of resources, feasibility of acquisition, quality of data, alongside available technical expertise, determines the accuracy of dengue risk maps and their applicability to the field of public health. A large number of unknowns, including effective entomological predictors, genetic diversity of circulating viruses, population serological profile, and human mobility, continue to pose challenges and to limit the ability to produce accurate and effective risk maps, and fail to support the development of early warning systems.7
Obj 7 Evaluate the outbreak predictive capability of climate variables (weather variability) and their operational utility for surveillance
Understanding the drivers of dengue is vital in controlling and preventing the disease spread. This study focused on quantifying the influence of one of the drivers of dengue by estimating the relationship between dengue incidence and weather variability, and describe its consistency over 10 Medical Officer of Health (MOH) divisions of the Kalutara district in Sri Lanka. Weekly weather and dengue data, measured at 10 MOH divisions in Kalutara from 2009 to 2013, were retrieved and analysed. Distributed lag non-linear model and hierarchical-analysis was used to estimate division specific and overall relationships between weather and dengue. We incorporated lags up to 12 weeks and evaluated models based on the Akaike Information Criterion.
More consistent exposure-response patterns between different geographical locations were observed for rainfall, showing increasing relative risk of dengue with increasing rainfall from 50 mm per week. The strongest association with dengue risk centred around 6 to 10 weeks following rainfalls of more than 300 mm per week. After heavy rainfall, we observed a reduction in dengue incidence in the first weeks followed by a strong net increase after 8 weeks. The overall relative risk of dengue associated with temperature increased steadily from lower temperatures towards the mean value of 29.80C starting from a lag of 4 weeks, and increased for temperatures above the mean value with a cumulative growing association with lags up to 12 weeks. We found, however, some considerable heterogeneity in the associations between the different MOH divisions studied (manuscript submitted, under review).
Obj 8 Develop a comprehensive laboratory-based sentinel disease surveillance system
In Sri Lanka, dengue poses a significant socio-economic and disease burden. The geographic spread, incidence and severity of disease has been increasing since the first dengue hemorrhagic fever epidemic occurred in 1989. Periodic epidemics have become progressively larger, peaking with the 2009-2014 epidemic with 28,000 to more than 40,000 cases reported each year (44,456 cases in 2012). During that same period, the disease dramatically expanded to the whole island.
During the EU funded 4 year period a total of 5436 febrile patients were recruited and blood samples were tested; we found that 3122 (57%) were found positive for dengue.
Summary of samples collected with positives for dengue: 2012 – 2015
Year Number of acute samples collected No. positive for dengue (%)
2012 1338 761 (57)
2013 1427 927 (65)
2014 1994 1144 (57)
2015 677 290 (43)
Total 5436 3122 (57)
Historically surveillance was passive, with mandatory dengue notifications based on clinical diagnosis with only limited laboratory confirmation. To obtain more accurate data on the disease burden of dengue, we set up a laboratory-based enhanced sentinel surveillance system in Colombo District. Here we describe the study design and report our findings of enhanced surveillance in the years 2012-2014.9 Three outpatient clinics and three government hospitals in Colombo District that covered most of the Colombo metropolitan area were selected for the sentinel surveillance system. Up to 60 patients per week presenting with an undifferentiated fever were enrolled. Acute blood samples from each patient were tested by dengue specific PCR, NS1 ELISA and IgM ELISA. A sub-set of samples was sent to Duke-NUS Singapore for quality assurance, virus isolation and serotyping. Trained medical research assistants used a standardized case report form to record clinical and epidemiological data. Clinical diagnoses by the clinicians-in-charge were recorded for hospitalized cases. Of 3,127 febrile cases, 43.6% were PCR and/or NS1 positive for dengue. A high proportion of lab confirmed dengue was observed from inpatients (IPD) (53.9%) compared to outpatient (clinics in hospitals and general practice) (7.6%). Dengue hemorrhagic fever (DHF) was diagnosed in 11% of patients at the time of first contact, and the median day of illness at time of presentation to the sentinel sites was 4. Dengue serotype 1 was responsible for 85% of the cases and serotype 4 for 15%. The sensitivity and specificity of the clinicians' presumptive diagnosis of dengue was 84% and 34%, respectively. CONCLUSION: DENV-1, and to a lesser degree DENV-4, infection were responsible for a high proportion of febrile illnesses in Colombo in the years 2012 to 2014. Clinicians' diagnoses were associated with high sensitivity, but laboratory confirmation is required to enhance specificity.
We documented a high proportion of DHF. In Sri Lanka, the 1997 WHO dengue case classification is used routinely by clinicians, hence we relied on the managing clinicians’ diagnosis using the classification they are familiar with rather than the 2009 TDR dengue case classification. Dengue hemorrhagic fever (DHF) was diagnosed in 11% of patients at the time of first contact. For hospitalized cases, 22% were discharged with the diagnosis of DHF (with 5 fatal outcomes due to DHF). A DHF proportion of 22% is unusually high, as most studies report a proportion of 5-10% underscoring that the current epidemic is associated with more severe disease. Patients who had signs and symptoms of DHF already at first encounter to our sentinel system presented much later compared to dengue fever or OFI, which could be one explanation why such cases have a worse outcome. 0.9% of the DHF cases died.
To assess whether costly diagnostic assays are indeed worthwhile in a sentinel surveillance system, we compared the clinicians’ presumptive clinical diagnoses with the laboratory confirmation. The clinicians’ diagnosis for dengue at time of admission had a sensitivity of 84.7% and specificity of 32.5%. The positive predictive value for the clinicians was 82.3% and the negative predictive value 36.4% at time of admission. The sensitivity and specificity was higher at discharge, most likely because the clinicians had by then all laboratory tests at hand and were familiar with the clinical evolution over time, even without knowing the results of our assays. The trained research assistants had a higher sensitivity and specificity compared to the clinicians who saw the patients as part of their daily clinical routine on busy days. The higher sensitivity by the research assistants who were all medically trained can be explained by the fact that they spent more time reviewing all the symptoms and signs as documented by our standardized case report form. This underscores that training does indeed improve sensitivity. However, having said this, the sensitivity for the routine clinicians-in-charge was still high, as Sri Lankan doctors are very familiar with the diagnosis and management of dengue patients. Furthermore, we were able to show that the sensitivity and specificity depends on the day of illness, with the highest sensitivity and specificity later in the course of illness. In other words, if patients presented later (after day 3-4 of illness), sensitivity increased, while specificity remained low. Mild respiratory illnesses and non-specific viral fevers are usually of shorter duration and hence present a large proportion of febrile patients seen in the first 3 days of illness. Patients with DHF were much more often correctly diagnosed as a laboratory confirmed dengue case. This finding is consistent with previous reports from Thailand, where the authors concluded that WHO case definition of DHF demonstrates 62% sensitivity and 92% specificity in identifying dengue illness requiring intervention without the need for laboratory confirmation of dengue virus infection in endemic areas. DHF presents clinically with a very characteristic constellation of clinical symptoms, signs and changes in leukocytes, platelets, and hematocrit; a constellation that is so pathognomonic for DHF that WHO in its 2009 revised dengue case classification does not require laboratory confirmation for severe disease but does require laboratory confirmation for dengue with or without warning signs). Overall, our findings show that the clinicians’ suspicion of dengue was very high as seen in the high sensitivity, but specificity was very low. To enhance specificity it is important to add laboratory confirmation of dengue.
Furthermore, we were able to assess three diagnostic essays (PCR, Dengue IgM ELISA and NS1 ELISA) in a sentinel surveillance setting. For dengue, day of illness determines the choice of diagnostic assay. During the viremic phase (up to day 4-5 of illness), molecular biological or virological approaches such as PCR or NS1 should be employed; after the viremic phase (>5 days), serological assays are indicated, with dengue IgM being the most frequently used assay 10. Our figures confirm the temporal changes in positive assays per day of illness, with PCR and NS1 being positive in the earlier phase, and IgM later. A combination of methods that target different time periods maximizes diagnostic sensitivity. Given the constraints of a large sentinel surveillance as ours, it was programmatically not feasible to take a convalescent serum at 14-21 days after discharge which would have helped in confirming the diagnosis – hence we lack a definitive “gold standard” diagnosis in those patients where we only have a single IgM result. In the absence of convalescent sera, we defined “dengue IgM, NS1 or PCR positive” as laboratory confirmed dengue. As NS1 based assays are increasingly used in endemic countries, we also particularly looked at the issues of NS1 as measured by ELISA. NS1 was positive for more days of illness compared with PCR. Indeed, NS1 can be found in the peripheral blood circulation for up to 9 days from illness onset, but can persist for up to 18 days for some cases. Hence NS1 offers a larger window of opportunity for diagnosis of dengue compared with virus isolation and PCR. Furthermore, the proportion of NS1 positive subjects in the first 5 days of illness was higher than that of PCR. Higher sensitivity of NS1 compared with PCR has been documented in some studies but not in others. It is important to note that we tested NS1 by ELISA and not with the cheaper rapid diagnostic (RDT) kits that are now widely available. Hunsperger et al showed that sensitivity of NS1 by ELISA is higher (60-75%) compared with NS1 RDT (38-71%). Hunsperger’s analysis also showed that NS1 was more sensitive in primary versus secondary infections, an observation that we can confirm with our findings.
Lastly, with the EU funded laboratory-enhanced surveillance project a dedicated government laboratory was set up to perform routine molecular testing for dengue. As a result, the capacity to do PCR and serotyping is now well established. Quality assurance with a subset of samples sent to the Duke-NUS Laboratory “Emerging Infectious Diseases Program” showed a high agreement. At Duke-NUS, we also did virus isolation. Although virus isolation is highly specific, the sensitivity is reported to be only approximately 40%. Virus isolation has the advantage of providing a virus isolate that can be used for further genome sequencing, or virus neutralization and other in vitro studies, but it requires highly trained operators, depends on a short viremia period, thus providing only a narrow window of opportunity from illness onset; in quintessence, it is not a diagnostic approach suitable for developing countries. Cost effectiveness studies are needed to evaluate the need for assays such as PCR on a routine basis in Sri Lanka compared to the more affordable NS1 only; and our sentinel surveillance can potentially serve as a basis for such studies.9
In terms of predictive capability, we did not find any predictive parameters for dengue peaks in terms of serotype, proportion of lab-confirmed dengue out of all febrile cases, except for seasonality. During our project, there were sustained high numbers of dengue notifications throughout the country with some seasonal variation but not inter-annual variations which could explain why we did not identify discriminatory parameters. The causative serotypes DENV 1 and to a lesser extent DENV-4 remained the same throughout, so that we could not identify serotypes/or genotypes associated with higher virulence.
We hence also explored internet-based media coverage to assess the extent of awareness of dengue and perceived severity of an outbreak at a national level as a proxy parameter for early outbreak detection. We compared internet references to dengue in Sri Lana with references to other diseases (malaria and influenza) in Sri Lanka and also compared Internet references to dengue in Sri Lanka with notified cases of dengue in Sri Lanka. We examined Internet-based news media articles on dengue queried from HealthMap for Sri Lanka, for the period January 2007 to November 2015. For comparative purposes, we compared hits on dengue with hits on influenza and malaria. There were 565 hits on dengue between 2007 and 2015, with a rapid rise in 2009 and followed by a rising trend ever since. These hits were highly correlated with the national epidemiological trend of dengue. The volume of digital media coverage of dengue was much higher than of influenza and malaria.
Dengue in Sri Lanka is receiving increasing media attention. Our findings underpin previous claims that digital media reports reflect national epidemiological trends, both in annual trends and inter-annual seasonal variation, thus acting as proxy biosurveillance to provide early warning and situation awareness of emerging infectious diseases.11
Obj 9: Study viral genomic sequence data and the potential role in causing outbreaks of more severe disease
Sri Lanka has experienced periodic dengue outbreaks since the 1960s, but recent epidemics have become progressively larger and associated with more severe disease. The 2012-2013 dengue epidemic in Sri Lanka was driven predominantly by DENV-1 with approximately 20% of dengue cases caused by DENV-4. DENV-4 transmission was first documented in Sri Lanka when it was isolated from a traveler in 1978, but has been comparatively uncommon since dengue surveillance began in in the early 1980s. To better understand the molecular epidemiology of DENV-4 infections in Sri Lanka, we conducted whole genome sequencing on dengue patient samples from two different geographic locations. Our E-gene analysis shows that two major lineages of DENV-4 have been observed in Sri Lanka since surveillance began, with data indicating that the currently circulating strain is a descendent of the Sri Lanka virus from 1978 (U18437). (Manuscript submitted). Phylogenetic analysis indicates that all sequenced DENV-4 strains belong to genotype 1 and are most closely related to DENV-4 viruses previously found in Sri Lanka and those more recently found to be circulating in India and Pakistan. There have been fewer DENV-4 outbreaks globally compared to the other three DENV serotypes and the cause of the unusually high number of DENV-4 cases in the ongoing Sri Lankan outbreak is unknown. One possibility is that genetic changes in the virus have led to an increase in the epidemic potential of this virus. Our analysis also suggest that this virus spread to India and/or Pakistan before re-emerging in Sri Lanka in 2009. Recent studies have shown that sequences outside of the E-gene can have a large impact on pathogenicity and epidemic potential. As such, we would like to emphasize the importance of moving towards the use of full genome sequencing rather than just the E-gene so that these other important regions can be examined as well. Given the recent technologic improvements and rapidly falling costs of sequencing, we feel this is an achievable goal. Combined with good epidemiological data, this knowledge has the potential to transform the current paradigm of retrospective study into one of active prediction and targeted, preventative action.
Obj 10: Determine the usefulness of the revised 2009 WHO/TDR case definition for surveillance purposes
The four serotypes of the dengue virus cause a spectrum of clinical disease ranging from self-limited dengue fever, usually accompanied by headache, myalgia and arthralgias, to more severe dengue associated with plasma leakage, hemorrhagic manifestations, thrombocytopenia, to severe shock and occasionally death. Sri Lanka still uses the old WHO classification with dengue fever/dengue haeemorrhagic fever Grade 1-2, Dengue shock syndrome (=DHF Grade 3-4). In 2009, WHO TDR published a revised dengue case classification consisting of dengue, dengue with warning signs and severe dengue. We found that very few patients presented with severe dengue at the first day of encounter to our sentinel sites, and that most patients that were clinically diagnosed as ‘severe dengue’ corresponded to the clinical diagnosis of Dengue Haemorrhagic Fever Grade 3 and 4. Due to small numbers of severe dengue at time of surveillance, we did not do further analysis. However, we looked into dengue warning signs which is more important at the time of first encounter with the health system as warning signs will determine whether such patients will be admitted or not. For many years, dengue appeared to mainly affect children, but increasingly adults and even elderly are also affected. However, the original dengue case classification and most of the WHO clinical guidelines were developed based on the clinical experiences with dengue in children. Clinical manifestations of dengue may differ in children versus adults, and this may also have implications on clinical management. We hence set out to study similarities and differences in clinical manifestations and clinical management of laboratory confirmed dengue infections in children versus adults.
Warning signs: at the time of first encounter, the following warning signs were present in children aged 2-12: Abdominal pain 38.8%, vomiting 2.5%, abdominal tenderness 31.3%, hepatomegaly 19.53%, clinical fluid accumulation 12.02%, mucosal bleeding 2.0%, petechiae 2.13%. The mean platelet count on the day of encounter with the sentinel system was 114,014.4 and hematocrit 37.99; mean ALT was 125.8 mean AST was 226.51.
For adults, the following warning signs were present: Abdominal pain 19.2%, vomiting 0.73%, abdominal tenderness 18.96%, hepatomegaly 3.17%, clinical fluid accumulation 18.71%, mucosal bleeding 4.64%, and petechiae 1.7%.
These results show that warning signs are clearly different between children aged 2-12 and adults aged 18 and above. The IDAMS study conducted by the other EU funded consortium will show which warning sign or combination thereof has the best prognostic value.
Obj 11: Describe the costs of novel strategies for dengue surveillance and prevention to their effect on the burden of the disease
Documenting the disease burden and the cost thereof to the individual but also to the public is important in particular in view of upcoming dengue vaccines to determine the cost effectiveness of dengue vaccines.12 Dengue is currently listed as a "neglected tropical disease" (NTD). We did a review to consider the criteria for the definition of an NTD, and documented the current research gaps and research activities and the adequacy of funding for dengue research and development (R&D) (2003-2013).13 Major research gaps exist in the area of integrated surveillance and vector control. Hence, although dengue differs from many of the NTDs, it still meets important criteria commonly used for NTDs. The current need for increased R&D spending, shared by dengue and other NTDs, is perhaps the key reason why dengue should continue to be considered an NTD.
The first dengue vaccine was licensed during the time period of DengueTools, and the Scientific Director of DengueTools Annelies Wilder-Smith was involved in many of the policy issues with WHO, the Dengue Vaccine Initiative and the global community.14-16 Yesim Tozan (Partner Heidelberg) explored the most immediate economic considerations of introducing a new dengue vaccine and evaluated the published economic analyses of dengue vaccination. Findings indicate that the current economic evidence base is of limited utility to support country-level decisions on dengue vaccine introduction. There are a handful of published cost-effectiveness studies and no country-specific costing studies to project the full resource requirements of dengue vaccine introduction. Country-level analytical expertise in economic analyses, another gap identified, needs to be strengthened to facilitate evidence-based decision-making on dengue vaccine introduction in endemic countries.12
In Sri Lanka, he Ministry of Health is responsible for controlling dengue and other disease outbreaks and associated health care. The substantial amount of public health staff in dengue control activities year-round and the provision of free medical care to dengue patients at secondary care hospitals place a formidable financial burden on the public health sector. We estimated the public sector costs of dengue control activities and the direct costs of hospitalizations in Colombo, the most heavily urbanized district in Sri Lanka, during the epidemic year of 2012 from the Ministry of Health's perspective.17 The financial costs borne by public health agencies and hospitals were collected using cost extraction tools designed specifically for the study and analysed retrospectively using a combination of activity-based and gross costing approaches. RESULTS: The total cost of dengue control and reported hospitalizations was estimated at US$3.45 million (US$1.50 per capita) in Colombo district in 2012. Personnel costs accounted for the largest shares of the total costs of dengue control activities (79%) and hospitalizations (46%). The results indicated a per capita cost of US$0.42 for dengue control activities. The average costs per hospitalization ranged between US$216-609 for pediatric cases and between US$196-866 for adult cases according to disease severity and treatment setting.
Research area 2: Develop novel tools for the prevention of dengue in school-aged children
Aedes aegypti, the principal vector for dengue transmission, mainly bites during the day. Children spend most of the day at school. Most children in dengue endemic countries wear school uniforms. We hypothesized that permethrin-impregnated school uniforms may reduce dengue infections. Insecticide-treated clothing has been used for many years by the military and in recreational activities as personal protection against bites from a variety of arthropods including ticks, chigger mites, sandflies and mosquitoes. Permethrin is the most commonly used active ingredient, but others, including bifenthrin, deltamethrin, cyfluthrin, DEET (N,N-diethyl-3-methylbenz-amide) and KBR3023, have also been trialled. Permethrin is a pyrethroid-based insecticide registered by the US Environmental Protection Agency (EPA) since 1977 that has been extensively used as insect repellent and insecticide with a documented safety record. Treatment is usually carried out by home or factory dipping. Permethrin can be bound to fabric fibres in clothing via different techniques such as micro-encapsulation and polymer coating. Indeed, field tests to assess the degree of protection from Aedes mosquito bites provided by wearing clothing treated with permethrin showed a reduction in the number of bites by 90%. Studies on the safety and effectiveness of the use of insecticide-treated clothing for pathogens other than Aedes-transmitted viral diseases suggest that it could potentially be a promising intervention for Aedes-transmitted diseases such as Zika, dengue, and chikungunya.
Obj 1: Conduct a literature review on the protective efficacy of impregnated clothing against vector-borne diseases
We reviewed evidence base for the use of insecticide-treated clothing for protection against bites from arthropods and its effect on arthropod-borne pathogen transmission. Although some studies do demonstrate protection against pathogen transmission, there are surprisingly few, and the level of protection provided varies according to the disease and the type of study conducted. For example, insecticide-treated clothing has been reported to give between 0% and 75% protection against malaria and between 0% and 79% protection against leishmaniasis. Studies vary in the type of treatment used, the age group of participants, the geographical location of the study, and the pathogen transmission potential. This makes it difficult to compare and assess intervention trials. Overall, there is substantial evidence that insecticide-treated clothing can provide protection against arthropod bites. Bite protection evidence suggests that insecticide-treated clothing may be useful in the prevention of pathogen transmission, but further investigations are required to accurately demonstrate transmission reduction.18
Obj 2: Determine the efficacy of impregnated school uniforms on the reduction of dengue incidence in school aged children
InsectShield manufactures permethrin-impregnated apparel for recreational and military purposes. InsectShield Repellent Apparel is registered by the US EPA; their approach is a polymer-coating technique which is claimed to withstand up to 70 washings. We tested our hypothesis by setting up a large community based trial to determine the reduction of laboratory-confirmed dengue infections as a result of permethrin-impregnated school uniforms (InsectShield).
We carefully designed a cluster randomized-controlled cross-over trial in Thailand and published the trial design prior to the start of the study.19 That publication also outlines the detailed sample size calculation, rhe rationale for a cross-over design and the detailed methods. The protocol of this school-based trial was reviewed and approved by the Mahidol University Institutional Review Board (MU-IRB 2009/357.1512). This trial was registered with ClinicalTrials.gov number: NCT01563640. We enrolled 10 schools in Thailand. The primary endpoint was laboratory-confirmed dengue infections, and for the secondary endpoints we measured the impregnated uniforms’ 1-hour knock-down and 24 hour mosquito mortality by standardised WHOPES bioassay cone tests at baseline and after repeated washing. Entomological assessments inside classrooms and in outside areas of schools were conducted.
Results: We enrolled 1,811 pupils aged 6-17 from 5 intervention and 5 control schools. Paired serum samples were obtained from 1,655 pupils. In the control schools, 24/641 (3.7%) and in the intervention schools 33/1,014 (3.3%) students had evidence of new dengue infections during one school term (5 months). There was no significant difference in proportions of students having incident dengue infections between the intervention and control schools, with adjustment for clustering by school. WHOPES cone tests showed a 100% knock down and mortality of Aedes aegypti mosquitoes exposed to impregnated clothing at baseline and up to 4 washes, but this efficacy rapidly declined to below 20% after 20 washes. Results of the entomological assessments showed that the mean number of Aedes aegypti mosquitoes caught inside the classrooms of the intervention schools was significantly reduced in the month following the introduction of the impregnated uniforms, compared to those collected in classrooms of the control schools (p =0.04)
Discussion of the results: Given the day-biting behaviour of Aedes mosquitoes, impregnated school uniforms could potentially be a simple novel tool to reduce mosquito-borne diseases and local vector populations. Our results from the WHOPES cone tests at the start of the trial underpin the potential for insecticide-treated uniforms to protect against dengue by reducing the populations of Aedes mosquitoes and hence mosquito-bites: knock-down effect and mortality immediately after impregnation of uniforms with permethrin by the InsectShield proprietary method were close to 100%, consistent with results obtained under laboratory conditions at the London School of Hygiene and Tropical Medicine.20 Furthermore, we documented a significant reduction in Aedes mosquito numbers in the classrooms of the intervention schools in the first month after the start of the trial at the time when the insecticidal activity of impregnated uniforms was still 100%.
However, our cluster- randomised controlled trial in ten Thai schools involving 1,811 children did not show serological evidence of a protective effect over the 5-month study period of one school term. Given the theoretical support for this strategy, we need to carefully examine plausible reasons for the apparent failure to protect in our trial, so that this intervention is not discarded as an ineffective strategy for control against Aedes-transmitted diseases such as dengue, Zika or chikungunya. The main reason for the negative result was the rapidly waning efficacy of InsectShield permethrin-impregnated clothes under field conditions. We chose InsectShield factory-impregnation over hand-dipping with permethrin because InsectShield impregnation (unlike hand-dipping with permethrin) results in odourless, well tolerated apparel—a fact that is important for a double-blind randomised trial where odour could otherwise had given away the allocation group. We had not anticipated rapid washing-out of permethrin prior to the study as InsectShield have consistently claimed that the insecticidal efficacy of their proprietary method of permethrin impregnation withstands up to 70 washes. However, we documented rapid declines in insecticidal activity after the first 4 washes. After 20 washes, the knock-down and mortality effects on mosquitoes were well below 20%. What might be the reasons for such rapid waning of insecticidal activity? Maybe the quality of already used cotton Thai school uniforms was inferior to clothing materials used by InsectShield for commercial purposes. With suboptimal cloth quality, the coating technique might have been less durable? Or maybe the washing conditions of the tropics, drying in the open air and ironing decreased the insecticidal effect more rapidly? However, we believe it was not just the potentially suboptimal cloth material of local Thai school uniforms, as a very recent study on laundering resistance of five commercially available, factory-treated permethrin-impregnated fabrics also found that permethrin content fell by 58.1 to 98.5 % after 100 defined machine launderings, with InsectShield showing the fastest loss. There is an urgent need for a standardised testing and licensing procedure for insecticide-impregnated commercial clothing to avoid misleading information.
We need to consider other potential causes for the lack of efficacy in our trial. Maybe compliance with the uniforms was not high? We conducted an acceptability study: Quantitative and qualitative tools were used in a mixed methods approach.21 Class-clustered randomised samples of school children enrolled in the RCT were selected and their parents completed 321 self-administered questionnaires. Descriptive statistics and logistic regression were used to analyse the quantitative data. Focus group discussions and individual semi-structured interviews were conducted with parents, teachers, and principals. Qualitative data analysis involved content analysis with coding and thematic development.
The community’s knowledge and experience of dengue was substantial. The acceptability of insecticide treated school uniforms (ISUs) was high. Parents (87.3%; 95% CI 82.9-90.8) would allow their child to wear an ISU and 59.9% (95% CI 53.7-65.9) of parents would incur additional costs for an ISU over a normal uniform. This was significantly associated with the total monthly income of a household and the educational level of the respondent. Parents (62.5%; 95% CI 56.6-68.1) indicated they would be willing to recommend ISUs to other parents.21
Although acceptability and compliance with the trial uniforms was high, school uniforms are not worn after school and over weekends. We did some simulation modelling and estimated a reduction of dengue infections by 47% if 60% of all mosquito bites occurred during school hours and 70% of the children wore treated uniforms, assuming that mosquito knock-down and mortality levels remained at baseline (without washing-out effect).22 A reduction of dengue infections by 47% would indeed be a major public health victory.
Because we used paired blood samples at baseline and at the end of the study period (5 months), we are unable to tease out the impact of impregnated clothing on dengue incidence in the first month of wearing the intervention uniforms, at a time when the efficacy in terms of knock-down and mortality effect on mosquitoes was still close to 100%. Nevertheless, the results of entomological assessments showed that the intervention had an impact on the number of Aedes mosquitoes inside intervention schools before insecticidal activity declined. This is an important finding that encourages continued research on the use of insecticide-treated clothing as a potential strategy for dengue prevention in school children.
Long-lasting insecticide-treated bednets were a major breakthrough in the control of malaria. However, bednets do not get washed so frequently. For insecticide-treated clothing to be a viable public health intervention it should withstand regular washing. If the rapid washing-out of permethrin can be overcome by novel technological approaches, insecticide-treated clothes would deserve to be re-evaluated as a potentially cost-effective and scalable intervention.
Obj 3: Determine the protective efficacy of school uniforms impregnated with repellent under different laboratory scenarios
Here we assessed the efficacy and durability of different types of insecticide-treated clothing on laboratory-reared Ae. aegypti.23 Standardised World Health Organisation Pesticide Evaluation Scheme (WHOPES) cone tests and arm-in-cage assays were used to assess knockdown (KD) and mortality of Ae. aegypti tested against factory-treated fabric, home-dipped fabric and microencapsulated fabric. Based on the testing of these three different treatment types, the most protective was selected for further analysis using arm-in cage assays with the effect of washing, ultra-violet light, and ironing investigated using high pressure liquid chromatography.
Results: Efficacy varied between the microencapsulated and factory dipped fabrics in cone testing. Factory-dipped clothing showed the greatest effect on KD (3 min 38.1%; 1 hour 96.5%) and mortality (97.1%) with no significant difference between this and the factory dipped school uniforms. Factory-dipped clothing was therefore selected for further testing. Factory dipped clothing provided 59% (95% CI = 49.2%- 66.9%) reduction in landing and a 100% reduction in biting in arm-in-cage tests. Washing duration and technique had a significant effect, with insecticidal longevity shown to be greater with machine washing (LW50 = 33.4) compared to simulated hand washing (LW50 = 17.6). Ironing significantly reduced permethrin content after 1 week of simulated use, with a 96.7% decrease after 3 months although UV exposure did not reduce permethrin content within clothing significantly after 3 months simulated use.
Conclusion: Permethrin-treated clothing may be a promising intervention in reducing dengue transmission. However, our findings also suggest that clothing may provide only short-term protection due to the effect of washing and ironing, highlighting the need for improved fabric treatment techniques.
In order to understand the findings from our school based trial better, we conducted several lab-based studies at the London School of Hygiene and Tropcial Medicine.
The next study focused on the effect of washing on treated clothing, skin coverage and protection against resistant and susceptible Ae. aegypti using modified WHO arm-in-cage assays.24 Coverage was further assessed using free-flight room tests to investigate the protective efficacy of unwashed factory-dipped permethrin-treated clothing. Clothing was worn as full coverage (long sleeves and trousers) and partial coverage (short sleeves and shorts). Residual permethrin on the skin and its effect on mosquitoes was measured using modified WHO cone assays and quantified using high-pressure liquid chromatography (HPLC) analysis.
Results: In the arm-in-cage assays, unwashed clothing reduced landing by 58.9% (95% CI 49.2-66.9) and biting by 28.5% (95% CI 22.5-34.0) but reduced to 18.5% (95% CI 14.7-22.3) and 11.1% (95% CI 8.5-13.8) respectively after 10 washes. Landing and biting for resistant and susceptible strains was not significantly different (p<0.05). In free-flight room tests, full coverage treated clothing reduced landing by 24.3% (95% CI 17.4-31.7) and biting by 91% (95% CI 82.2-95.9) with partial coverage reducing landing and biting by 26.4% (95% CI 20.3-31.2) and 49.3% (95% CI 42.1-59.1) respectively with coverage type having no significant difference on landing (p<0.05). Residual permethrin was present on the skin in low amounts (0.0041mg/cm2) but still produced a KD of >80% one hour after wearing treated clothing.
Conclusion: Whilst partially covering the body with permethrin-treated clothing provided some protection against biting, wearing treated clothing with long sleeves and trousers provided the highest form of protection. This finding may explain some of the absence of protection that we saw in the Thai trial. After all, school uniforms are short-sleeved tops and shorts. Furthermore, washing treated clothing dramatically reduced protection provided. An encouraging finding was that permethrin-treated clothing could provide protection to individuals from Ae. aegypti that show permethrin resistance. Additionally, it could continue to provide protection even after the clothing has been worn.24
With the extension of DengueTools, The London School of Hygiene and Tropical Medicine under Dr James Cook took on testing a number of different candidate repellent compounds which could be impregnated into clothing, instead of permethrin. These compounds can be used on their own or in combination with the permethrin in treated clothing. The aim was to increase protection provided by the clothing by reducing biting and landing rates. The results have shown a number of candidate repellents which could be incorporated into fabrics to provide protection from mosquito bites with protection of up to 45%. The London school also successfully approached commercial companies with these compound and have shown these compounds can be incorporated into fabrics and garments.
Obj 4: Develop a cost-effectiveness framework and model for the school-based preventive intervention of insecticide treated school uniforms
Schools where children spend most of their day is proposed as an ideal setting to implement preventive strategies against day-biting Aedes mosquitoes. The use of insecticide-treated school uniforms is a promising strategy currently under investigation. Using a decision-analytic model, we evaluated the cost-effectiveness of the use of insecticide-treated school uniforms for prevention of dengue, compared with a "do-nothing" alternative, in schoolchildren from the societal perspective. We explored how the potential economic value of the intervention varied under various scenarios of intervention effectiveness and cost, as well as dengue infection risk in school-aged children, using data specific to Thailand.
Results: At an average dengue incidence rate of 5.8% per year in school-aged children, the intervention was cost-effective (ICER≤$16,440) in a variety of scenarios when the intervention cost per child was $5.3 or less and the intervention effectiveness was 50% or higher. In fact, the intervention was cost saving (ICER<0) in all scenarios in which the intervention cost per child was $2.9 or less per year and the intervention effectiveness was 50% or higher. The results suggested that this intervention would be of no interest to Thai policy makers when the intervention cost per child was $10.6 or higher per year regardless of intervention effectiveness (ICER>$16,440). Our results present the potential economic value of the use of insecticide-treated uniforms for prevention of dengue in schoolchildren in a typical dengue endemic setting and highlight the urgent need for additional research on this intervention.
Obj 5: Produce policy briefs and engage with policy makers to ensure the scalability of such school intervention programs
We produced a policy brief, but as the conclusion was that at this stage insecticide treated uniforms are not yet scalable, we did not meet up with policy makers but instead met up with the Ministry of Science, Bangkok. The pressing next step is to develop wearable repellent clothing that can withstand frequent washing, ironing and exposure to sun. Once such a repellent/insecticide has been developed, a new proof of principle study needs to be conducted, and if the data support a reduction of dengue infections, even if only by 30%, then this would be an approach that would warrant being scaled up to national and international levels as a school-based strategy.
Research area 3: Risk of introduction of dengue into naïve areas including Europe
Obj 1: Describe the trends of dengue importations into Europe
Totally unplanned of course, during the DengueTools project period, there were two dengue outbreaks in previously naïve areas/countries, which gave additional impetus to the research in research area 3: the Madeira outbreak with > 2000 cases in 2012/13; and Japan in the year 2014, both in temperate climates. These outbreaks were also picked up in our sentinel surveillance (WP 6).
Our sentinel surveillance system recruited 242 returning dengue viremic travellers through travel medicine providers from TropNet; these 242 who presented during the viremic phase (eg within 5 days after infection) corresponded to 36% of all returning travellers seen at TropNet sites during that period, underpinning that a significant number of dengue infected travellers are viremic which poses a risk for dengue introduction and autochthonous transmission in European regions where Aedes albopictus is prevalent. The most likely place for dengue virus infection for travellers was Asia, followed by the Americas, then Western Pacific region, and the smallest number from Africa.25
We aimed to determine the attack rate of dengue in Swedish travellers and analyse the trends over time and the geographical variation. 26 METHODS: We obtained the following data from the Swedish Institute for Communicable Disease Control for the y 1995-2010: number of Swedish residents with confirmed dengue, the country and year of infection. We also obtained registers on the Swedish annual air traveller arrivals to dengue endemic areas from the United Nations World Tourist Organization for the time period. We estimated attack rates with 95% confidence intervals (CI). RESULTS: In total, 925 Swedish travellers with confirmed dengue were reported. We found an increasing trend over time for most destinations. The majority of the dengue cases were acquired in Thailand (492 out of 925 travellers; 53%), with an attack rate of 13.6 (95% CI 12.7 14.4) per 100,000 travellers. However, the 2 highest attack rates per 100,000 travellers were found for Sri Lanka (45.3 95% CI 34.3 56.4) and Bangladesh (42.6 95% CI 23.8 61.5).26 CONCLUSIONS: Information on attack rates in travellers is more helpful in guiding travel medicine practitioners than reports of absolute numbers, as the latter reflect travel preferences rather than the true risk. Although the majority of dengue infections in Swedish travellers were acquired in Thailand, the attack rates for dengue in travellers to Sri Lanka and Bangladesh were much higher. These data aid in refining information on the risk of dengue in travellers.
Obj 2: Conduct dengue sequencing and produce phylogenetic trees on imported dengue viruses to Europe
The following findings highlight how travellers can unmask an ongoing outbreak or identify outbreaks in areas with poor surveillance or reporting.
First, we detected circulation of dengue serotype 3 (genotype III) in Cuba in 2013 and 2014, despite the fact that PAHO did not report any dengue during that time period. The last detection of dengue 3 (genotype III) in Cuba was during a big epidemic occurring in 2001-2002.
Second, we identified dengue virus circulating from countries in Africa that hitherto have rarely reported dengue. We isolated a genotype III DENV-3 strain from a traveller returning from Togo and Burkina Faso, a region where circulation of dengue had not been reported. We also identified a dengue virus strain imported during the dengue outbreak in Angola in 2013. Our dengue sequencing analysis points towards an importation of a dengue virus from Brazil. We also show that Portugal and South Africa are most likely at the highest risk of importation of dengue from Angola due to the large number of air passengers between Angola and these countries.27 Furthermore, we isolated DENV from travellers returning from Tanzania during the dengue outbreak affecting Dar es Salaam and neighboring regions in 2014. The isolated virus strains cluster in a lineage different from the strains introduced in Africa in the early 1980s, suggesting recent introduction from Asia.
Third, our sentinel surveillance also identified travellers from the first major dengue outbreak in Europe: the 2012/2013 outbreak in Madeira, Portugal. Dengue sequencing done by our partners in Madrid and Singapore showed that the virus was imported from Venezuela.28,29 Studying IATA data, we found that there were 22,948 air travellers to Madeira in 2012, originating from twenty-nine dengue-endemic countries; 89.6% of these international travellers originated from Venezuela and Brazil.29 We developed an importation index that takes into account both travel volume and the extent of dengue incidence in the country of origin.30 Venezuela and Brazil had by far the highest importation indices compared with all other dengue-endemic countries. The importation index for Venezuela was twice as high as that for Brazil. When taking into account seasonality in the months preceding the onset of the Madeira outbreak, this index was even seven times higher for Venezuela than for Brazil during this time.
Fourth, another country that has not seen a dengue outbreak for more than 70 years was picked up through our sentinel surveillance system: the genotype I (Asian) DENV-1 isolate we isolated from a traveller returning from Japan in September 2014 was linked to a local outbreak with ~160 reported autochthonous cases affecting Tokyo in August-September 2014, despite Japan's temperate climate. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam.31 With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come. Despite Japan's temperate climate, the increasing travel interconnectivity to Japan, combined with more suitable climate and environmental drivers for dengue epidemics, made such an outbreak possible.31
In conclusion, our data demonstrate that epidemiological and virological data obtained from dengue infected international travellers can add an important additional layer to global dengue surveillance efforts.
Obj 3: Compare clinical manifestations and the usefulness of the 2009 WHO dengue case classification of dengue in travellers (non-endemic populations) versus endemic populations (Sri Lanka)
We were not able to achieve this comparison within the timeframe, but analyses have now started.
Obj 4: Explore reasons for the resurgence and geographic spread of dengue
Dengue has increased exponentially over the past decades, although accurate data remain elusive.32 We did a thorough literature review and identified the following factors as main drivers for dengue resurgence: globalisation with increasing interconnectivity, ecological habitat encroachment, socioeconomic changes, climate change, virus evolution, and urbanization.33
In Singapore, the frequency and magnitude of dengue epidemics have increased significantly over the past 40 years. Singapore has excellent documentation of data over decades, and is probably the best place to untangle which of the drivers as listed above have caused the rapid increase in dengue incidence. We studied the relative contributions of putative drivers for the rise of dengue in Singapore: population growth, climate parameters and international air passenger arrivals from dengue endemic countries, for the time period of 1974 until 2011. We used multivariable Poisson regression models with the following predictors: Annual Population Size; Aedes Premises Index; Mean Annual Temperature; Minimum and Maximum Temperature Recorded in each year; Annual Precipitation and Annual Number of Air Passengers arriving from dengue-endemic South-East Asia to Singapore.34 The relative risk (RR) of the increase in dengue incidence due to population growth over the study period was 42.7 while the climate variables (mean and minimum temperature) together explained an RR of 7.1 (RR defined as risk at the end of the time period relative to the beginning and goodness of fit associated with the model leading to these estimates assessed by pseudo-R2 equal to 0.83). 34 Estimating the extent of the contribution of these individual factors on the increasing dengue incidence, we found that population growth contributed to 86% while the residual 14% was explained by increase in temperature. We found no correlation with incoming air passenger arrivals into Singapore from dengue endemic countries. Our findings have significant implications for predicting future trends of the dengue epidemics given the rapid urbanization with population growth in many dengue endemic countries. It is time for policy-makers and the scientific community alike to pay more attention to the negative impact of urbanization and urban climate on diseases such as dengue.
Obj 5: Estimate and model dengue virus introductions via travellers
We first developed a simple importation index, as described above, with the main purpose to identify the country of origin for importation.30 This index is founded on the notion that the extent of importation depends on the interconnectivity between the exporting and receiving country AND the extent of dengue endemicity in the exporting country. However, this index is crude, does not allow to quantify the risk, and can only rank countries at highest risk. To estimate the risk, the probability of being infected at the time of travel and the duration of viremia also needs to be taken into account, so PhD student Mikkel Quam (Partner UmU) developed a more complex importation model. Given that Italy experienced the first ever outbreak of chikungunya in Europe in 2007, we set out to estimate the extent of dengue virus importations into Italy via air travelers. We found that from 2005 to 2012, more than 7.3 million air passengers departing from 100 dengue-endemic countries arrived in Rome. Our Importation Model, which included air traveler volume, estimated the incidence of dengue infections in the countries of disembarkation, and the probability of infection coinciding with travel accounted for an average of 2,320 (1,621-3,255) imported dengue virus infections per year, of which 572 (381-858) were "apparent" dengue infections and 1,747 (1,240-2,397) "inapparent."35 Between 2005 and 2012, we found an increasing trend of dengue virus infections imported into Rome via air travel, which may pose a potential threat for future emergence of dengue in Italy, given that the reoccurring pattern of peak importations corresponds seasonally with periods of relevant mosquito vector activity.
We then developed an even more complex model: In the first model (disease importation), susceptible individuals travel from their disease-free home country to the endemic country and come back after some weeks. The risk of infection spreading in their home country is then estimated supposing the visitors are submitted to the same force of infection as the local population but do not contribute to it. In the second model (disease exportation), it is calculated the probability that an individual from the endemic (or epidemic) country travels to a disease-free country in the condition of latent infected and eventually introduces the infection there. The input of both models is the force of infection at the visited/source country, assumed known. The models are deterministic, but a preliminary stochastic formulation is presented as an appendix of our publication.36
Using mathematical modelling, we set out to estimate the risk of non-immune persons acquiring dengue when travelling to Thailand. The model is deterministic with stochastic parameters and assumes a Poisson distribution for the mosquitoes' biting rate and a Gamma distribution for the probability of acquiring dengue from an infected mosquito. From the force of infection we calculated the risk of dengue acquisition for travellers to Thailand arriving in a typical year (averaged over a 17-year period) in the high season of transmission. A traveller arriving in the high season of transmission and remaining for 7 days has a risk of acquiring dengue of 0.2% (95% CI 0.16-0.23) whereas the risk for travel of 15 and 30 days' duration is 0.46% (95% CI 0.41-0.50) and 0.81% (95% CI 0.76-0.87) respectively.37 Our data highlight that the risk of non-immune travellers acquiring dengue in Thailand is substantial. The incidence of 0.81% after a 1-month stay is similar to that reported in prospective seroconversion studies in Israeli travellers to Thailand, highlighting that our models are consistent with actual data. Risk estimates based on mathematical modelling offer more detailed information depending on various travel scenarios, and will help the travel medicine provider give better evidence-based advice for travellers to dengue-endemic countries.
We also applied these models that we had originally developed for dengue to polio38,39 and Zika.40,41
Obj 6: Estimate the risk of dengue in travellers to the World Cup in 2014 and to the Summer Olympics in Rio de Janeiro in 2016
We had not planned this objective originally in our application, but felt it would be appropriate to add this objective, given the high topicality of the risk of dengue in both the World Cup in 2014 and now also the Summer Olympics in 2016, both in Brazil. As our modeler is from Sao Paolo University Brazil (Partner FUSP), these research questions were even more pertinent. Brazil hosted the FIFA World Cup, the biggest single-event competition in the world, from June 12-July 13 2014 in 12 cities. This event draws an estimated 600,000 international visitors. We calculated the risk of dengue acquisition to non-immune international travellers to Brazil, depending on the football match schedules, considering locations and dates of such matches for June and July 2014.42 We estimated the average per-capita risk and expected number of dengue cases for each host-city and each game schedule chosen based on reported dengue cases to the Brazilian Ministry of Health for the period between 2010-2013. On the average, the expected number of cases among the 600,000 foreigner tourists during the World Cup is 33, varying from 3-59.43 We published these findings prior to the FIFA World Cup—and interestingly, our predictions were found to be true. Only three dengue cases were reported in visitors to the World Cup.
About 400,000 non-immune foreign tourists are expected to attend the 2016 Olympic games in August this year (after the DengueTools project period). As Brazil is the country with the highest number of dengue cases worldwide, concern about the risk of dengue for travelers is justified. A system of differential equation models the spread of dengue amongst the resident population and a stochastic approximation is used to assess the risk to tourists. Historical reported dengue time series in Rio de Janeiro for the years 2000-2015 is used to find out the time dependent force of infection, which is then used to estimate the potential risks to a large tourist cohort. The worst outbreak of dengue occurred in 2012 and this and the other years in the history of Dengue in Rio are used to discuss potential risks to tourists amongst visitors to the forthcoming Rio Olympics. RESULTS: The individual risk to be infected by dengue is very much dependent on the ratio asymptomatic/symptomatic considered but independently of this the worst month of August in the period studied in terms of dengue transmission, occurred in 2007. CONCLUSIONS: If dengue returns in 2016 with the pattern observed in the worst month of August in history (2007), the expected number of symptomatic and asymptomatic dengue cases among tourists will be 23 and 206 cases, respectively. This worst-case scenario would have an incidence of 5.75 (symptomatic) and 51.5 (asymptomatic) per 100,000 individuals.44
Obj 7: Model vectorial capacity for Aedes mosquitoes in temperate climate zones and globally under current and future climate scenarios
Dengue infections are climate sensitive, so it is important to better understand how changing climate factors affect the potential for geographic spread and future dengue epidemics. Vectorial capacity (VC) describes a vector's propensity to transmit dengue taking into account human, virus, and vector interactions. VC is highly temperature dependent, but most dengue models only take mean temperature values into account. Recent evidence shows that diurnal temperature range (DTR) plays an important role in influencing the behavior of the primary dengue vector Aedes aegypti. In this study, we used relative VC to estimate dengue epidemic potential (DEP) based on the temperature and DTR dependence of the parameters of A. aegypti. We found a strong temperature dependence of DEP; it peaked at a mean temperature of 29.3 degrees C when DTR was 0 degrees C and at 20 degrees C when DTR was 20 degrees C. Increasing average temperatures up to 29 degrees C led to an increased DEP, but temperatures above 29 degrees C reduced DEP. In tropical areas where the mean temperatures are close to 29 degrees C, a small DTR increased DEP while a large DTR reduced it. In cold to temperate or extremely hot climates where the mean temperatures are far from 29 degrees C, increasing DTR was associated with increasing DEP.45 Incorporating these findings using historical and predicted temperature and DTR over a two hundred year period (1901-2099), we found an increasing trend of global DEP in temperate regions. Small increases in DEP were observed over the last 100 years and large increases are expected by the end of this century in temperate Northern Hemisphere regions using climate change projections. These findings illustrate the importance of including DTR when mapping DEP based on VC.
As warming temperatures may increase the geographic spread of vector-borne diseases into temperate areas, we calculated temperature-dependent VC for Europe, highlighting 10 European cities and three non-European reference cities. Compared with the tropics, Europe shows pronounced seasonality and geographical heterogeneity. Although low, VC during summer is currently sufficient for dengue outbreaks in Southern Europe to commence-if sufficient vector populations (either Ae. aegypti and Ae. albopictus) were active and virus were introduced. Under various climate change scenarios, the seasonal peak and time window for dengue epidemic potential increases during the 21st century. Our study maps dengue epidemic potential in Europe and identifies seasonal time windows when major cities are most conducive for dengue transmission from 1901 to 2099. Our findings illustrate, that besides vector control, mitigating greenhouse gas emissions crucially reduces the future epidemic potential of dengue in Europe.45
Obj 8: Describe typical breeding sites for Aedes albopictus in Southern France
Aedes albopictus is generally considered a secondary vector of dengue because it feeds indiscriminately on many species of vertebrate yet humans and primates are the only hosts for the virus. For this reason the species received relatively little attention until its appearance in the United States in 1982. Since then it has spread globally with unprecedented speed: it is now established throughout much of the Americas, at least four countries in Africa and 26 countries in Europe and the Middle East. Nevertheless, the role of Ae. albopictus in dengue transmission remains unresolved; in particular its role in Europe and little attention has been devoted to methods for emergency control. Our field inspections indicated that garden-watering by residents was an important source of breeding sites during the long, dry Mediterranean summer. We also observed that oviposition and adult capture rates were influenced by the degree of vegetation-cover (Spearman r = -0.47 p < 0.05) (manuscript submitted). In this sense, Ae. albopictus differs from Ae. aegypti (considered the most important urban vector of dengue) in that it is primarily exophilic; the association with vegetation cover is an important factor in the efficacy of space sprays for adult control.
Obj 9: Evaluate the impact of ultra-low volume (ULV) aerosols on Ae.albopictus in Southern France
As a pre-requisite to field evaluations of the efficacy of insecticidal spray machines on wild Ae. albopictus, we undertook a study of insecticide susceptibility of males and females. Results demonstrated that the local Ae. albopictus population was highly susceptible to Deltamethrin but that females required a significantly higher dosage than males: DL50 0.00075 (0.00058- 0.00097) and 0.00273 (0.00228- 0.00325). The susceptibility of the local strains of Ae. albopictus to deltamethrin is a welcome finding; a high level of DDT/pyrethroid resistance is widespread in populations of Ae. aegypti worldwide.
We anticipated that Ae. albopictus might be more susceptible to ULV treatments than Ae. aegypti because it is largely exophilic, whereas the indoor resting activity of Ae. aegypti protects it from the aerosol. We used infusion-baited ovitraps and BG-sentinel adult traps to monitor the population for at least four days before treatment and at least five afterwards. During the 2013 season we completed three field trials. In two applications of ULV-deltamethrin there was limited impact on female mosquitoes captures in all age cohorts, as well as on the rate of oviposition. On the other hand, males appeared much more vulnerable; they were virtually eliminated. In 2014 we ran two more trials of the same treatment in a second treatment area, including one in which we made two consecutive applications, with similar results: males were far more vulnerable than females. (Manuscript submitted)
We decided to mount small-scale evaluations of a second application method: a hand-held thermal fogger (Pulsfog®). Results were much more encouraging: ca. 90% reduction of both males and females. From these observations we conclude that degree of cover is a critical factor in the efficacy of the two methods: foliage and other degrees of shelter reduce the probability that the aerosol particles will encounter their target; in this context, males may be more vulnerable because they rest at more exposed sites and may be more prone to flight in their quest for females.
As an alternative to ULV treatments we made an exploratory study of the efficacy of 5 residual formulations of Inesfly®, a resinous paint that incorporates micro-encapsulated insecticide granules. Persistence of the most efficient product was estimated at T0, T12 months and T24 months. Delayed 24-hour mortality at T0 was 100% (p < 10-5) for both surfaces porous and non-porous. Twelve months later (T12 m) OP-based paint applied on cement with one layer showed a decrease in the lethal effect on females Ae. albopictus. Delayed 24-hour mortality at T24 m was 100% plastic surfaces and cement with an under-layer of control paint application, however cement plates treated without the under-layer control paint became completely unlethal (0%/ 24h) (manuscript in preparation).
Obj 10: Determine the susceptibility of Aedes albopictus populations to DENV under different temperature regimes (in comparison to chikungunya virus)
At 200C, our data suggest a high rate of transmission of chikungunya at low temperatures, and that, on viral criteria, epidemic transmission at low temperatures is less likely for DENV.
Additional objectives for Zika in response to the public health emergency
1) Develop RPA for Zika virus
Real time Quantitative Reverse Transcriptase Polymerase Chain (qRT-PCR) Reaction Assay
A real time qRT-PCR assay was developed and used to quantify the Zika virus RNA template copy number needed for the optimization of the Zika virus RT-RPA primers.
RPA primer optimization
A series of RPA primers and fluorescence probes specific to the Zika virus genome were designed. From the list of RPA primers and fluorescence probes, optimal pairs were chosen. The optimal primer pairs detected the ZIKV RNA up to 1000 genome copies. Subsequent cross-reactivity assessments performed using viral RNA for DENV (DENV-1, DENV-2, DENV-3, and DENV-4), chikungunya virus (CHIKV) and Getah virus (GETV) found no cross-reacting amplification. There was no cross-reactivity between the primers/probes to the six arboviruses tested, hence, the RT-RPA assay was specific for ZIKV RNA
2) Determine the Zika incidence in febrile patients recruited from the laboratory enhanced sentinel study in Sri Lanka
Of 204 samples obtained from patients with an undifferentiated fever in 2014/2015 in Sri Lanka, none were found to be Zika virus positive.
3) Determine the risk of introduction and establishment of Zika virus in Europe based on vectorial capacity of Aedes albopictus in Europe and interconnectivity
The explosive Zika virus epidemic in the Americas is amplifying spread of this emerging pathogen into Europe.46 Given the interconnectivity of Brazil with the rest of the world, Zika virus (ZIKV) infections have the potential to spread rapidly around the world via viremic travellers. The extent of spread depends on the travel volume and the endemicity in the exporting country. In the absence of reliable surveillance data, we did mathematical modelling to estimate the number of importations of ZIKV from Brazil into Europe. DESIGN: We applied a previously developed mathematical model on importations of dengue to estimate the number of ZIKV importations into Europe, based on the travel volume, the probability of being infected at the time of travel, the population size of Brazil, and the estimated incidence of ZIKV infections. RESULTS: Our model estimated between 508 and 1,778 imported infections into Europe in 2016, of which we would expect between 116 and 355 symptomatic Zika infections; with the highest number of importations being into France, Portugal and Italy. CONCLUSIONS: Our model identified high-risk countries in Europe. Such data can assist policymakers and public health professionals in estimating the extent of importations in order to prepare for the scale up of laboratory diagnostic assays and estimate the occurrence of Guillain-Barre Syndrome, potential sexual transmission, and infants with congenital ZIKV syndrome.41 While Aedes albopictus has proven to be a vector for the transmission of dengue and chikungunya viruses in Europe, there is growing experimental and ecological evidence to suggest that it may also be competent for Zika virus. We analyzed and overlaid the monthly flows of airline travellers arriving into European cities from Zika affected areas across the Americas, the predicted monthly estimates of the basic reproduction number of Zika virus in areas where Aedes mosquito populations reside in Europe (Aedes aegypti in Madeira, Portugal and Ae. albopictus in continental Europe), and human populations living within areas where mosquito-borne transmission of Zika virus may be possible. We highlight specific geographic areas and timing of risk for Zika virus introduction and possible spread within Europe to inform the efficient use of human disease surveillance, vector surveillance and control, and public education resources.47
4) Determining the risk of Zika infection during the Olympics 2016 in Brazil
We modelled the risk of acquiring Zika infections in visitors and athletes to the Summer Olympics in Rio de Janeiro in August 2016 and estimated only 9 in 1,000,000, about 15 times lower than that for dengue.40,48
Literature list of DengueTools publications, all referenced above
1. Wilder-Smith A, Renhorn KE, Tissera H, et al. DengueTools: innovative tools and strategies for the surveillance and control of dengue. Global health action 2012; 5.
2. Jaenisch T, Idams, Sakuntabhai A, Denfree, Wilder-Smith A, DengueTools. Dengue research funded by the European commission-scientific strategies of three European dengue research consortia. PLoS Negl Trop Dis 2013; 7(12): e2320.
3. Teoh BT, Sam SS, Tan KK, et al. Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification. BMC Infect Dis 2013; 13: 387.
4. Teoh BT, Sam SS, Tan KK, et al. Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification. J Clin Microbiol 2015; 53(3): 830-7.
5. Teoh BT, Sam SS, Tan KK, et al. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen. Sci Rep 2016; 6: 27663.
6. Harris CL, Sanchez-Vargas IJ, Olson KE, Alphey L, Fu G. Polymerase chain displacement reaction. Biotechniques 2013; 54(2): 93-7.
7. Louis VR, Phalkey R, Horstick O, et al. Modeling tools for dengue risk mapping - a systematic review. Int J Health Geogr 2014; 13: 50.
8. Louis VR, Montenegro Quinonez CA, Kusumawathie P, et al. Characteristics of and factors associated with dengue vector breeding sites in the City of Colombo, Sri Lanka. Pathogens and global health 2016; 110(2): 79-86.
9. Tissera H, Amarasinghe A, Gunasena S, et al. Laboratory-Enhanced Dengue Sentinel Surveillance in Colombo District, Sri Lanka: 2012-2014. PLoS Negl Trop Dis 2016; 10(2): e0004477.
10. Tang KF, Ooi EE. Diagnosis of dengue: an update. Expert Rev Anti Infect Ther 2012; 10(8): 895-907.
11. Wilder-Smith A, Cohn E, Lloyd DC, Tozan Y, Brownstein JS. Internet-based media coverage on dengue in Sri Lanka between 2007 and 2015. Glob Health Action 2016; 9: 31620.
12. Tozan Y. Current issues in the economics of vaccination against dengue. Expert Rev Vaccines 2016; 15(4): 519-28.
13. Horstick O, Tozan Y, Wilder-Smith A. Reviewing Dengue: Still a Neglected Tropical Disease? PLoS Negl Trop Dis 2015; 9(4): e0003632.
14. Wilder-Smith A. Dengue vaccines: dawning at last? Lancet 2014; 384(9951): 1327-9.
15. Wilder-Smith A, Gubler DJ. PUBLIC HEALTH. Dengue vaccines at a crossroad. Science 2015; 350(6261): 626-7.
16. Wilder-Smith A, Massad E. Age specific differences in efficacy and safety for the CYD-tetravalent dengue vaccine. Expert Rev Vaccines 2016.
17. Thalagala N, Tissera H, Palihawadana P, et al. Costs of Dengue Control Activities and Hospitalizations in the Public Health Sector during an Epidemic Year in Urban Sri Lanka. PLoS Negl Trop Dis 2016; 10(2): e0004466.
18. Banks SD, Murray N, Wilder-Smith A, Logan JG. Insecticide-treated clothes for the control of vector-borne diseases: a review on effectiveness and safety. Med Vet Entomol 2014; 28 Suppl 1: 14-25.
19. Wilder-Smith A, Byass P, Olanratmanee P, et al. The impact of insecticide-treated school uniforms on dengue infections in school-aged children: study protocol for a randomised controlled trial in Thailand. Trials 2012; 13: 212.
20. Banks S, Orsborne J, Wilder-Smith A, Logan J. Permethrin-treated clothing as protection against the dengue vector, Aedes aegypti: extent and duration of protection. PLoS NTD 2015; in print.
21. Murray N, Jansarikij S, Olanratmanee P, et al. Acceptability of impregnated school uniforms for dengue control in Thailand: a mixed methods approach. Glob Health Action 2014; 7: 24887.
22. Massad E, Amaku M, Coutinho FA, Kittayapong P, Wilder-Smith A. Theoretical impact of insecticide-impregnated school uniforms on dengue incidence in Thai children. Glob Health Action 2013; 6: 20473.
23. DeRaedt Banks S, Orsborne J, Gezan SA, et al. Permethrin-Treated Clothing as Protection against the Dengue Vector, Aedes aegypti: Extent and Duration of Protection. PLoS Negl Trop Dis 2015; 9(10): e0004109.
24. Orsborne J, DeRaedt Banks S, Hendy A, et al. Personal Protection of Permethrin-Treated Clothing against Aedes aegypti, the Vector of Dengue and Zika Virus, in the Laboratory. PLoS One 2016; 11(5): e0152805.
25. Neumayr AM, J. Schunk, M. Genton, B. Wilder-Smith, A. Hatz, C. Franco L. Sentinel surveillance of imported dengue via travelers to Europe 2012 - 2014: TropNet
data from the DengueTools Research Initiative. Euro Surveill 2016; in print.
26. Rocklov J, Lohr W, Hjertqvist M, Wilder-Smith A. Attack rates of dengue fever in Swedish travellers. Scand J Infect Dis 2014; 46(6): 412-7.
27. Sessions OM, Khan K, Hou Y, et al. Exploring the origin and potential for spread of the 2013 dengue outbreak in Luanda, Angola. Global health action 2013; 6: 21822.
28. Franco L, Pagan I, Serre Del Cor N, et al. Molecular epidemiology suggests Venezuela as the origin of the dengue outbreak in Madeira, Portugal in 2012-2013. Clin Microbiol Infect 2015; 21(7): 713 e5-8.
29. Wilder-Smith A, Quam M, Sessions O, et al. The 2012 dengue outbreak in Madeira: exploring the origins. Euro Surveill 2014; 19(8): 20718.
30. Quam MB, Wilder-Smith A. Importation index of dengue to determine the most probable origin of importation. J Travel Med 2015; 22(1): 72.
31. Quam MB, Sessions O, Kamaraj US, Rocklov J, Wilder-Smith A. Dissecting Japan's Dengue Outbreak in 2014. Am J Trop Med Hyg 2016; 94(2): 409-12.
32. Wilder-Smith A, Byass P. The elusive global burden of dengue. Lancet Infect Dis 2016.
33. Murray NE, Quam MB, Wilder-Smith A. Epidemiology of dengue: past, present and future prospects. Clin Epidemiol 2013; 5: 299-309.
34. Struchiner CJ, Rocklov J, Wilder-Smith A, Massad E. Increasing Dengue Incidence in Singapore over the Past 40 Years: Population Growth, Climate and Mobility. PLoS One 2015; 10(8): e0136286.
35. Quam MB, Khan K, Sears J, Hu W, Rocklov J, Wilder-Smith A. Estimating Air Travel-Associated Importations of Dengue Virus Into Italy. J Travel Med 2015.
36. Lopez LF, Amaku M, Coutinho FA, et al. Modeling Importations and Exportations of Infectious Diseases via Travelers. Bull Math Biol 2016; 78(2): 185-209.
37. Massad E, Rocklov J, Wilder-Smith A. Dengue infections in non-immune travellers to Thailand. Epidemiol Infect 2013; 141(2): 412-7.
38. Quam M, Massad E, Wilder-Smith A. Effects of India's new polio policy on travellers. Lancet 2014; 383(9929): 1632.
39. Wilder-Smith A, Leong WY, Lopez LF, et al. Potential for international spread of wild poliovirus via travelers. BMC Med 2015; 13: 133.
40. Massad E, Coutinho FA, Wilder-Smith A. Is Zika a substantial risk for visitors to the Rio de Janeiro Olympic Games? Lancet 2016.
41. Massad E, Tan SH, Khan K, Wilder-Smith A. Estimated Zika virus importations to Europe by travellers from Brazil. Glob Health Action 2016; 9: 31669.
42. Massad E, Burattini MN, Ximenes R, Amaku M, Wilder-Smith A. Dengue outlook for the World Cup in Brazil. Lancet Infect Dis 2014; 14(7): 552-3.
43. Massad E, Wilder-Smith A, Ximenes R, et al. Risk of symptomatic dengue for foreign visitors to the 2014 FIFA World Cup in Brazil. Mem Inst Oswaldo Cruz 2014; 109(3): 394-7.
44. Ximenes R, Amaku M, Lopez LF, et al. The risk of dengue for non-immune foreign visitors to the 2016 summer olympic games in Rio de Janeiro, Brazil. BMC Infect Dis 2016; 16(1): 186.
45. Liu-Helmersson J, Stenlund H, Wilder-Smith A, Rocklov J. Vectorial capacity of Aedes aegypti: effects of temperature and implications for global dengue epidemic potential. PLoS One 2014; 9(3): e89783.
46. Musso D, Gubler DJ. Zika Virus. Clin Microbiol Rev 2016; 29(3): 487-524.
47. Rocklov JQ, M. Sudre, B., Brady, O. Kraemer, M. Bogoch,.I Liu-Helmersson, J. Wilder-Smith, A. Semenza, J. Khan, K. Assessing Seasonal Risks for the Introduction and Mosquito-borne Spread of Zika Virus
in Europe. EBioMedicine 2016; in print.
48. Burattini MN, Coutinho FA, Lopez LF, et al. Potential exposure to Zika virus for foreign tourists during the 2016 Carnival and Olympic Games in Rio de Janeiro, Brazil. Epidemiol Infect 2016; 144(9): 1904-6.
Potential Impact:
Research area 1: Surveillance in a dengue endemic country
Background:
The dramatic global spread and increased frequency and magnitude of epidemic dengue/dengue hemorrhagic fever (DEN/DHF) in the past 40 years underscores the critical need for more effective surveillance, prevention and control of this disease. Most endemic countries do not have adequate surveillance to monitor the disease, let alone mosquito control or vaccine prevention programmes. Using a well-designed population-based system to monitor the spatial and temporal distribution of dengue in its different clinical expressions is of enormous public health importance for determining the priority areas for intervention as well as to identify variables for triggering outbreak alerts. For surveillance to effectively provide early warning for epidemic transmission, it must be active, laboratory-based, and comprehensive in its coverage of the spectrum of clinical illness and the factors that influence transmission dynamics.
Scientific impact:
We were able to document the significant dengue burden in Colombo District over three years in more than 5000 patients recruited: almost 50% of all patients with an undifferentiated fever had lab confirmed dengue. Furthermore, we assessed three diagnostic essays (PCR, Dengue IgM ELISA and NS1 ELISA) and their utility in a sentinel surveillance setting, plus dengue sequencing. NS1 was more sensitive and remained positive for a longer time period than PCR, an advantage which was however off-set by the finding that NS1 was less sensitive in secondary dengue infections. PCR allows for further serotyping. Clinical acumen was high, in terms of high sensitivity, but associated with low specificity.
We also developed an integrated surveillance that included climate and ecological parameters in additional to behavioural practices in the community and socioeconomic factors. The strongest association with dengue risk centred around 6 to 10 weeks following rainfalls of more than 300 mm per week. The overall relative risk of dengue associated with temperature increased steadily from lower temperatures towards the mean value of 29.80C starting from a lag of 4 weeks, and increased for temperatures above the mean value with a cumulative growing association with lags up to 12 weeks. We found, however, some considerable heterogeneity in the associations highlighting that other factors also play a role in triggering new outbreaks, in particular the introduction of new virus genotypes. Perhaps our most interesting finding was the high awareness of dengue in the population combined with a high proportion of appropriate community based practices that resulted in low vector indices- this was in stark contrast to our findings in public buildings such as construction sites, factories and in particular also schools. Given that schools are the sites where the most vulnerable age group for dengue spends most of the day, these findings need to be translated into new policies on where to prioritize vector control measures and public health education.
Our results suggest that the RT-Recombinase Polymerase Amplification technology (RPA) is superior over LAMP. RPA is a rapid diagnostic method for detection of dengue viruses with diagnostic sensitivity similar or comparable to the reference qRT-PCR assay, however, it has several advantages over PCR, namely user-friendliness, speed of test performance, affordability, independence of outdoor temperatures (of particular importance in tropical countries). However, in comparison to the rapid diagnostic tests such as NS1, RPA – although it can be used as a point of care test—requires an equipment to read the results, the Twistdx equipment. In other words, although the assay itself is very cheap (< 1USD), the equipment still needs to be purchased. The easiness of performing the RT-RPA assay was evaluated among new users with the user usability test and RPA was found to be highly user-friendly.
The performance of the RT-RPA test in actual diagnostic setting was evaluated in the arbovirus surveillance laboratory of the WHO Collaborating Centre for Arbovirus Reference & Research (Dengue/Severe Dengue) at the University of Malaya. The diagnostic utility of the RT-RPA assay was assessed so favorably that the WHO CC for Arbovirus in Malaysia decided to role out RPA for all their screening of samples from blood donors, as RPA is so highly sensitive, rapid and cheap (if the Twistdx reader is in place).
Societal and policy impact:
With the EU funded laboratory-enhanced surveillance project a dedicated government laboratory was set up in Sri Lanka to perform routine molecular testing for dengue. As a result, the capacity to do PCR and serotyping is now well established. Quality assurance with a subset of samples sent to the Duke-NUS Laboratory “Emerging Infectious Diseases Program” showed a high agreement, underpinning successful local capacity building. Technology transfer was achieved. Although costs for PCR are higher than for IgM and NS1, PCR would still need to be done in a randomly selected sub-group to allow for serotyping—a recommendation that we gave to the Ministry of Health. In terms of cost effectiveness, the most appropriate test in a clinical setting is NS1—but for sentinel surveillance sites, the combination of PCR with serotyping remains a priority. Sequencing should only be done in research settings, as the cost is prohibitive, and the predictive value of genotypic changes remains unresolved. Based on our findings we shared our recommendations with the Ministry of Health in Sri Lanka to continue enhancing national reference laboratories throughout the country, transfer technologies to peripheral cities for PCR and serotyping, apply lessons learned through our sentinel surveillance, and implement random selection of febrile patients both from in-and outpatient settings. The sentinel sites in Sri Lanka will continue to be used for laboratory enhanced dengue surveillance even after the funding period of DengueTools, underpinning that we set up a sustainable new system.
The high amount of public health staff in dengue control activities year-round combined with free medical care to dengue patients at secondary care hospitals place a formidable financial burden on the public health sector. We calculated the total cost of dengue control and reported hospitalizations to be USD 3.45 million (US$1.50 per capita) in Colombo district in 2012. This analysis was a first attempt to assess the economic burden of dengue response in the public health sector in Sri Lanka. Country-specific evidence is needed for setting public health priorities and deciding about the deployment of existing or new technologies. Such data will help future cost-effectiveness studies when the time comes for dengue vaccine introduction into Sri Lanka.
Strategic impact:
We did biobanking of more than 5,000 blood samples obtained during the enhanced sentinel surveillance in 6 different sites in Colombo District which has already led to various spin-off projects.
First of all, we secured an additional grant from the National Medical Research Council in Singapore to do further detailed sequencing. This information will help us to understand the potential origins of this virus and, combined with current international travel data, where the virus is most likely to go next. Recently, we have also identified regions in non-protein coding regions of the genome that are responsible for increases in epidemic potential. With full genome sequencing, we will specifically look for these genetic signatures as well as other areas of the genome that may be responsible for this epidemic.
Second, we secured more funding to benefit from a subset of 100 patients with more detailed daily clinical information, using a similar questionnaire as used by DENCO. In this subset, we took daily blood samples for biomarker testing using metabolomics, proteomic and lipidomic approaches in order to identify prognostic markers that indicate development of more severe disease.
Third, the set up of the sentinel sites and the cohort study in Ward 33 (=sub-district of Colombo) has attracted the interest of the industry. Takeda has selected this ward for a large Phase 3 trial for their dengue vaccine, leveraging on the relationships built with the community, the scientific community and the Ministry of Health.
Fourth, the promising results of RT-RPA motivated us to develop primers for Zika diagnostics. RT-RPA for Zika was highly specific, in other words we did not find cross-reactivity between the primers/probes to the six arboviruses tested. These results will lead to further work on optimizing the primers and evaluating the assay against a panel of more flaviviruses, and for the duration to positivity compared to PCR.
Fifth, our stored dengue-negative samples can be used to describe the cause of non-dengue fever.
Research area 2:
Scientific Impact
There is an urgent need for integrated and complementary population-based strategies to protect vulnerable children. Insecticide-treated bednets were a breakthrough in community based, scalable measures to reduce malaria infections that are mainly acquired during dawn and dusk and evening/night hours. Insecticide-treated clothing is an analogue idea that could be used during the day-time for day-time biting Aedes mosquitoes that transmit dengue viruses. Impregnated school uniforms to reduce dengue infections in children would be a simple, safe and cost-effective intervention, that is sustainable through community-based programmes; and our laboratory-based studies confirm that this approach is promising. Knockdown rates and mortality of mosquitoes landing on impregnated clothing was very high. Even partial body coverage (short sleeves, shorts) had an impact. However, our studies also showed rapid waning of initial close to 100% efficacy to below 20% after just 20 washes. UV light and ironing also had deleterious effects on the longevity of the impregnation. The results of entomological assessments showed that the intervention had an impact on the number of Aedes mosquitoes inside intervention schools before insecticidal activity declined. This is an important finding that encourages continued research on the use of insecticide-treated clothing as a potential strategy for dengue prevention in school children.
Long-lasting insecticide-treated bednets were a major breakthrough in the control of malaria. However, bednets do not get washed so frequently. For insecticide-treated clothing to be a viable public health intervention it should withstand regular washing. If the rapid washing-out of permethrin can be overcome by novel technological approaches, insecticide-treated clothes would deserve to be re-evaluated as a potentially cost-effective and scalable intervention.
Policy impact:
A very recent study on laundering resistance of five commercially available, factory-treated permethrin-impregnated fabrics also found that permethrin content fell by 58.1 to 98.5 % after 100 defined machine launderings, with InsectShield showing the fastest loss. InsectShield itself claims efficacy for up to 70 washes. Currently InsectShield and other companies that produce insecticide treated clothing are advertising their products in the context of protective measures against Zika. There is an urgent need for a standardised testing and licensing procedure for insecticide-impregnated commercial clothing to avoid misleading information.
Strategic impact:
Despite the urgency of the current Zika outbreak associated with serious pregnancy outcomes, we should not rush into recommending factory-impregnated clothing to pregnant women in Zika affected areas, until standardised testing and licensing procedures for insecticide-treated materials are implemented, with defined cut-off values for initial maximum and post-laundering minimum concentrations of permethrin as well as data on toxicity, homogeneity on fabrics, residual activity, and laundering resistance. Failing to do this could generate a dangerous sense of security among Zika-exposed pregnant women using impregnated clothing, since the wearer has no means of judging insecticidal efficacy. Given the increasingly epidemic proportions of Aedes-transmitted viral infections, we hope that the findings from this trial will provide strong impetus to fund research to develop appropriate and safe technologies for long-lasting insecticide-treated clothing materials that can be used for school uniforms, work place uniforms and maternity clothing alike.
The rapid wash-out effect of permethrin-impregnated uniforms have also motivated the Ministry of Science, Thailand, together with Mahidol University, to develop alternative approaches. School vests (light cotton covers over the school uniforms) could potentially also be impregnated and would have the following advantages over school uniforms: they do not get washed so frequently, they are not directly on the skin (to avoid any direct skin contact with permethrin), and there would be only one type (whereas for school uniforms, we found that there were 4 types that all needed to be impregnated: regular uniforms, sport uniforms, scout and cultural uniforms). We added acceptability studies for such vests and found the acceptability to be surprisingly high. We also added semi-field trials to evaluate the repellency effect of impregnated vests. This work investigated the ability for insecticide treated clothing to provide a "protective" or “diversion” effect to individuals not wearing the protective clothing. The repellent effect of the treated vests was low but mortality of the mosquitoes which came into contact with the treated vests was high. The use of a compound with better repellent properties may increase the protection provide to the individual as well as provide a protective effect to others in close proximity.
Another spin-off from this project was to investigate further on technologies that would overcome the rapid-wash out effect. We are pleased that our application under Horizon 2020 was successful—this is a true continuation and expansion of what we did in DengueTools to develop wearable protective strategies. One idea is to develop novel detergents containing repellents that can be used during laundry to allow active repellents to be applied to every day clothing during each wash for protection against Aedes mosquitoes. Furthermore, this project will be expanded to investigate new wash-resistant technologies, including novel silica-shell, polymer fibres and microencapsulated formulations to determine whether repellent active ingredients can be retained in fabrics for multiple washes.
Socio-economic relevance
At an average dengue incidence rate of 5.8% per year in school-aged children in Thailand, we calculated that any intervention would be cost-effective (ICER≤$16,440) if the intervention cost per child was $5.3 or less and the intervention effectiveness was 50% or higher. In fact, intervention was cost saving (ICER<0) in all scenarios in which the intervention cost per child was $2.9 or less per year and the intervention effectiveness was 50% or higher. The results suggested that the impregnated school uniform intervention would be of no interest to Thai policy makers when the intervention cost per child was $10.6 or higher per year regardless of intervention effectiveness (ICER>$16,440). Our results present the potential economic value of the use of insecticide-treated uniforms for prevention of dengue in schoolchildren in a typical dengue endemic setting.
Exploitation
With the extension of DengueTools, we investigated compounds beyond permethrin and we found a number of candidate repellents which can be incorporated into fabrics to provide protection from mosquito bites with protection of up to 45%. The London school also successfully approached commercial companies with these compounds and have demonstrated that such compounds can be successfully incorporated into fabrics and garments.
Research area 3:
Here we explored the risk of geographic spread of dengue and the risk of introduction of dengue virus into Europe. We took into account mobility data and climate change, analysed the Madeira outbreak, and studied various parameters related to Aedes albopictus in Europe with a particular focus on vector control measures for Aedes albopictus in temperate France.
Scientific impact:
We documented a convergence of various factors that put Europe increasingly at risk of dengue introduction AND establishment. We found an increasing trend of dengue virus infections imported into Europe via air passengers. The observed increasing annual trends of dengue importation and the consistent peaks in late summer underpin the urgency in determining the threshold levels for the vector and infected human populations that could facilitate novel autochthonous transmission of dengue in Europe. We calculated temperature-dependent vectorial capacity (VC) for Europe, highlighting 10 European cities and three non-European reference cities. Compared with the tropics, Europe shows pronounced seasonality and geographical heterogeneity. Although low, VC during summer is currently sufficient for dengue outbreaks in Southern Europe to commence-if sufficient vector populations (either Ae. aegypti and Ae. albopictus) were active and virus were introduced. Under various climate change scenarios, the seasonal peak and time window for dengue epidemic potential increases during the 21st century. Our study mapped dengue epidemic potential in Europe and identified seasonal time windows when major cities are most conducive for dengue transmission from 1901 to 2099. Our findings illustrate, that besides vector control, mitigating greenhouse gas emissions crucially reduces the future epidemic potential of dengue in Europe. A spin-off of these models was to remodel the risk for Zika introduction and establishment.
We developed a novel importation index, and a de-novo mathematical model, the first of its kind, to estimate the risk of importation and exportation of dengue. In the absence of data, mathematical models are important to estimate the extent of geographic spread, and the risk of importation. Such models also help the travel medicine provider give better evidence-based advice for travellers to dengue-endemic countries, and we calculated the risk for dengue in travellers to Thailand, as well as attack rates in European travellers to dengue endemic countries—such data are important for future dengue vaccine introduction, also in the travel medicine context. The scientific impact of these models went beyond dengue, and we applied them for modeling the spread of poliomyelitis in travellers and for the risk of dengue in visitors to the FIFA World Cup.
Political Impact
Our mathematical models that we developed for dengue were sought after during the Zika outbreak. We were consulted to model the risk of Zika infections in visitors to the Summer Olympics in Rio de Janeiro in August 2016, as many demanded the cancellation or postponement of these Games. A Wilder-Smith was invited as Advisor to the WHO IHR Emergency Committee for Zika led by David Heymann to advise exactly on this issue. For the WHO IHR Emergency Committee on Polio, A Wilder-Smith was also invited to be member and shared the results on polio exportation risk using the models developed under DengueTools.
Strategic and policy impact:
For dengue endemic countries we explored different drivers to tease out the main ones. We found that urbanization with population growth and increased population densities were the main drivers for dengue over the past 40 years. Our findings have significant implications for predicting future trends of the dengue epidemics given the rapid urbanization with population growth in many dengue endemic countries. It is time for policy-makers and the scientific community alike to pay more attention to the negative impact of urbanization and urban climate on diseases such as dengue.
Scientific and policy impact of our studies on Aedes albopictus in Europe
Aedes albopictus is the main vector for dengue in Europe. It differs from Ae. aegypti (considered the most important urban vector of dengue) in that it is primarily exophilic; the association with vegetation cover is an important factor in the efficacy of space sprays for adult control. Control measures taken for Aedes aegypti in tropical countries may hence not necessarily be the same as for Aedes albopictus in temperate countries. Aedes albopictus is an invasive species that is present—often abundant—in 27 European countries from Spain to Romania and is spreading rapidly northwards. Given its cold-hardiness and marked winter diapause, it could conceivably become established as far north as Scandinavia.
In our field studies, in contrast to previous evaluations of space-sprays—nearly all of which have relied on the mortality of caged mosquitoes—we assessed the impact of such treatments by monitoring the wild mosquito population on a daily (24-hour) basis. In four large-scale trials in a residential area of Nice we did not detect any change in the oviposition rate, nor the catch of adult female mosquitoes, nor any change in the parous rate. We believe that this lack of efficacy is due to lack of interaction between the target mosquitoes and the aerosol. By contrast, we observed a >90% reduction of oviposition rate and adult catch in small-scale treatments with hand-held thermal fog. We conclude that in the event of autochthonous transmission, thermal fogging may be the control method of choice despite being highly labor-intensive. This is an absolutely important finding with immediate implications for environmental agencies and policy makers.
We had observed that male Ae. albopictus were significantly more susceptible to deltamethrin than females, both by the standard WHO tube test and by topical application. When dosages were corrected for weight of the insects the difference was eliminated, i.e. the difference in susceptibility was entirely due to the smaller size of the males.
Transmission studies: We found that titres of DENV in saliva compatible with high transmission rates occurred in all strains at 28°C but none at 20°C. By contrast, infective titres
of chikungunya virus (a virus that caused its first outbreak in Europe, in Italy, in 2008) was much higher. Mean temperatures of 20°C and above are normal for several months in much of Europe. We conclude that if temperature is the key environmental factor limiting transmission, then autochthonous chikungunya virus transmission, but not so much dengue virus, is feasible in much of Europe.
We tested several different paints marketed as slow-release formulations for residual application. An organophosphate-based formulation proved the most effective. Paint applied directly to cement had poor persistence but when the surface was pre-treated with a primer there was still 100% kill after 2 years. These results can be exploited for further commercialization.
Dissemination
Key stakeholders:
Engagement with stakeholders and policy makers started early in the project to ensure the potential for scaling up our recommendations. This was particularly the case for Sri Lanka where the Ministry of Health showed great interest in the laboratory training, the setting up of a separate room for PCR testing, and the sentinel surveillance results as a result of increased PCR testing. It was then also no surprise that the President of Sri Lanka honoured our dissemination in conference in February 2016 with his presence during the inauguration ceremony. We had almost annual meetings with policy makers at the highest level in Sri Lanka including the Director of Medical Services to share about lessons learnt during the set-up of the sentinel sites. The Director of Medical Services assured us that the Sri Lankan Ministry of Health will continue funding the laboratory enhanced surveillance in Sri Lanka beyond the EU DengueTools funding period.
The key stakeholders of the dissemination conference included international organizations such as the World Health Organization (WHO), the Centre for Global Health Research, University of Umeå, Sweden, Partnership for Dengue Control (PDC), France, and the Epidemiology Unit, Ministry of Health Sri Lanka among others. We had several invited speakers from US universities. All industry partners participated in the meeting, in particular Takeda, Sanofi Pasteur and GSK.
Scientific dissemination:
48 scientific publications have been the result so far, with 5 more currently under review, and 6 in preparation for submission.
As a result of DengueTools, we had 15 PhD Students and 7 Msc students.
The scientific dissemination meeting was at the 9th European Congress on Tropical Medicine and International Health (ECTMIH) 6-10 September. The Organizing Committee had invited Prof Wilder-Smith to be the Convener of one 90 minute symposium on dengue, and we invited the other two EU funded dengue consortia (Denfree and IDAMS) to join under the topic of “Dengue Control”. During ECTMIH, we were invited to give 8 oral presentations, and 9 posters. There were several conferences where DengueTools funded research was presented:
No Conference Abbreviated Name Venue Date Oral/Poster presentation
1 Singapore International Conference on Dengue and Emerging Infections - Singapore 21-23 Nov 2012 1 poster WP 2
2 Research Colloquium: Building on Heritage - University of Malaya, Malaysia 21-23 Jan 2013 1 poster WP 2
3 8th European Congress on Tropical Medicine and International Health 2013 ECTMIH Copenhagen, Denmark 10-13 Sept 2013 6 posters & 5 oral WP 2, WP 4, WP 8 & WP 10
4 The 2nd International Society for Neglected Tropical Diseases ISNTD Bites London, UK 15 October 2013 1 poster WP 2
5 62nd Annual Meeting of the American Society of Tropical Medicine and Hygiene ASTMH Washington, DC 13-17 Nov 2013 2 posters & 1 oral WP 2, WP 4 & WP 6
6 MOSTI Commercialization Conference and Exhibition 2014 MCCE Selangor, Malaysia 13-15 August 2014 1 poster WP 2
7 Fifth European Congress of Virology ECV Lyon, France 11-14 Sept 2013 1 poster WP 6
8 Annual Consortium of DengueTools Project - London 01-02 Sept 2014 1 oral WP 7
9 (EDENext), Genes, Ecosystems and risk of infection, congress 2015 GERI Heraklion, Crete, Greece 21-23 April 2015 1 oral
WP 7
10 Impact of Environmental Changes on Infectious Diseases 2015 IECID Sitges, Spain 23-25 Mar 2015 2 posters WP 8
11 International Symposium on Mathematical and Computational Biology BIOMAT Toronto, Canada 04-08 Nov 2013 1 oral WP 8
12 International Forum on Climate Change and Health - Beijing, China 26-28 Nov 2013 1 oral WP 8
13 Dengue: The Way Forward 2013 - Colombo, Sri Lanka 16 Oct 2013 7 oral -
14 Dengue – to stem the tide 2016 - Colombo, Sri Lanka 24-26 Feb 2016 7 oral -
Total Number of oral presentations: 24
Total Number of poster presentations: 15
Collaboration with other two EU funded dengue consortia
We had a planning meeting with the leaders of the other 2 EU funded dengue consortia in London in 2011, followed by a meeting with members of all three consortia on dengue mapping in France. DengueTools organized a working group meeting with all the mathematical modellers from the three EU funded dengue consortia. Furthermore, all three consortia presented their scientific findings at the ECTMIH conference in Basel where also the EU scientific officer attended, organized by DengueTools as convener. At the DengueTools final dissemination event in Sri Lanka, representatives from the other two consortia were invited and shared their work.
List of Websites:
www.denguetools.net
contact: Prof Annelies Wilder-Smith, annelies.wilder-smith@umu.se
Dr. Raman Preet, raman.preet@umu.se
Dr. Karl-Erik Renhorn, karl-erik.renhorn@umu.se
Research area 1: Develop a comprehensive early warning surveillance system that has predictive capability for epidemic dengue using novel tools for laboratory diagnosis and vector monitoring
We successfully set up a laboratory enhanced sentinel surveillance system in Colombo District, Sri Lanka, recruited 5436 febrile patients, documented the high dengue disease burden, and achieved effective technology transfer for PCR and serotyping. We integrated climate variables, socioeconomic factors, and mapping of breeding sites to assess the predictive value. Rainfall and increasing mean temperatures were highly predictive for dengue activity, but the results showed considerable heterogeneity. We found that schools were associated with more breeding sites, and these results were replicated by cluster analyses in Thailand that confirmed clustering of dengue infections around schools and within classrooms. Thus, schools should be targeted for intensified vector control strategies. We documented the economic burden of dengue in Sri Lanka: the annual total cost of dengue control and reported hospitalizations to be USD 3.45 million (US$1.50 per capita) in Colombo district.
We developed primers and optimized RT-Recombinase Polymerase Amplification technology (RPA); our results suggest that RPA is superior over Loop-mediated Amplification Isothermal Amplification. RPA’s diagnostic sensitivity is comparable to qRT-PCR assays, however, it has several advantages over PCR, namely user-friendliness, speed of test performance, affordability, and temperature independence.
Research area 2: Develop novel strategies to prevent dengue infections in children
Effective scalable control strategies to protect children are lacking. Given the day-biting nature of Aedes mosquitoes, we hypothesize that insecticide treated school uniforms may be a target for school based intervention to reduce the incidence of dengue in school children.
We conducted a cluster-randomized controlled trial with 1,811 pupils over a school-term in 10 schools in Thailand. Laboratory confirmed dengue incidence was 3.7 versus 3.3% for intervention versus control groups (no significant difference). WHOPES cone tests showed a 100% knock down and mortality of Aedes aegypti mosquitoes exposed to impregnated clothing at baseline, but this efficacy rapidly declined to below 20% after 20 washes. Entomological assessments showed that the mean number of Aedes aegypti mosquitoes caught inside the classrooms of the intervention schools was significantly reduced in the month following the introduction of the impregnated uniforms (p =0.04). We conclude that if the rapid washing-out of permethrin can be overcome by novel technological approaches, insecticide-treated clothes would deserve to be re-evaluated as a potentially cost-effective and scalable intervention.
Research area 3: Geographic spread and risk of introduction into new areas including Europe
We found that urbanization with increased population densities is the main driver for dengue in endemic countries. Our findings have significant implications for predicting future trends; policy-makers and the scientific community should heed more attention to the negative impact of urbanization on diseases such as dengue. We documented a convergence of various factors that put Europe increasingly at risk of dengue introduction and establishment. By calculating temperature-dependent vectorial capacity for Europe, we highlighted seasonal dengue epidemic potential in ten European cities and predicted. Our field trials showed that thermal fogging may be the control method of choice in Europe despite being highly labor-intensive. De-novo mathematical models were developed to quantify the risk of dengue introduction (and Zika for that matter) into Europe.
Project Context and Objectives:
Dengue is a major international public health concern and one of the most important arthropod-borne diseases. Approximately 2.5 billion people – over 40% of world’s population in over 100 countries - are at risk of dengue virus (DENV) infection. Every year, an estimated 390 million people in more than 100 countries are infected with dengue. Infection with any of its four serotypes of dengue virus may result in a mild nonspecific febrile illness, classic dengue fever, or severe dengue manifested by plasma leakage, hemorrhagic tendencies, shock and possibly death. Dengue often requires hospitalization, thereby challenging already fragile health care systems. The often unpredictable nature of dengue epidemics further compounds the public health impact.
Dengue has increased more than 30 fold in the past 40 years and is being introduced to non-endemic areas such as the United States, Southern Europe and Australia with increasing frequency. Current surveillance systems and control efforts are clearly insufficient to combat dengue in endemic countries and to prevent spread to previously uninfected areas (including Europe). We lack understanding of individual or combined roles of viral, entomological, ecological, and environmental and climate factors that influence dengue transmission dynamics and their respective outbreak predictive capability and the most cost-effective approach for surveillance and early warning systems. For surveillance to effectively provide early warning for epidemic transmission, it must be active, laboratory-based, and comprehensive in its coverage of the spectrum of clinical illness and the factors that influence transmission dynamics. Early diagnostic assays at point-of-care that are affordable and can be used in the field are also missing. Vector surveillance in particular has many shortcomings, in particular the lack of sensitive, reliable and simple field methods for vector surveillance.
Furthermore, children are the most vulnerable group for dengue. Effective control strategies to protect children are lacking, in particular simple, cost-effective and scalable strategies. Given that Aedes mosquitoes mainly bite during the day, at a time when children spend most of their time at schools, we set out to (1) determine whether schools are target sites for infections (WP 1 and WP 4 and 10) and (2) to test our hypothesis that insecticide treated school uniforms may be a target for school-based interventions to reduce the incidence of dengue in school children (WP 4).
Lastly, gaps in understanding the risk of introduction of dengue to non-infected areas, including Europe, hampers effective preventive strategies. We currently have insufficient data on the magnitude and trends of importation and virus evolution over time and by geographic origin. We also only have a poor understanding of the vectorial capacity of Aedes in temperate climates that are needed for predictive models under changing climate conditions. It is important to develop a theoretical framework for dengue emergence in naive areas with a particular focus on Europe.
To address these knowledge gaps, we developed three research areas around surveillance (including diagnostics and predictive models) in endemic countries (research area 1), novel strategies for the prevention of dengue in children (research area 2) and dedicated the third research area to the risk of dengue in Europe (importation via returning travellers, control of Aedes albopictus in Europe, and predictive climate dependent models for the risk of introduction and establishment of dengue in Europe).
The objectives were the following, and these were also published in our “DengueTools Design paper”:
Objectives:
Research area 1: Develop a comprehensive, early warning, surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring
Research area 2: Develop novel strategies to prevent dengue in children
Research area 3: Describe and predict the risk of global spread of dengue in particular the risk of introduction and establishment in Europe in the context of parameters of vectorial capacity, global mobility and climate change.
Specific scientific and technical objectives
The Consortium collaborated on the following specific objectives:
Research area 1: Develop a comprehensive, early warning, surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring
1. Develop and validate novel diagnostic assays for point-of-care use
2. Field-test novel diagnostic assays
3. Develop improved field devices and attractants for vector monitoring
4. Develop novel assays for virus detection and characterisation in Aedes mosquitoes
5. Study entomological and environmental indicators that may influence dengue transmission dynamics
6. Develop geo-spatial modelling and risk maps
7. Evaluate the outbreak predictive capability of climate variables (weather variability) and their operational utility for surveillance
8. Develop a comprehensive, early warning, laboratory-based sentinel disease surveillance system
9. Study viral genomic sequence data and the potential role in causing outbreaks of more severe disease
10. Determine the usefulness of the revised 2009 WHO/TDR case definition for surveillance purposes
11. Describe the costs of novel strategies for dengue surveillance and prevention to their effect on the burden of the disease.
Research area 2: Develop novel strategies to prevent dengue in children
1. Conduct a literature review on the protective efficacy of impregnated clothing against vector-borne diseases
2. Determine the efficacy of impregnated school uniforms on the reduction of dengue incidence in school aged children
3. Determine the protection efficacy of school uniforms impregnated with repellent under different laboratory scenarios
4. Develop a cost-effectiveness framework and model for the school-based preventive intervention of insecticide treated school uniforms
5. Produce policy briefs and engage with policy makers to ensure the scalability of such school intervention programs
Research area 3: Describe and predict the risk of global spread of dengue in particular the risk of introduction and establishment in Europe in the context of parameters of vectorial capacity, global mobility and climate change.
1. Describe the trends of dengue importation into Europe
2. Conduct dengue sequencing and produce phylogenetic trees on imported dengue viruses to Europe
3. Compare clinical manifestations and the usefulness of the 2009 WHO dengue case of dengue in travellers (non-endemic populations) versus endemic populations (Sri Lanka)
4. Explore reasons for the resurgence and geographic spread of dengue
5. Estimate and model dengue virus introductions via travellers
6. Estimate the risk of dengue in travellers to the World Cup in 2014 and to the Summer Olympics in Rio de Janeiro in 2016
7. Model vectorial capacity for Aedes mosquitoes in temperate climate zones and globally under current and future climate scenarios
8. Describe typical breeding sites for Ae. albopictus in Southern France
9. Evaluate the impact of ultra-low volume (ULV) aerosols on Ae. albopictus in Sothern France
10. Determine the susceptibility of Ae. albopictus populations to DENV under different temperature regimes
Project Results:
In the following, we will describe the main findings and foreground by research area, whereby the contributions of all work packages will be addressed within the appropriate research areas, as outlined in our design papers.1,2 The findings are ordered by overarching objectives, and not by work packages. Only publications are cited that are a direct result of our DengueTools collaborative work.
Research area 1: Develop a comprehensive, early warning, surveillance system that has predictive capability for epidemic dengue and benefits from novel tools for laboratory diagnosis and vector monitoring
Obj 1: Develop and validate novel diagnostic assays for point-of-care use
The main objective was to develop a rapid (15 minute) nucleic acid test for dengue virus that can be undertaken in a low resource setting using Recombinase Polymerase Amplification (RPA) in comparison with LAMP methods.
A single-tube reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay with a set of nine primers was developed for the detection of all four DENV serotypes and their different genotypes.3 The clinical applicability of RT-LAMP assay for detection of DENV RNA was assessed in a total of 305 sera of clinically-suspected dengue patients. Acute DENV infection was confirmed in 171 samples (n = 305); 43.3% (74/171) and 46.8% (80/171) of the samples were positive for DENV using RT-LAMP and qRT-PCR, respectively. The combination of RT-LAMP with the dengue IgM and IgG ELISA increased detection of acute DENV infection to 97.7% (167/171), in comparison to only 70.8% (121/171) when dengue IgM and IgG ELISA alone were used. The RT-LAMP assays showed high concordance (κ = 0.939) with the qRT-PCR. The RT-LAMP assay detected up to 10 copies of virus RNA within an hour but 100% reproducibility (12/12) was achieved with 100 copies. There was no cross reactivity of RT-LAMP with other closely related arboviruses. The RT-LAMP assay developed in this study is sensitive, specific and simple to perform. The assay improved the detection of dengue when used in combination with serological methods. We then compared this with a new technique:
A prototype reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed.4 The assay detected DENV RNA in <20 min without the need for thermocycling amplification. The assay enabled the detection of as few as 10 copies of DENV RNA. The designed RT-RPA primers and exo probe detected the DENV genome of at least 12 genotypes of DENV circulating globally without cross-reacting with other arboviruses. We assessed the diagnostic performance of the RT-RPA assay for the detection of DENV RNA in 203 serum samples of patients with clinically suspected dengue. The sera were simultaneously tested for DENV using a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay, quantitative RT-PCR (qRT-PCR), and IgM- and IgG-capture enzyme-linked immunosorbent assays (ELISA). Acute DENV infection was confirmed in 130 samples and 61 of the samples (46.9%) were classified as viremic with qRT-PCR. The RT-RPA assay showed good concordance (κ of ≥0.723) with the RT-LAMP and qRT-PCR assays in detecting the dengue viremic samples. When used in combination with ELISA, both the RT-RPA and RT-LAMP assays increased the detection of acute DENV infection to ≥95.7% (≥45/47) in samples obtained within 5 days of illness. The results from the study suggest that the RT-RPA assay is the most rapid molecular diagnostic tool available for the detection of DENV. The only down-side is that it still needs the Twistdx equipment, which means also it is simple, affordable, user-friendly, independent of outside temperature, for field-use it is only of use if one has purchased the Twistdx diagnostic equipment.
In another study, we compared the usefulness and applicability of NS1 RDT (NS1 Ag Strip) and qRT-PCR tests in complementing the IgM ELISA for dengue diagnosis on single serum specimen (n =375). 5The NS1 Ag Strip and qRT-PCR showed a fair concordance. While the NS1 Ag Strip showed higher positivity than qRT-PCR for acute (97.8% vs. 84.8%) and post-acute samples (94.8% vs. 71.8%) of primary infection, qRT-PCR showed higher positivity for acute (58.1% vs. 48.4%) and post-acute (50.0% vs.41.4%) samples in secondary infection. IgM ELISA showed higher positivity in samples from secondary dengue (74.2–94.8%) than in those from primary dengue (21.7–64.1%). More primary dengue samples showed positive with combined NS1 Ag Strip/IgM ELISA (99.0% vs. 92.8%) whereas more secondary samples showed positive with combined qRT-PCR/IgM ELISA (99.4% vs. 96.2%). Combined NS1 Ag Strip/IgM ELISA is a suitable combination tests for timely and accurate dengue diagnosis on single serum specimen. If complemented with qRT-PCR, combined NS1 Ag Strip/IgM ELISA would improve detection of secondary dengue samples.
Obj 2: Field-test novel diagnostic assays
The diagnostic utility of the RT-RPA assay was assessed surveillance laboratory of the WHO Collaborating Centre for Arbovirus Reference & Research (Dengue/Severe Dengue) at the University of Malaya (UM). The easiness of performing the RT-RPA assay was evaluated among new users using the user usability test. 84.2% (16/19) of the participants performed the assay with only 1 to 5 deviations from the provided written protocol. 88.2% of the test results obtained by the participants were similar to the reference results. The user satisfaction towards the RT-RPA protocol and interpretation of results were 100% and 90%, respectively. The improved RT-RPA is now routinely used for detection of dengue at the WHO Collaborating Center for Arbovirus Research and Reference (Dengue/Severe dengue) at UM. In an analysis of
429 dengue virus IgM negative serum samples, 196 samples tested positive for dengue virus NS1 antigen using the rapid strip test (RDT). By using the RT-RPA assays, 238 samples were tested positive. All the in-house RT-PCR-positive serum samples were also positive by the RT-RPA assay. By using the RT-RPA assay, detection of positive samples among the dengue IgM negative samples increased from 39 (8.7%) to 238 (53%).
Obj 3: Develop improved field devices and attractants for vector monitoring
Novel traps that were developed for Panama were used again in the field in Sri Lanka with less success than in Panama. However, we caught sufficient numbers to feed obj 4
Obj 4: Develop novel assays for virus detection and characterisation in Aedes mosquitoes
We describe a new technique, polymerase chain displacement reaction (PCDR), which uses multiple nested primers in a rapid, capped, one-tube reaction that increases the sensitivity of normal quantitative PCR (qPCR) assays. Sensitivity was increased by approximately 10-fold in a proof-of-principle test on dengue virus sequence. In PCDR, when extension occurs from the outer primer, it displaces the extension strand produced from the inner primer by utilizing a polymerase that has strand displacement activity. This allows a greater than 2-fold increase of amplification product for each amplification cycle and therefore increased sensitivity and speed over conventional PCR. Increased sensitivity in PCDR would be useful in nucleic acid detection for viral diagnostics.6
Obj 5: Study entomological and environmental indicators that may influence dengue transmission dynamics
We did an extensive survey in a sub-district in Sri Lanka to to assess Aedes mosquito breeding sites and the prevention practices of community members in a heavily urbanized part of Colombo. Hence, we did a cross-sectional entomological survey from April to June 2013 in 1469 premises located in a subdistrict of the City of Colombo.8 Types of breeding sites and their infestation with larvae or pupae were recorded. Furthermore, a questionnaire was administered to the occupants of these premises to record current practices of dengue vector control. The surveyed premises consisted of 1341 residential premises and 110 non-residential premises (11 schools, 99 work or public sites), 5 open lands, and 13 non-specified. In these 1469 premises, 15447 potential breeding sites suitable to host larvae of pupae were found; of these sites18.0% contained water. Among the 2775 potential breeding sites that contained water, 452 (16.3%) were positive for larvae and/or pupae. Schools were associated with the proportionally highest number of breeding sites; 85 out of 133 (63.9%) breeding sites were positive for larvae and/or pupae in schools compared with 338 out of 2288 (14.8%) in residential premises. The odds ratio (OR) for schools and work or public sites for being infested with larvae and/or pupae was 2.77 (95% CI 1.58 4.86) when compared to residential premises. Occupants of 80.8% of the residential premises, 54.5% of the schools and 67.7% of the work or public sites reported using preventive measures. The main prevention practices were coverage of containers and elimination of mosquito breeding places. Occupants of residential premises were much more likely to practice preventive measures than were those of non-residential premises (OR 2.23; 1.49 3.36). In conclusion, schools and working sites were associated with the highest numbers of breeding sites and lacked preventive measures for vector control. In addition to pursuing vector control measures at residential level, public health strategies should be expanded in schools and work places.
As we identified schools to be the sites with the highest number of Aedes breeding sites in Sri Lanka, we also investigated whether schools or even classrooms would be at higher risk for dengue incidence. To answer this question, we did a study in schools in Thailand. This manuscript is now under minor revision before final publication: Ratanawong P, Kittayapong P, Olanratmanee, P, Wilder-Smith A, Byass P, Tozan Y, Dambach P, Montenegro Quiñonez CA, Louis VR. Are schools important sites for dengue transmission in semi-rural and semi-urban Thailand? 2016 PLOS One. A cohort of 1,811 students from 10 schools in semi-rural and semi-urban Thailand participated in this study (WP 4). Seroconversion data and location of participants’ residences and schools were recorded to determine spatial patterns of dengue cases. Blood samples were taken to confirm dengue infections in participants. Entomological surveys recorded adult mosquito density and breeding sites of Aedes vectors in schools. Clustering analyses were performed to detect spatial aggregation of dengue cases among participants.
Results: A total of 57 dengue seroconversions were detected among the 1,655 participants who provided paired blood samples. Of the 57 confirmed dengue infections, 23 (40.0%) occurred in students from 6 (6.8%) of the 88 classrooms in 10 schools. Dengue cases did not show significant clustering by residential location in the study area. A total of 66 Aedes aegypti mosquitoes were identified from 278 mosquitoes caught using a portable vacuum aspirator in 88 classrooms. In a follow-up survey of breeding sites one year after the study, 484 out of 2,399 water containers surveyed (20.2%) were identified as active mosquito breeding sites.
Our findings suggest that dengue infections were clustered among schools and among classrooms within schools. The schools studied were found to contain a large number of different types of breeding sites. Aedes vector densities in schools were correlated with dengue infections and breeding sites in those schools. Given that only a small proportion of breeding sites in the schools were subjected to vector control measures (11%), this study emphasizes the urgent need to implement vector control strategies at schools, while maintaining efforts at the household level.
Obj 6 Develop geo-spatial modelling and risk maps
A systematic search was undertaken, using the PubMed, Web of Science, WHOLIS, Centers for Disease Control and Prevention (CDC) and OvidSP databases for published citations.7 A manual search of the titles and abstracts was carried out using predefined criteria, notably the inclusion of dengue cases. Data were extracted for pre-identified variables, including the type of predictors and the type of modeling approach used for risk mapping.
Our review revelealed that a wide variety of both predictors and modeling approaches was used to create dengue risk maps. No specific patterns could be identified in the combination of predictors or models across studies. The most important and commonly used predictors for the category of demographic and socio-economic variables were age, gender, education, housing conditions and level of income. Among environmental variables, precipitation and air temperature were often significant predictors. Remote sensing provided a source of varied land cover data that could act as a proxy for other predictor categories. Descriptive maps showing dengue case hotspots were useful for identifying high-risk areas. Predictive maps based on more complex methodology facilitated advanced data analysis and visualization, but their applicability in public health contexts remains to be established.
Availability of resources, feasibility of acquisition, quality of data, alongside available technical expertise, determines the accuracy of dengue risk maps and their applicability to the field of public health. A large number of unknowns, including effective entomological predictors, genetic diversity of circulating viruses, population serological profile, and human mobility, continue to pose challenges and to limit the ability to produce accurate and effective risk maps, and fail to support the development of early warning systems.7
Obj 7 Evaluate the outbreak predictive capability of climate variables (weather variability) and their operational utility for surveillance
Understanding the drivers of dengue is vital in controlling and preventing the disease spread. This study focused on quantifying the influence of one of the drivers of dengue by estimating the relationship between dengue incidence and weather variability, and describe its consistency over 10 Medical Officer of Health (MOH) divisions of the Kalutara district in Sri Lanka. Weekly weather and dengue data, measured at 10 MOH divisions in Kalutara from 2009 to 2013, were retrieved and analysed. Distributed lag non-linear model and hierarchical-analysis was used to estimate division specific and overall relationships between weather and dengue. We incorporated lags up to 12 weeks and evaluated models based on the Akaike Information Criterion.
More consistent exposure-response patterns between different geographical locations were observed for rainfall, showing increasing relative risk of dengue with increasing rainfall from 50 mm per week. The strongest association with dengue risk centred around 6 to 10 weeks following rainfalls of more than 300 mm per week. After heavy rainfall, we observed a reduction in dengue incidence in the first weeks followed by a strong net increase after 8 weeks. The overall relative risk of dengue associated with temperature increased steadily from lower temperatures towards the mean value of 29.80C starting from a lag of 4 weeks, and increased for temperatures above the mean value with a cumulative growing association with lags up to 12 weeks. We found, however, some considerable heterogeneity in the associations between the different MOH divisions studied (manuscript submitted, under review).
Obj 8 Develop a comprehensive laboratory-based sentinel disease surveillance system
In Sri Lanka, dengue poses a significant socio-economic and disease burden. The geographic spread, incidence and severity of disease has been increasing since the first dengue hemorrhagic fever epidemic occurred in 1989. Periodic epidemics have become progressively larger, peaking with the 2009-2014 epidemic with 28,000 to more than 40,000 cases reported each year (44,456 cases in 2012). During that same period, the disease dramatically expanded to the whole island.
During the EU funded 4 year period a total of 5436 febrile patients were recruited and blood samples were tested; we found that 3122 (57%) were found positive for dengue.
Summary of samples collected with positives for dengue: 2012 – 2015
Year Number of acute samples collected No. positive for dengue (%)
2012 1338 761 (57)
2013 1427 927 (65)
2014 1994 1144 (57)
2015 677 290 (43)
Total 5436 3122 (57)
Historically surveillance was passive, with mandatory dengue notifications based on clinical diagnosis with only limited laboratory confirmation. To obtain more accurate data on the disease burden of dengue, we set up a laboratory-based enhanced sentinel surveillance system in Colombo District. Here we describe the study design and report our findings of enhanced surveillance in the years 2012-2014.9 Three outpatient clinics and three government hospitals in Colombo District that covered most of the Colombo metropolitan area were selected for the sentinel surveillance system. Up to 60 patients per week presenting with an undifferentiated fever were enrolled. Acute blood samples from each patient were tested by dengue specific PCR, NS1 ELISA and IgM ELISA. A sub-set of samples was sent to Duke-NUS Singapore for quality assurance, virus isolation and serotyping. Trained medical research assistants used a standardized case report form to record clinical and epidemiological data. Clinical diagnoses by the clinicians-in-charge were recorded for hospitalized cases. Of 3,127 febrile cases, 43.6% were PCR and/or NS1 positive for dengue. A high proportion of lab confirmed dengue was observed from inpatients (IPD) (53.9%) compared to outpatient (clinics in hospitals and general practice) (7.6%). Dengue hemorrhagic fever (DHF) was diagnosed in 11% of patients at the time of first contact, and the median day of illness at time of presentation to the sentinel sites was 4. Dengue serotype 1 was responsible for 85% of the cases and serotype 4 for 15%. The sensitivity and specificity of the clinicians' presumptive diagnosis of dengue was 84% and 34%, respectively. CONCLUSION: DENV-1, and to a lesser degree DENV-4, infection were responsible for a high proportion of febrile illnesses in Colombo in the years 2012 to 2014. Clinicians' diagnoses were associated with high sensitivity, but laboratory confirmation is required to enhance specificity.
We documented a high proportion of DHF. In Sri Lanka, the 1997 WHO dengue case classification is used routinely by clinicians, hence we relied on the managing clinicians’ diagnosis using the classification they are familiar with rather than the 2009 TDR dengue case classification. Dengue hemorrhagic fever (DHF) was diagnosed in 11% of patients at the time of first contact. For hospitalized cases, 22% were discharged with the diagnosis of DHF (with 5 fatal outcomes due to DHF). A DHF proportion of 22% is unusually high, as most studies report a proportion of 5-10% underscoring that the current epidemic is associated with more severe disease. Patients who had signs and symptoms of DHF already at first encounter to our sentinel system presented much later compared to dengue fever or OFI, which could be one explanation why such cases have a worse outcome. 0.9% of the DHF cases died.
To assess whether costly diagnostic assays are indeed worthwhile in a sentinel surveillance system, we compared the clinicians’ presumptive clinical diagnoses with the laboratory confirmation. The clinicians’ diagnosis for dengue at time of admission had a sensitivity of 84.7% and specificity of 32.5%. The positive predictive value for the clinicians was 82.3% and the negative predictive value 36.4% at time of admission. The sensitivity and specificity was higher at discharge, most likely because the clinicians had by then all laboratory tests at hand and were familiar with the clinical evolution over time, even without knowing the results of our assays. The trained research assistants had a higher sensitivity and specificity compared to the clinicians who saw the patients as part of their daily clinical routine on busy days. The higher sensitivity by the research assistants who were all medically trained can be explained by the fact that they spent more time reviewing all the symptoms and signs as documented by our standardized case report form. This underscores that training does indeed improve sensitivity. However, having said this, the sensitivity for the routine clinicians-in-charge was still high, as Sri Lankan doctors are very familiar with the diagnosis and management of dengue patients. Furthermore, we were able to show that the sensitivity and specificity depends on the day of illness, with the highest sensitivity and specificity later in the course of illness. In other words, if patients presented later (after day 3-4 of illness), sensitivity increased, while specificity remained low. Mild respiratory illnesses and non-specific viral fevers are usually of shorter duration and hence present a large proportion of febrile patients seen in the first 3 days of illness. Patients with DHF were much more often correctly diagnosed as a laboratory confirmed dengue case. This finding is consistent with previous reports from Thailand, where the authors concluded that WHO case definition of DHF demonstrates 62% sensitivity and 92% specificity in identifying dengue illness requiring intervention without the need for laboratory confirmation of dengue virus infection in endemic areas. DHF presents clinically with a very characteristic constellation of clinical symptoms, signs and changes in leukocytes, platelets, and hematocrit; a constellation that is so pathognomonic for DHF that WHO in its 2009 revised dengue case classification does not require laboratory confirmation for severe disease but does require laboratory confirmation for dengue with or without warning signs). Overall, our findings show that the clinicians’ suspicion of dengue was very high as seen in the high sensitivity, but specificity was very low. To enhance specificity it is important to add laboratory confirmation of dengue.
Furthermore, we were able to assess three diagnostic essays (PCR, Dengue IgM ELISA and NS1 ELISA) in a sentinel surveillance setting. For dengue, day of illness determines the choice of diagnostic assay. During the viremic phase (up to day 4-5 of illness), molecular biological or virological approaches such as PCR or NS1 should be employed; after the viremic phase (>5 days), serological assays are indicated, with dengue IgM being the most frequently used assay 10. Our figures confirm the temporal changes in positive assays per day of illness, with PCR and NS1 being positive in the earlier phase, and IgM later. A combination of methods that target different time periods maximizes diagnostic sensitivity. Given the constraints of a large sentinel surveillance as ours, it was programmatically not feasible to take a convalescent serum at 14-21 days after discharge which would have helped in confirming the diagnosis – hence we lack a definitive “gold standard” diagnosis in those patients where we only have a single IgM result. In the absence of convalescent sera, we defined “dengue IgM, NS1 or PCR positive” as laboratory confirmed dengue. As NS1 based assays are increasingly used in endemic countries, we also particularly looked at the issues of NS1 as measured by ELISA. NS1 was positive for more days of illness compared with PCR. Indeed, NS1 can be found in the peripheral blood circulation for up to 9 days from illness onset, but can persist for up to 18 days for some cases. Hence NS1 offers a larger window of opportunity for diagnosis of dengue compared with virus isolation and PCR. Furthermore, the proportion of NS1 positive subjects in the first 5 days of illness was higher than that of PCR. Higher sensitivity of NS1 compared with PCR has been documented in some studies but not in others. It is important to note that we tested NS1 by ELISA and not with the cheaper rapid diagnostic (RDT) kits that are now widely available. Hunsperger et al showed that sensitivity of NS1 by ELISA is higher (60-75%) compared with NS1 RDT (38-71%). Hunsperger’s analysis also showed that NS1 was more sensitive in primary versus secondary infections, an observation that we can confirm with our findings.
Lastly, with the EU funded laboratory-enhanced surveillance project a dedicated government laboratory was set up to perform routine molecular testing for dengue. As a result, the capacity to do PCR and serotyping is now well established. Quality assurance with a subset of samples sent to the Duke-NUS Laboratory “Emerging Infectious Diseases Program” showed a high agreement. At Duke-NUS, we also did virus isolation. Although virus isolation is highly specific, the sensitivity is reported to be only approximately 40%. Virus isolation has the advantage of providing a virus isolate that can be used for further genome sequencing, or virus neutralization and other in vitro studies, but it requires highly trained operators, depends on a short viremia period, thus providing only a narrow window of opportunity from illness onset; in quintessence, it is not a diagnostic approach suitable for developing countries. Cost effectiveness studies are needed to evaluate the need for assays such as PCR on a routine basis in Sri Lanka compared to the more affordable NS1 only; and our sentinel surveillance can potentially serve as a basis for such studies.9
In terms of predictive capability, we did not find any predictive parameters for dengue peaks in terms of serotype, proportion of lab-confirmed dengue out of all febrile cases, except for seasonality. During our project, there were sustained high numbers of dengue notifications throughout the country with some seasonal variation but not inter-annual variations which could explain why we did not identify discriminatory parameters. The causative serotypes DENV 1 and to a lesser extent DENV-4 remained the same throughout, so that we could not identify serotypes/or genotypes associated with higher virulence.
We hence also explored internet-based media coverage to assess the extent of awareness of dengue and perceived severity of an outbreak at a national level as a proxy parameter for early outbreak detection. We compared internet references to dengue in Sri Lana with references to other diseases (malaria and influenza) in Sri Lanka and also compared Internet references to dengue in Sri Lanka with notified cases of dengue in Sri Lanka. We examined Internet-based news media articles on dengue queried from HealthMap for Sri Lanka, for the period January 2007 to November 2015. For comparative purposes, we compared hits on dengue with hits on influenza and malaria. There were 565 hits on dengue between 2007 and 2015, with a rapid rise in 2009 and followed by a rising trend ever since. These hits were highly correlated with the national epidemiological trend of dengue. The volume of digital media coverage of dengue was much higher than of influenza and malaria.
Dengue in Sri Lanka is receiving increasing media attention. Our findings underpin previous claims that digital media reports reflect national epidemiological trends, both in annual trends and inter-annual seasonal variation, thus acting as proxy biosurveillance to provide early warning and situation awareness of emerging infectious diseases.11
Obj 9: Study viral genomic sequence data and the potential role in causing outbreaks of more severe disease
Sri Lanka has experienced periodic dengue outbreaks since the 1960s, but recent epidemics have become progressively larger and associated with more severe disease. The 2012-2013 dengue epidemic in Sri Lanka was driven predominantly by DENV-1 with approximately 20% of dengue cases caused by DENV-4. DENV-4 transmission was first documented in Sri Lanka when it was isolated from a traveler in 1978, but has been comparatively uncommon since dengue surveillance began in in the early 1980s. To better understand the molecular epidemiology of DENV-4 infections in Sri Lanka, we conducted whole genome sequencing on dengue patient samples from two different geographic locations. Our E-gene analysis shows that two major lineages of DENV-4 have been observed in Sri Lanka since surveillance began, with data indicating that the currently circulating strain is a descendent of the Sri Lanka virus from 1978 (U18437). (Manuscript submitted). Phylogenetic analysis indicates that all sequenced DENV-4 strains belong to genotype 1 and are most closely related to DENV-4 viruses previously found in Sri Lanka and those more recently found to be circulating in India and Pakistan. There have been fewer DENV-4 outbreaks globally compared to the other three DENV serotypes and the cause of the unusually high number of DENV-4 cases in the ongoing Sri Lankan outbreak is unknown. One possibility is that genetic changes in the virus have led to an increase in the epidemic potential of this virus. Our analysis also suggest that this virus spread to India and/or Pakistan before re-emerging in Sri Lanka in 2009. Recent studies have shown that sequences outside of the E-gene can have a large impact on pathogenicity and epidemic potential. As such, we would like to emphasize the importance of moving towards the use of full genome sequencing rather than just the E-gene so that these other important regions can be examined as well. Given the recent technologic improvements and rapidly falling costs of sequencing, we feel this is an achievable goal. Combined with good epidemiological data, this knowledge has the potential to transform the current paradigm of retrospective study into one of active prediction and targeted, preventative action.
Obj 10: Determine the usefulness of the revised 2009 WHO/TDR case definition for surveillance purposes
The four serotypes of the dengue virus cause a spectrum of clinical disease ranging from self-limited dengue fever, usually accompanied by headache, myalgia and arthralgias, to more severe dengue associated with plasma leakage, hemorrhagic manifestations, thrombocytopenia, to severe shock and occasionally death. Sri Lanka still uses the old WHO classification with dengue fever/dengue haeemorrhagic fever Grade 1-2, Dengue shock syndrome (=DHF Grade 3-4). In 2009, WHO TDR published a revised dengue case classification consisting of dengue, dengue with warning signs and severe dengue. We found that very few patients presented with severe dengue at the first day of encounter to our sentinel sites, and that most patients that were clinically diagnosed as ‘severe dengue’ corresponded to the clinical diagnosis of Dengue Haemorrhagic Fever Grade 3 and 4. Due to small numbers of severe dengue at time of surveillance, we did not do further analysis. However, we looked into dengue warning signs which is more important at the time of first encounter with the health system as warning signs will determine whether such patients will be admitted or not. For many years, dengue appeared to mainly affect children, but increasingly adults and even elderly are also affected. However, the original dengue case classification and most of the WHO clinical guidelines were developed based on the clinical experiences with dengue in children. Clinical manifestations of dengue may differ in children versus adults, and this may also have implications on clinical management. We hence set out to study similarities and differences in clinical manifestations and clinical management of laboratory confirmed dengue infections in children versus adults.
Warning signs: at the time of first encounter, the following warning signs were present in children aged 2-12: Abdominal pain 38.8%, vomiting 2.5%, abdominal tenderness 31.3%, hepatomegaly 19.53%, clinical fluid accumulation 12.02%, mucosal bleeding 2.0%, petechiae 2.13%. The mean platelet count on the day of encounter with the sentinel system was 114,014.4 and hematocrit 37.99; mean ALT was 125.8 mean AST was 226.51.
For adults, the following warning signs were present: Abdominal pain 19.2%, vomiting 0.73%, abdominal tenderness 18.96%, hepatomegaly 3.17%, clinical fluid accumulation 18.71%, mucosal bleeding 4.64%, and petechiae 1.7%.
These results show that warning signs are clearly different between children aged 2-12 and adults aged 18 and above. The IDAMS study conducted by the other EU funded consortium will show which warning sign or combination thereof has the best prognostic value.
Obj 11: Describe the costs of novel strategies for dengue surveillance and prevention to their effect on the burden of the disease
Documenting the disease burden and the cost thereof to the individual but also to the public is important in particular in view of upcoming dengue vaccines to determine the cost effectiveness of dengue vaccines.12 Dengue is currently listed as a "neglected tropical disease" (NTD). We did a review to consider the criteria for the definition of an NTD, and documented the current research gaps and research activities and the adequacy of funding for dengue research and development (R&D) (2003-2013).13 Major research gaps exist in the area of integrated surveillance and vector control. Hence, although dengue differs from many of the NTDs, it still meets important criteria commonly used for NTDs. The current need for increased R&D spending, shared by dengue and other NTDs, is perhaps the key reason why dengue should continue to be considered an NTD.
The first dengue vaccine was licensed during the time period of DengueTools, and the Scientific Director of DengueTools Annelies Wilder-Smith was involved in many of the policy issues with WHO, the Dengue Vaccine Initiative and the global community.14-16 Yesim Tozan (Partner Heidelberg) explored the most immediate economic considerations of introducing a new dengue vaccine and evaluated the published economic analyses of dengue vaccination. Findings indicate that the current economic evidence base is of limited utility to support country-level decisions on dengue vaccine introduction. There are a handful of published cost-effectiveness studies and no country-specific costing studies to project the full resource requirements of dengue vaccine introduction. Country-level analytical expertise in economic analyses, another gap identified, needs to be strengthened to facilitate evidence-based decision-making on dengue vaccine introduction in endemic countries.12
In Sri Lanka, he Ministry of Health is responsible for controlling dengue and other disease outbreaks and associated health care. The substantial amount of public health staff in dengue control activities year-round and the provision of free medical care to dengue patients at secondary care hospitals place a formidable financial burden on the public health sector. We estimated the public sector costs of dengue control activities and the direct costs of hospitalizations in Colombo, the most heavily urbanized district in Sri Lanka, during the epidemic year of 2012 from the Ministry of Health's perspective.17 The financial costs borne by public health agencies and hospitals were collected using cost extraction tools designed specifically for the study and analysed retrospectively using a combination of activity-based and gross costing approaches. RESULTS: The total cost of dengue control and reported hospitalizations was estimated at US$3.45 million (US$1.50 per capita) in Colombo district in 2012. Personnel costs accounted for the largest shares of the total costs of dengue control activities (79%) and hospitalizations (46%). The results indicated a per capita cost of US$0.42 for dengue control activities. The average costs per hospitalization ranged between US$216-609 for pediatric cases and between US$196-866 for adult cases according to disease severity and treatment setting.
Research area 2: Develop novel tools for the prevention of dengue in school-aged children
Aedes aegypti, the principal vector for dengue transmission, mainly bites during the day. Children spend most of the day at school. Most children in dengue endemic countries wear school uniforms. We hypothesized that permethrin-impregnated school uniforms may reduce dengue infections. Insecticide-treated clothing has been used for many years by the military and in recreational activities as personal protection against bites from a variety of arthropods including ticks, chigger mites, sandflies and mosquitoes. Permethrin is the most commonly used active ingredient, but others, including bifenthrin, deltamethrin, cyfluthrin, DEET (N,N-diethyl-3-methylbenz-amide) and KBR3023, have also been trialled. Permethrin is a pyrethroid-based insecticide registered by the US Environmental Protection Agency (EPA) since 1977 that has been extensively used as insect repellent and insecticide with a documented safety record. Treatment is usually carried out by home or factory dipping. Permethrin can be bound to fabric fibres in clothing via different techniques such as micro-encapsulation and polymer coating. Indeed, field tests to assess the degree of protection from Aedes mosquito bites provided by wearing clothing treated with permethrin showed a reduction in the number of bites by 90%. Studies on the safety and effectiveness of the use of insecticide-treated clothing for pathogens other than Aedes-transmitted viral diseases suggest that it could potentially be a promising intervention for Aedes-transmitted diseases such as Zika, dengue, and chikungunya.
Obj 1: Conduct a literature review on the protective efficacy of impregnated clothing against vector-borne diseases
We reviewed evidence base for the use of insecticide-treated clothing for protection against bites from arthropods and its effect on arthropod-borne pathogen transmission. Although some studies do demonstrate protection against pathogen transmission, there are surprisingly few, and the level of protection provided varies according to the disease and the type of study conducted. For example, insecticide-treated clothing has been reported to give between 0% and 75% protection against malaria and between 0% and 79% protection against leishmaniasis. Studies vary in the type of treatment used, the age group of participants, the geographical location of the study, and the pathogen transmission potential. This makes it difficult to compare and assess intervention trials. Overall, there is substantial evidence that insecticide-treated clothing can provide protection against arthropod bites. Bite protection evidence suggests that insecticide-treated clothing may be useful in the prevention of pathogen transmission, but further investigations are required to accurately demonstrate transmission reduction.18
Obj 2: Determine the efficacy of impregnated school uniforms on the reduction of dengue incidence in school aged children
InsectShield manufactures permethrin-impregnated apparel for recreational and military purposes. InsectShield Repellent Apparel is registered by the US EPA; their approach is a polymer-coating technique which is claimed to withstand up to 70 washings. We tested our hypothesis by setting up a large community based trial to determine the reduction of laboratory-confirmed dengue infections as a result of permethrin-impregnated school uniforms (InsectShield).
We carefully designed a cluster randomized-controlled cross-over trial in Thailand and published the trial design prior to the start of the study.19 That publication also outlines the detailed sample size calculation, rhe rationale for a cross-over design and the detailed methods. The protocol of this school-based trial was reviewed and approved by the Mahidol University Institutional Review Board (MU-IRB 2009/357.1512). This trial was registered with ClinicalTrials.gov number: NCT01563640. We enrolled 10 schools in Thailand. The primary endpoint was laboratory-confirmed dengue infections, and for the secondary endpoints we measured the impregnated uniforms’ 1-hour knock-down and 24 hour mosquito mortality by standardised WHOPES bioassay cone tests at baseline and after repeated washing. Entomological assessments inside classrooms and in outside areas of schools were conducted.
Results: We enrolled 1,811 pupils aged 6-17 from 5 intervention and 5 control schools. Paired serum samples were obtained from 1,655 pupils. In the control schools, 24/641 (3.7%) and in the intervention schools 33/1,014 (3.3%) students had evidence of new dengue infections during one school term (5 months). There was no significant difference in proportions of students having incident dengue infections between the intervention and control schools, with adjustment for clustering by school. WHOPES cone tests showed a 100% knock down and mortality of Aedes aegypti mosquitoes exposed to impregnated clothing at baseline and up to 4 washes, but this efficacy rapidly declined to below 20% after 20 washes. Results of the entomological assessments showed that the mean number of Aedes aegypti mosquitoes caught inside the classrooms of the intervention schools was significantly reduced in the month following the introduction of the impregnated uniforms, compared to those collected in classrooms of the control schools (p =0.04)
Discussion of the results: Given the day-biting behaviour of Aedes mosquitoes, impregnated school uniforms could potentially be a simple novel tool to reduce mosquito-borne diseases and local vector populations. Our results from the WHOPES cone tests at the start of the trial underpin the potential for insecticide-treated uniforms to protect against dengue by reducing the populations of Aedes mosquitoes and hence mosquito-bites: knock-down effect and mortality immediately after impregnation of uniforms with permethrin by the InsectShield proprietary method were close to 100%, consistent with results obtained under laboratory conditions at the London School of Hygiene and Tropical Medicine.20 Furthermore, we documented a significant reduction in Aedes mosquito numbers in the classrooms of the intervention schools in the first month after the start of the trial at the time when the insecticidal activity of impregnated uniforms was still 100%.
However, our cluster- randomised controlled trial in ten Thai schools involving 1,811 children did not show serological evidence of a protective effect over the 5-month study period of one school term. Given the theoretical support for this strategy, we need to carefully examine plausible reasons for the apparent failure to protect in our trial, so that this intervention is not discarded as an ineffective strategy for control against Aedes-transmitted diseases such as dengue, Zika or chikungunya. The main reason for the negative result was the rapidly waning efficacy of InsectShield permethrin-impregnated clothes under field conditions. We chose InsectShield factory-impregnation over hand-dipping with permethrin because InsectShield impregnation (unlike hand-dipping with permethrin) results in odourless, well tolerated apparel—a fact that is important for a double-blind randomised trial where odour could otherwise had given away the allocation group. We had not anticipated rapid washing-out of permethrin prior to the study as InsectShield have consistently claimed that the insecticidal efficacy of their proprietary method of permethrin impregnation withstands up to 70 washes. However, we documented rapid declines in insecticidal activity after the first 4 washes. After 20 washes, the knock-down and mortality effects on mosquitoes were well below 20%. What might be the reasons for such rapid waning of insecticidal activity? Maybe the quality of already used cotton Thai school uniforms was inferior to clothing materials used by InsectShield for commercial purposes. With suboptimal cloth quality, the coating technique might have been less durable? Or maybe the washing conditions of the tropics, drying in the open air and ironing decreased the insecticidal effect more rapidly? However, we believe it was not just the potentially suboptimal cloth material of local Thai school uniforms, as a very recent study on laundering resistance of five commercially available, factory-treated permethrin-impregnated fabrics also found that permethrin content fell by 58.1 to 98.5 % after 100 defined machine launderings, with InsectShield showing the fastest loss. There is an urgent need for a standardised testing and licensing procedure for insecticide-impregnated commercial clothing to avoid misleading information.
We need to consider other potential causes for the lack of efficacy in our trial. Maybe compliance with the uniforms was not high? We conducted an acceptability study: Quantitative and qualitative tools were used in a mixed methods approach.21 Class-clustered randomised samples of school children enrolled in the RCT were selected and their parents completed 321 self-administered questionnaires. Descriptive statistics and logistic regression were used to analyse the quantitative data. Focus group discussions and individual semi-structured interviews were conducted with parents, teachers, and principals. Qualitative data analysis involved content analysis with coding and thematic development.
The community’s knowledge and experience of dengue was substantial. The acceptability of insecticide treated school uniforms (ISUs) was high. Parents (87.3%; 95% CI 82.9-90.8) would allow their child to wear an ISU and 59.9% (95% CI 53.7-65.9) of parents would incur additional costs for an ISU over a normal uniform. This was significantly associated with the total monthly income of a household and the educational level of the respondent. Parents (62.5%; 95% CI 56.6-68.1) indicated they would be willing to recommend ISUs to other parents.21
Although acceptability and compliance with the trial uniforms was high, school uniforms are not worn after school and over weekends. We did some simulation modelling and estimated a reduction of dengue infections by 47% if 60% of all mosquito bites occurred during school hours and 70% of the children wore treated uniforms, assuming that mosquito knock-down and mortality levels remained at baseline (without washing-out effect).22 A reduction of dengue infections by 47% would indeed be a major public health victory.
Because we used paired blood samples at baseline and at the end of the study period (5 months), we are unable to tease out the impact of impregnated clothing on dengue incidence in the first month of wearing the intervention uniforms, at a time when the efficacy in terms of knock-down and mortality effect on mosquitoes was still close to 100%. Nevertheless, the results of entomological assessments showed that the intervention had an impact on the number of Aedes mosquitoes inside intervention schools before insecticidal activity declined. This is an important finding that encourages continued research on the use of insecticide-treated clothing as a potential strategy for dengue prevention in school children.
Long-lasting insecticide-treated bednets were a major breakthrough in the control of malaria. However, bednets do not get washed so frequently. For insecticide-treated clothing to be a viable public health intervention it should withstand regular washing. If the rapid washing-out of permethrin can be overcome by novel technological approaches, insecticide-treated clothes would deserve to be re-evaluated as a potentially cost-effective and scalable intervention.
Obj 3: Determine the protective efficacy of school uniforms impregnated with repellent under different laboratory scenarios
Here we assessed the efficacy and durability of different types of insecticide-treated clothing on laboratory-reared Ae. aegypti.23 Standardised World Health Organisation Pesticide Evaluation Scheme (WHOPES) cone tests and arm-in-cage assays were used to assess knockdown (KD) and mortality of Ae. aegypti tested against factory-treated fabric, home-dipped fabric and microencapsulated fabric. Based on the testing of these three different treatment types, the most protective was selected for further analysis using arm-in cage assays with the effect of washing, ultra-violet light, and ironing investigated using high pressure liquid chromatography.
Results: Efficacy varied between the microencapsulated and factory dipped fabrics in cone testing. Factory-dipped clothing showed the greatest effect on KD (3 min 38.1%; 1 hour 96.5%) and mortality (97.1%) with no significant difference between this and the factory dipped school uniforms. Factory-dipped clothing was therefore selected for further testing. Factory dipped clothing provided 59% (95% CI = 49.2%- 66.9%) reduction in landing and a 100% reduction in biting in arm-in-cage tests. Washing duration and technique had a significant effect, with insecticidal longevity shown to be greater with machine washing (LW50 = 33.4) compared to simulated hand washing (LW50 = 17.6). Ironing significantly reduced permethrin content after 1 week of simulated use, with a 96.7% decrease after 3 months although UV exposure did not reduce permethrin content within clothing significantly after 3 months simulated use.
Conclusion: Permethrin-treated clothing may be a promising intervention in reducing dengue transmission. However, our findings also suggest that clothing may provide only short-term protection due to the effect of washing and ironing, highlighting the need for improved fabric treatment techniques.
In order to understand the findings from our school based trial better, we conducted several lab-based studies at the London School of Hygiene and Tropcial Medicine.
The next study focused on the effect of washing on treated clothing, skin coverage and protection against resistant and susceptible Ae. aegypti using modified WHO arm-in-cage assays.24 Coverage was further assessed using free-flight room tests to investigate the protective efficacy of unwashed factory-dipped permethrin-treated clothing. Clothing was worn as full coverage (long sleeves and trousers) and partial coverage (short sleeves and shorts). Residual permethrin on the skin and its effect on mosquitoes was measured using modified WHO cone assays and quantified using high-pressure liquid chromatography (HPLC) analysis.
Results: In the arm-in-cage assays, unwashed clothing reduced landing by 58.9% (95% CI 49.2-66.9) and biting by 28.5% (95% CI 22.5-34.0) but reduced to 18.5% (95% CI 14.7-22.3) and 11.1% (95% CI 8.5-13.8) respectively after 10 washes. Landing and biting for resistant and susceptible strains was not significantly different (p<0.05). In free-flight room tests, full coverage treated clothing reduced landing by 24.3% (95% CI 17.4-31.7) and biting by 91% (95% CI 82.2-95.9) with partial coverage reducing landing and biting by 26.4% (95% CI 20.3-31.2) and 49.3% (95% CI 42.1-59.1) respectively with coverage type having no significant difference on landing (p<0.05). Residual permethrin was present on the skin in low amounts (0.0041mg/cm2) but still produced a KD of >80% one hour after wearing treated clothing.
Conclusion: Whilst partially covering the body with permethrin-treated clothing provided some protection against biting, wearing treated clothing with long sleeves and trousers provided the highest form of protection. This finding may explain some of the absence of protection that we saw in the Thai trial. After all, school uniforms are short-sleeved tops and shorts. Furthermore, washing treated clothing dramatically reduced protection provided. An encouraging finding was that permethrin-treated clothing could provide protection to individuals from Ae. aegypti that show permethrin resistance. Additionally, it could continue to provide protection even after the clothing has been worn.24
With the extension of DengueTools, The London School of Hygiene and Tropical Medicine under Dr James Cook took on testing a number of different candidate repellent compounds which could be impregnated into clothing, instead of permethrin. These compounds can be used on their own or in combination with the permethrin in treated clothing. The aim was to increase protection provided by the clothing by reducing biting and landing rates. The results have shown a number of candidate repellents which could be incorporated into fabrics to provide protection from mosquito bites with protection of up to 45%. The London school also successfully approached commercial companies with these compound and have shown these compounds can be incorporated into fabrics and garments.
Obj 4: Develop a cost-effectiveness framework and model for the school-based preventive intervention of insecticide treated school uniforms
Schools where children spend most of their day is proposed as an ideal setting to implement preventive strategies against day-biting Aedes mosquitoes. The use of insecticide-treated school uniforms is a promising strategy currently under investigation. Using a decision-analytic model, we evaluated the cost-effectiveness of the use of insecticide-treated school uniforms for prevention of dengue, compared with a "do-nothing" alternative, in schoolchildren from the societal perspective. We explored how the potential economic value of the intervention varied under various scenarios of intervention effectiveness and cost, as well as dengue infection risk in school-aged children, using data specific to Thailand.
Results: At an average dengue incidence rate of 5.8% per year in school-aged children, the intervention was cost-effective (ICER≤$16,440) in a variety of scenarios when the intervention cost per child was $5.3 or less and the intervention effectiveness was 50% or higher. In fact, the intervention was cost saving (ICER<0) in all scenarios in which the intervention cost per child was $2.9 or less per year and the intervention effectiveness was 50% or higher. The results suggested that this intervention would be of no interest to Thai policy makers when the intervention cost per child was $10.6 or higher per year regardless of intervention effectiveness (ICER>$16,440). Our results present the potential economic value of the use of insecticide-treated uniforms for prevention of dengue in schoolchildren in a typical dengue endemic setting and highlight the urgent need for additional research on this intervention.
Obj 5: Produce policy briefs and engage with policy makers to ensure the scalability of such school intervention programs
We produced a policy brief, but as the conclusion was that at this stage insecticide treated uniforms are not yet scalable, we did not meet up with policy makers but instead met up with the Ministry of Science, Bangkok. The pressing next step is to develop wearable repellent clothing that can withstand frequent washing, ironing and exposure to sun. Once such a repellent/insecticide has been developed, a new proof of principle study needs to be conducted, and if the data support a reduction of dengue infections, even if only by 30%, then this would be an approach that would warrant being scaled up to national and international levels as a school-based strategy.
Research area 3: Risk of introduction of dengue into naïve areas including Europe
Obj 1: Describe the trends of dengue importations into Europe
Totally unplanned of course, during the DengueTools project period, there were two dengue outbreaks in previously naïve areas/countries, which gave additional impetus to the research in research area 3: the Madeira outbreak with > 2000 cases in 2012/13; and Japan in the year 2014, both in temperate climates. These outbreaks were also picked up in our sentinel surveillance (WP 6).
Our sentinel surveillance system recruited 242 returning dengue viremic travellers through travel medicine providers from TropNet; these 242 who presented during the viremic phase (eg within 5 days after infection) corresponded to 36% of all returning travellers seen at TropNet sites during that period, underpinning that a significant number of dengue infected travellers are viremic which poses a risk for dengue introduction and autochthonous transmission in European regions where Aedes albopictus is prevalent. The most likely place for dengue virus infection for travellers was Asia, followed by the Americas, then Western Pacific region, and the smallest number from Africa.25
We aimed to determine the attack rate of dengue in Swedish travellers and analyse the trends over time and the geographical variation. 26 METHODS: We obtained the following data from the Swedish Institute for Communicable Disease Control for the y 1995-2010: number of Swedish residents with confirmed dengue, the country and year of infection. We also obtained registers on the Swedish annual air traveller arrivals to dengue endemic areas from the United Nations World Tourist Organization for the time period. We estimated attack rates with 95% confidence intervals (CI). RESULTS: In total, 925 Swedish travellers with confirmed dengue were reported. We found an increasing trend over time for most destinations. The majority of the dengue cases were acquired in Thailand (492 out of 925 travellers; 53%), with an attack rate of 13.6 (95% CI 12.7 14.4) per 100,000 travellers. However, the 2 highest attack rates per 100,000 travellers were found for Sri Lanka (45.3 95% CI 34.3 56.4) and Bangladesh (42.6 95% CI 23.8 61.5).26 CONCLUSIONS: Information on attack rates in travellers is more helpful in guiding travel medicine practitioners than reports of absolute numbers, as the latter reflect travel preferences rather than the true risk. Although the majority of dengue infections in Swedish travellers were acquired in Thailand, the attack rates for dengue in travellers to Sri Lanka and Bangladesh were much higher. These data aid in refining information on the risk of dengue in travellers.
Obj 2: Conduct dengue sequencing and produce phylogenetic trees on imported dengue viruses to Europe
The following findings highlight how travellers can unmask an ongoing outbreak or identify outbreaks in areas with poor surveillance or reporting.
First, we detected circulation of dengue serotype 3 (genotype III) in Cuba in 2013 and 2014, despite the fact that PAHO did not report any dengue during that time period. The last detection of dengue 3 (genotype III) in Cuba was during a big epidemic occurring in 2001-2002.
Second, we identified dengue virus circulating from countries in Africa that hitherto have rarely reported dengue. We isolated a genotype III DENV-3 strain from a traveller returning from Togo and Burkina Faso, a region where circulation of dengue had not been reported. We also identified a dengue virus strain imported during the dengue outbreak in Angola in 2013. Our dengue sequencing analysis points towards an importation of a dengue virus from Brazil. We also show that Portugal and South Africa are most likely at the highest risk of importation of dengue from Angola due to the large number of air passengers between Angola and these countries.27 Furthermore, we isolated DENV from travellers returning from Tanzania during the dengue outbreak affecting Dar es Salaam and neighboring regions in 2014. The isolated virus strains cluster in a lineage different from the strains introduced in Africa in the early 1980s, suggesting recent introduction from Asia.
Third, our sentinel surveillance also identified travellers from the first major dengue outbreak in Europe: the 2012/2013 outbreak in Madeira, Portugal. Dengue sequencing done by our partners in Madrid and Singapore showed that the virus was imported from Venezuela.28,29 Studying IATA data, we found that there were 22,948 air travellers to Madeira in 2012, originating from twenty-nine dengue-endemic countries; 89.6% of these international travellers originated from Venezuela and Brazil.29 We developed an importation index that takes into account both travel volume and the extent of dengue incidence in the country of origin.30 Venezuela and Brazil had by far the highest importation indices compared with all other dengue-endemic countries. The importation index for Venezuela was twice as high as that for Brazil. When taking into account seasonality in the months preceding the onset of the Madeira outbreak, this index was even seven times higher for Venezuela than for Brazil during this time.
Fourth, another country that has not seen a dengue outbreak for more than 70 years was picked up through our sentinel surveillance system: the genotype I (Asian) DENV-1 isolate we isolated from a traveller returning from Japan in September 2014 was linked to a local outbreak with ~160 reported autochthonous cases affecting Tokyo in August-September 2014, despite Japan's temperate climate. We dissected this dengue outbreak based on phylogenetic analysis, travel interconnectivity, and environmental drivers for dengue epidemics. Comparing the available dengue virus 1 (DENV1) E gene sequence from this outbreak with 3,282 unique DENV1 sequences in National Center for Biotechnology Information suggested that the DENV might have been imported from China, Indonesia, Singapore, or Vietnam.31 With travelers arriving into Japan, Guangzhou (China) may have been the source of DENV introduction, given that Guangzhou also reported a large-scale dengue outbreak in 2014. Coinciding with the 2014 outbreak, Tokyo's climate conditions permitted the amplification of Aedes vectors and the annual peak of vectorial capacity. Given suitable vectors and climate conditions in addition to increasing interconnectivity with endemic areas of Asia, Tokyo's 2014 outbreak did not come as a surprise and may foretell more to come. Despite Japan's temperate climate, the increasing travel interconnectivity to Japan, combined with more suitable climate and environmental drivers for dengue epidemics, made such an outbreak possible.31
In conclusion, our data demonstrate that epidemiological and virological data obtained from dengue infected international travellers can add an important additional layer to global dengue surveillance efforts.
Obj 3: Compare clinical manifestations and the usefulness of the 2009 WHO dengue case classification of dengue in travellers (non-endemic populations) versus endemic populations (Sri Lanka)
We were not able to achieve this comparison within the timeframe, but analyses have now started.
Obj 4: Explore reasons for the resurgence and geographic spread of dengue
Dengue has increased exponentially over the past decades, although accurate data remain elusive.32 We did a thorough literature review and identified the following factors as main drivers for dengue resurgence: globalisation with increasing interconnectivity, ecological habitat encroachment, socioeconomic changes, climate change, virus evolution, and urbanization.33
In Singapore, the frequency and magnitude of dengue epidemics have increased significantly over the past 40 years. Singapore has excellent documentation of data over decades, and is probably the best place to untangle which of the drivers as listed above have caused the rapid increase in dengue incidence. We studied the relative contributions of putative drivers for the rise of dengue in Singapore: population growth, climate parameters and international air passenger arrivals from dengue endemic countries, for the time period of 1974 until 2011. We used multivariable Poisson regression models with the following predictors: Annual Population Size; Aedes Premises Index; Mean Annual Temperature; Minimum and Maximum Temperature Recorded in each year; Annual Precipitation and Annual Number of Air Passengers arriving from dengue-endemic South-East Asia to Singapore.34 The relative risk (RR) of the increase in dengue incidence due to population growth over the study period was 42.7 while the climate variables (mean and minimum temperature) together explained an RR of 7.1 (RR defined as risk at the end of the time period relative to the beginning and goodness of fit associated with the model leading to these estimates assessed by pseudo-R2 equal to 0.83). 34 Estimating the extent of the contribution of these individual factors on the increasing dengue incidence, we found that population growth contributed to 86% while the residual 14% was explained by increase in temperature. We found no correlation with incoming air passenger arrivals into Singapore from dengue endemic countries. Our findings have significant implications for predicting future trends of the dengue epidemics given the rapid urbanization with population growth in many dengue endemic countries. It is time for policy-makers and the scientific community alike to pay more attention to the negative impact of urbanization and urban climate on diseases such as dengue.
Obj 5: Estimate and model dengue virus introductions via travellers
We first developed a simple importation index, as described above, with the main purpose to identify the country of origin for importation.30 This index is founded on the notion that the extent of importation depends on the interconnectivity between the exporting and receiving country AND the extent of dengue endemicity in the exporting country. However, this index is crude, does not allow to quantify the risk, and can only rank countries at highest risk. To estimate the risk, the probability of being infected at the time of travel and the duration of viremia also needs to be taken into account, so PhD student Mikkel Quam (Partner UmU) developed a more complex importation model. Given that Italy experienced the first ever outbreak of chikungunya in Europe in 2007, we set out to estimate the extent of dengue virus importations into Italy via air travelers. We found that from 2005 to 2012, more than 7.3 million air passengers departing from 100 dengue-endemic countries arrived in Rome. Our Importation Model, which included air traveler volume, estimated the incidence of dengue infections in the countries of disembarkation, and the probability of infection coinciding with travel accounted for an average of 2,320 (1,621-3,255) imported dengue virus infections per year, of which 572 (381-858) were "apparent" dengue infections and 1,747 (1,240-2,397) "inapparent."35 Between 2005 and 2012, we found an increasing trend of dengue virus infections imported into Rome via air travel, which may pose a potential threat for future emergence of dengue in Italy, given that the reoccurring pattern of peak importations corresponds seasonally with periods of relevant mosquito vector activity.
We then developed an even more complex model: In the first model (disease importation), susceptible individuals travel from their disease-free home country to the endemic country and come back after some weeks. The risk of infection spreading in their home country is then estimated supposing the visitors are submitted to the same force of infection as the local population but do not contribute to it. In the second model (disease exportation), it is calculated the probability that an individual from the endemic (or epidemic) country travels to a disease-free country in the condition of latent infected and eventually introduces the infection there. The input of both models is the force of infection at the visited/source country, assumed known. The models are deterministic, but a preliminary stochastic formulation is presented as an appendix of our publication.36
Using mathematical modelling, we set out to estimate the risk of non-immune persons acquiring dengue when travelling to Thailand. The model is deterministic with stochastic parameters and assumes a Poisson distribution for the mosquitoes' biting rate and a Gamma distribution for the probability of acquiring dengue from an infected mosquito. From the force of infection we calculated the risk of dengue acquisition for travellers to Thailand arriving in a typical year (averaged over a 17-year period) in the high season of transmission. A traveller arriving in the high season of transmission and remaining for 7 days has a risk of acquiring dengue of 0.2% (95% CI 0.16-0.23) whereas the risk for travel of 15 and 30 days' duration is 0.46% (95% CI 0.41-0.50) and 0.81% (95% CI 0.76-0.87) respectively.37 Our data highlight that the risk of non-immune travellers acquiring dengue in Thailand is substantial. The incidence of 0.81% after a 1-month stay is similar to that reported in prospective seroconversion studies in Israeli travellers to Thailand, highlighting that our models are consistent with actual data. Risk estimates based on mathematical modelling offer more detailed information depending on various travel scenarios, and will help the travel medicine provider give better evidence-based advice for travellers to dengue-endemic countries.
We also applied these models that we had originally developed for dengue to polio38,39 and Zika.40,41
Obj 6: Estimate the risk of dengue in travellers to the World Cup in 2014 and to the Summer Olympics in Rio de Janeiro in 2016
We had not planned this objective originally in our application, but felt it would be appropriate to add this objective, given the high topicality of the risk of dengue in both the World Cup in 2014 and now also the Summer Olympics in 2016, both in Brazil. As our modeler is from Sao Paolo University Brazil (Partner FUSP), these research questions were even more pertinent. Brazil hosted the FIFA World Cup, the biggest single-event competition in the world, from June 12-July 13 2014 in 12 cities. This event draws an estimated 600,000 international visitors. We calculated the risk of dengue acquisition to non-immune international travellers to Brazil, depending on the football match schedules, considering locations and dates of such matches for June and July 2014.42 We estimated the average per-capita risk and expected number of dengue cases for each host-city and each game schedule chosen based on reported dengue cases to the Brazilian Ministry of Health for the period between 2010-2013. On the average, the expected number of cases among the 600,000 foreigner tourists during the World Cup is 33, varying from 3-59.43 We published these findings prior to the FIFA World Cup—and interestingly, our predictions were found to be true. Only three dengue cases were reported in visitors to the World Cup.
About 400,000 non-immune foreign tourists are expected to attend the 2016 Olympic games in August this year (after the DengueTools project period). As Brazil is the country with the highest number of dengue cases worldwide, concern about the risk of dengue for travelers is justified. A system of differential equation models the spread of dengue amongst the resident population and a stochastic approximation is used to assess the risk to tourists. Historical reported dengue time series in Rio de Janeiro for the years 2000-2015 is used to find out the time dependent force of infection, which is then used to estimate the potential risks to a large tourist cohort. The worst outbreak of dengue occurred in 2012 and this and the other years in the history of Dengue in Rio are used to discuss potential risks to tourists amongst visitors to the forthcoming Rio Olympics. RESULTS: The individual risk to be infected by dengue is very much dependent on the ratio asymptomatic/symptomatic considered but independently of this the worst month of August in the period studied in terms of dengue transmission, occurred in 2007. CONCLUSIONS: If dengue returns in 2016 with the pattern observed in the worst month of August in history (2007), the expected number of symptomatic and asymptomatic dengue cases among tourists will be 23 and 206 cases, respectively. This worst-case scenario would have an incidence of 5.75 (symptomatic) and 51.5 (asymptomatic) per 100,000 individuals.44
Obj 7: Model vectorial capacity for Aedes mosquitoes in temperate climate zones and globally under current and future climate scenarios
Dengue infections are climate sensitive, so it is important to better understand how changing climate factors affect the potential for geographic spread and future dengue epidemics. Vectorial capacity (VC) describes a vector's propensity to transmit dengue taking into account human, virus, and vector interactions. VC is highly temperature dependent, but most dengue models only take mean temperature values into account. Recent evidence shows that diurnal temperature range (DTR) plays an important role in influencing the behavior of the primary dengue vector Aedes aegypti. In this study, we used relative VC to estimate dengue epidemic potential (DEP) based on the temperature and DTR dependence of the parameters of A. aegypti. We found a strong temperature dependence of DEP; it peaked at a mean temperature of 29.3 degrees C when DTR was 0 degrees C and at 20 degrees C when DTR was 20 degrees C. Increasing average temperatures up to 29 degrees C led to an increased DEP, but temperatures above 29 degrees C reduced DEP. In tropical areas where the mean temperatures are close to 29 degrees C, a small DTR increased DEP while a large DTR reduced it. In cold to temperate or extremely hot climates where the mean temperatures are far from 29 degrees C, increasing DTR was associated with increasing DEP.45 Incorporating these findings using historical and predicted temperature and DTR over a two hundred year period (1901-2099), we found an increasing trend of global DEP in temperate regions. Small increases in DEP were observed over the last 100 years and large increases are expected by the end of this century in temperate Northern Hemisphere regions using climate change projections. These findings illustrate the importance of including DTR when mapping DEP based on VC.
As warming temperatures may increase the geographic spread of vector-borne diseases into temperate areas, we calculated temperature-dependent VC for Europe, highlighting 10 European cities and three non-European reference cities. Compared with the tropics, Europe shows pronounced seasonality and geographical heterogeneity. Although low, VC during summer is currently sufficient for dengue outbreaks in Southern Europe to commence-if sufficient vector populations (either Ae. aegypti and Ae. albopictus) were active and virus were introduced. Under various climate change scenarios, the seasonal peak and time window for dengue epidemic potential increases during the 21st century. Our study maps dengue epidemic potential in Europe and identifies seasonal time windows when major cities are most conducive for dengue transmission from 1901 to 2099. Our findings illustrate, that besides vector control, mitigating greenhouse gas emissions crucially reduces the future epidemic potential of dengue in Europe.45
Obj 8: Describe typical breeding sites for Aedes albopictus in Southern France
Aedes albopictus is generally considered a secondary vector of dengue because it feeds indiscriminately on many species of vertebrate yet humans and primates are the only hosts for the virus. For this reason the species received relatively little attention until its appearance in the United States in 1982. Since then it has spread globally with unprecedented speed: it is now established throughout much of the Americas, at least four countries in Africa and 26 countries in Europe and the Middle East. Nevertheless, the role of Ae. albopictus in dengue transmission remains unresolved; in particular its role in Europe and little attention has been devoted to methods for emergency control. Our field inspections indicated that garden-watering by residents was an important source of breeding sites during the long, dry Mediterranean summer. We also observed that oviposition and adult capture rates were influenced by the degree of vegetation-cover (Spearman r = -0.47 p < 0.05) (manuscript submitted). In this sense, Ae. albopictus differs from Ae. aegypti (considered the most important urban vector of dengue) in that it is primarily exophilic; the association with vegetation cover is an important factor in the efficacy of space sprays for adult control.
Obj 9: Evaluate the impact of ultra-low volume (ULV) aerosols on Ae.albopictus in Southern France
As a pre-requisite to field evaluations of the efficacy of insecticidal spray machines on wild Ae. albopictus, we undertook a study of insecticide susceptibility of males and females. Results demonstrated that the local Ae. albopictus population was highly susceptible to Deltamethrin but that females required a significantly higher dosage than males: DL50 0.00075 (0.00058- 0.00097) and 0.00273 (0.00228- 0.00325). The susceptibility of the local strains of Ae. albopictus to deltamethrin is a welcome finding; a high level of DDT/pyrethroid resistance is widespread in populations of Ae. aegypti worldwide.
We anticipated that Ae. albopictus might be more susceptible to ULV treatments than Ae. aegypti because it is largely exophilic, whereas the indoor resting activity of Ae. aegypti protects it from the aerosol. We used infusion-baited ovitraps and BG-sentinel adult traps to monitor the population for at least four days before treatment and at least five afterwards. During the 2013 season we completed three field trials. In two applications of ULV-deltamethrin there was limited impact on female mosquitoes captures in all age cohorts, as well as on the rate of oviposition. On the other hand, males appeared much more vulnerable; they were virtually eliminated. In 2014 we ran two more trials of the same treatment in a second treatment area, including one in which we made two consecutive applications, with similar results: males were far more vulnerable than females. (Manuscript submitted)
We decided to mount small-scale evaluations of a second application method: a hand-held thermal fogger (Pulsfog®). Results were much more encouraging: ca. 90% reduction of both males and females. From these observations we conclude that degree of cover is a critical factor in the efficacy of the two methods: foliage and other degrees of shelter reduce the probability that the aerosol particles will encounter their target; in this context, males may be more vulnerable because they rest at more exposed sites and may be more prone to flight in their quest for females.
As an alternative to ULV treatments we made an exploratory study of the efficacy of 5 residual formulations of Inesfly®, a resinous paint that incorporates micro-encapsulated insecticide granules. Persistence of the most efficient product was estimated at T0, T12 months and T24 months. Delayed 24-hour mortality at T0 was 100% (p < 10-5) for both surfaces porous and non-porous. Twelve months later (T12 m) OP-based paint applied on cement with one layer showed a decrease in the lethal effect on females Ae. albopictus. Delayed 24-hour mortality at T24 m was 100% plastic surfaces and cement with an under-layer of control paint application, however cement plates treated without the under-layer control paint became completely unlethal (0%/ 24h) (manuscript in preparation).
Obj 10: Determine the susceptibility of Aedes albopictus populations to DENV under different temperature regimes (in comparison to chikungunya virus)
At 200C, our data suggest a high rate of transmission of chikungunya at low temperatures, and that, on viral criteria, epidemic transmission at low temperatures is less likely for DENV.
Additional objectives for Zika in response to the public health emergency
1) Develop RPA for Zika virus
Real time Quantitative Reverse Transcriptase Polymerase Chain (qRT-PCR) Reaction Assay
A real time qRT-PCR assay was developed and used to quantify the Zika virus RNA template copy number needed for the optimization of the Zika virus RT-RPA primers.
RPA primer optimization
A series of RPA primers and fluorescence probes specific to the Zika virus genome were designed. From the list of RPA primers and fluorescence probes, optimal pairs were chosen. The optimal primer pairs detected the ZIKV RNA up to 1000 genome copies. Subsequent cross-reactivity assessments performed using viral RNA for DENV (DENV-1, DENV-2, DENV-3, and DENV-4), chikungunya virus (CHIKV) and Getah virus (GETV) found no cross-reacting amplification. There was no cross-reactivity between the primers/probes to the six arboviruses tested, hence, the RT-RPA assay was specific for ZIKV RNA
2) Determine the Zika incidence in febrile patients recruited from the laboratory enhanced sentinel study in Sri Lanka
Of 204 samples obtained from patients with an undifferentiated fever in 2014/2015 in Sri Lanka, none were found to be Zika virus positive.
3) Determine the risk of introduction and establishment of Zika virus in Europe based on vectorial capacity of Aedes albopictus in Europe and interconnectivity
The explosive Zika virus epidemic in the Americas is amplifying spread of this emerging pathogen into Europe.46 Given the interconnectivity of Brazil with the rest of the world, Zika virus (ZIKV) infections have the potential to spread rapidly around the world via viremic travellers. The extent of spread depends on the travel volume and the endemicity in the exporting country. In the absence of reliable surveillance data, we did mathematical modelling to estimate the number of importations of ZIKV from Brazil into Europe. DESIGN: We applied a previously developed mathematical model on importations of dengue to estimate the number of ZIKV importations into Europe, based on the travel volume, the probability of being infected at the time of travel, the population size of Brazil, and the estimated incidence of ZIKV infections. RESULTS: Our model estimated between 508 and 1,778 imported infections into Europe in 2016, of which we would expect between 116 and 355 symptomatic Zika infections; with the highest number of importations being into France, Portugal and Italy. CONCLUSIONS: Our model identified high-risk countries in Europe. Such data can assist policymakers and public health professionals in estimating the extent of importations in order to prepare for the scale up of laboratory diagnostic assays and estimate the occurrence of Guillain-Barre Syndrome, potential sexual transmission, and infants with congenital ZIKV syndrome.41 While Aedes albopictus has proven to be a vector for the transmission of dengue and chikungunya viruses in Europe, there is growing experimental and ecological evidence to suggest that it may also be competent for Zika virus. We analyzed and overlaid the monthly flows of airline travellers arriving into European cities from Zika affected areas across the Americas, the predicted monthly estimates of the basic reproduction number of Zika virus in areas where Aedes mosquito populations reside in Europe (Aedes aegypti in Madeira, Portugal and Ae. albopictus in continental Europe), and human populations living within areas where mosquito-borne transmission of Zika virus may be possible. We highlight specific geographic areas and timing of risk for Zika virus introduction and possible spread within Europe to inform the efficient use of human disease surveillance, vector surveillance and control, and public education resources.47
4) Determining the risk of Zika infection during the Olympics 2016 in Brazil
We modelled the risk of acquiring Zika infections in visitors and athletes to the Summer Olympics in Rio de Janeiro in August 2016 and estimated only 9 in 1,000,000, about 15 times lower than that for dengue.40,48
Literature list of DengueTools publications, all referenced above
1. Wilder-Smith A, Renhorn KE, Tissera H, et al. DengueTools: innovative tools and strategies for the surveillance and control of dengue. Global health action 2012; 5.
2. Jaenisch T, Idams, Sakuntabhai A, Denfree, Wilder-Smith A, DengueTools. Dengue research funded by the European commission-scientific strategies of three European dengue research consortia. PLoS Negl Trop Dis 2013; 7(12): e2320.
3. Teoh BT, Sam SS, Tan KK, et al. Detection of dengue viruses using reverse transcription-loop-mediated isothermal amplification. BMC Infect Dis 2013; 13: 387.
4. Teoh BT, Sam SS, Tan KK, et al. Early detection of dengue virus by use of reverse transcription-recombinase polymerase amplification. J Clin Microbiol 2015; 53(3): 830-7.
5. Teoh BT, Sam SS, Tan KK, et al. The Use of NS1 Rapid Diagnostic Test and qRT-PCR to Complement IgM ELISA for Improved Dengue Diagnosis from Single Specimen. Sci Rep 2016; 6: 27663.
6. Harris CL, Sanchez-Vargas IJ, Olson KE, Alphey L, Fu G. Polymerase chain displacement reaction. Biotechniques 2013; 54(2): 93-7.
7. Louis VR, Phalkey R, Horstick O, et al. Modeling tools for dengue risk mapping - a systematic review. Int J Health Geogr 2014; 13: 50.
8. Louis VR, Montenegro Quinonez CA, Kusumawathie P, et al. Characteristics of and factors associated with dengue vector breeding sites in the City of Colombo, Sri Lanka. Pathogens and global health 2016; 110(2): 79-86.
9. Tissera H, Amarasinghe A, Gunasena S, et al. Laboratory-Enhanced Dengue Sentinel Surveillance in Colombo District, Sri Lanka: 2012-2014. PLoS Negl Trop Dis 2016; 10(2): e0004477.
10. Tang KF, Ooi EE. Diagnosis of dengue: an update. Expert Rev Anti Infect Ther 2012; 10(8): 895-907.
11. Wilder-Smith A, Cohn E, Lloyd DC, Tozan Y, Brownstein JS. Internet-based media coverage on dengue in Sri Lanka between 2007 and 2015. Glob Health Action 2016; 9: 31620.
12. Tozan Y. Current issues in the economics of vaccination against dengue. Expert Rev Vaccines 2016; 15(4): 519-28.
13. Horstick O, Tozan Y, Wilder-Smith A. Reviewing Dengue: Still a Neglected Tropical Disease? PLoS Negl Trop Dis 2015; 9(4): e0003632.
14. Wilder-Smith A. Dengue vaccines: dawning at last? Lancet 2014; 384(9951): 1327-9.
15. Wilder-Smith A, Gubler DJ. PUBLIC HEALTH. Dengue vaccines at a crossroad. Science 2015; 350(6261): 626-7.
16. Wilder-Smith A, Massad E. Age specific differences in efficacy and safety for the CYD-tetravalent dengue vaccine. Expert Rev Vaccines 2016.
17. Thalagala N, Tissera H, Palihawadana P, et al. Costs of Dengue Control Activities and Hospitalizations in the Public Health Sector during an Epidemic Year in Urban Sri Lanka. PLoS Negl Trop Dis 2016; 10(2): e0004466.
18. Banks SD, Murray N, Wilder-Smith A, Logan JG. Insecticide-treated clothes for the control of vector-borne diseases: a review on effectiveness and safety. Med Vet Entomol 2014; 28 Suppl 1: 14-25.
19. Wilder-Smith A, Byass P, Olanratmanee P, et al. The impact of insecticide-treated school uniforms on dengue infections in school-aged children: study protocol for a randomised controlled trial in Thailand. Trials 2012; 13: 212.
20. Banks S, Orsborne J, Wilder-Smith A, Logan J. Permethrin-treated clothing as protection against the dengue vector, Aedes aegypti: extent and duration of protection. PLoS NTD 2015; in print.
21. Murray N, Jansarikij S, Olanratmanee P, et al. Acceptability of impregnated school uniforms for dengue control in Thailand: a mixed methods approach. Glob Health Action 2014; 7: 24887.
22. Massad E, Amaku M, Coutinho FA, Kittayapong P, Wilder-Smith A. Theoretical impact of insecticide-impregnated school uniforms on dengue incidence in Thai children. Glob Health Action 2013; 6: 20473.
23. DeRaedt Banks S, Orsborne J, Gezan SA, et al. Permethrin-Treated Clothing as Protection against the Dengue Vector, Aedes aegypti: Extent and Duration of Protection. PLoS Negl Trop Dis 2015; 9(10): e0004109.
24. Orsborne J, DeRaedt Banks S, Hendy A, et al. Personal Protection of Permethrin-Treated Clothing against Aedes aegypti, the Vector of Dengue and Zika Virus, in the Laboratory. PLoS One 2016; 11(5): e0152805.
25. Neumayr AM, J. Schunk, M. Genton, B. Wilder-Smith, A. Hatz, C. Franco L. Sentinel surveillance of imported dengue via travelers to Europe 2012 - 2014: TropNet
data from the DengueTools Research Initiative. Euro Surveill 2016; in print.
26. Rocklov J, Lohr W, Hjertqvist M, Wilder-Smith A. Attack rates of dengue fever in Swedish travellers. Scand J Infect Dis 2014; 46(6): 412-7.
27. Sessions OM, Khan K, Hou Y, et al. Exploring the origin and potential for spread of the 2013 dengue outbreak in Luanda, Angola. Global health action 2013; 6: 21822.
28. Franco L, Pagan I, Serre Del Cor N, et al. Molecular epidemiology suggests Venezuela as the origin of the dengue outbreak in Madeira, Portugal in 2012-2013. Clin Microbiol Infect 2015; 21(7): 713 e5-8.
29. Wilder-Smith A, Quam M, Sessions O, et al. The 2012 dengue outbreak in Madeira: exploring the origins. Euro Surveill 2014; 19(8): 20718.
30. Quam MB, Wilder-Smith A. Importation index of dengue to determine the most probable origin of importation. J Travel Med 2015; 22(1): 72.
31. Quam MB, Sessions O, Kamaraj US, Rocklov J, Wilder-Smith A. Dissecting Japan's Dengue Outbreak in 2014. Am J Trop Med Hyg 2016; 94(2): 409-12.
32. Wilder-Smith A, Byass P. The elusive global burden of dengue. Lancet Infect Dis 2016.
33. Murray NE, Quam MB, Wilder-Smith A. Epidemiology of dengue: past, present and future prospects. Clin Epidemiol 2013; 5: 299-309.
34. Struchiner CJ, Rocklov J, Wilder-Smith A, Massad E. Increasing Dengue Incidence in Singapore over the Past 40 Years: Population Growth, Climate and Mobility. PLoS One 2015; 10(8): e0136286.
35. Quam MB, Khan K, Sears J, Hu W, Rocklov J, Wilder-Smith A. Estimating Air Travel-Associated Importations of Dengue Virus Into Italy. J Travel Med 2015.
36. Lopez LF, Amaku M, Coutinho FA, et al. Modeling Importations and Exportations of Infectious Diseases via Travelers. Bull Math Biol 2016; 78(2): 185-209.
37. Massad E, Rocklov J, Wilder-Smith A. Dengue infections in non-immune travellers to Thailand. Epidemiol Infect 2013; 141(2): 412-7.
38. Quam M, Massad E, Wilder-Smith A. Effects of India's new polio policy on travellers. Lancet 2014; 383(9929): 1632.
39. Wilder-Smith A, Leong WY, Lopez LF, et al. Potential for international spread of wild poliovirus via travelers. BMC Med 2015; 13: 133.
40. Massad E, Coutinho FA, Wilder-Smith A. Is Zika a substantial risk for visitors to the Rio de Janeiro Olympic Games? Lancet 2016.
41. Massad E, Tan SH, Khan K, Wilder-Smith A. Estimated Zika virus importations to Europe by travellers from Brazil. Glob Health Action 2016; 9: 31669.
42. Massad E, Burattini MN, Ximenes R, Amaku M, Wilder-Smith A. Dengue outlook for the World Cup in Brazil. Lancet Infect Dis 2014; 14(7): 552-3.
43. Massad E, Wilder-Smith A, Ximenes R, et al. Risk of symptomatic dengue for foreign visitors to the 2014 FIFA World Cup in Brazil. Mem Inst Oswaldo Cruz 2014; 109(3): 394-7.
44. Ximenes R, Amaku M, Lopez LF, et al. The risk of dengue for non-immune foreign visitors to the 2016 summer olympic games in Rio de Janeiro, Brazil. BMC Infect Dis 2016; 16(1): 186.
45. Liu-Helmersson J, Stenlund H, Wilder-Smith A, Rocklov J. Vectorial capacity of Aedes aegypti: effects of temperature and implications for global dengue epidemic potential. PLoS One 2014; 9(3): e89783.
46. Musso D, Gubler DJ. Zika Virus. Clin Microbiol Rev 2016; 29(3): 487-524.
47. Rocklov JQ, M. Sudre, B., Brady, O. Kraemer, M. Bogoch,.I Liu-Helmersson, J. Wilder-Smith, A. Semenza, J. Khan, K. Assessing Seasonal Risks for the Introduction and Mosquito-borne Spread of Zika Virus
in Europe. EBioMedicine 2016; in print.
48. Burattini MN, Coutinho FA, Lopez LF, et al. Potential exposure to Zika virus for foreign tourists during the 2016 Carnival and Olympic Games in Rio de Janeiro, Brazil. Epidemiol Infect 2016; 144(9): 1904-6.
Potential Impact:
Research area 1: Surveillance in a dengue endemic country
Background:
The dramatic global spread and increased frequency and magnitude of epidemic dengue/dengue hemorrhagic fever (DEN/DHF) in the past 40 years underscores the critical need for more effective surveillance, prevention and control of this disease. Most endemic countries do not have adequate surveillance to monitor the disease, let alone mosquito control or vaccine prevention programmes. Using a well-designed population-based system to monitor the spatial and temporal distribution of dengue in its different clinical expressions is of enormous public health importance for determining the priority areas for intervention as well as to identify variables for triggering outbreak alerts. For surveillance to effectively provide early warning for epidemic transmission, it must be active, laboratory-based, and comprehensive in its coverage of the spectrum of clinical illness and the factors that influence transmission dynamics.
Scientific impact:
We were able to document the significant dengue burden in Colombo District over three years in more than 5000 patients recruited: almost 50% of all patients with an undifferentiated fever had lab confirmed dengue. Furthermore, we assessed three diagnostic essays (PCR, Dengue IgM ELISA and NS1 ELISA) and their utility in a sentinel surveillance setting, plus dengue sequencing. NS1 was more sensitive and remained positive for a longer time period than PCR, an advantage which was however off-set by the finding that NS1 was less sensitive in secondary dengue infections. PCR allows for further serotyping. Clinical acumen was high, in terms of high sensitivity, but associated with low specificity.
We also developed an integrated surveillance that included climate and ecological parameters in additional to behavioural practices in the community and socioeconomic factors. The strongest association with dengue risk centred around 6 to 10 weeks following rainfalls of more than 300 mm per week. The overall relative risk of dengue associated with temperature increased steadily from lower temperatures towards the mean value of 29.80C starting from a lag of 4 weeks, and increased for temperatures above the mean value with a cumulative growing association with lags up to 12 weeks. We found, however, some considerable heterogeneity in the associations highlighting that other factors also play a role in triggering new outbreaks, in particular the introduction of new virus genotypes. Perhaps our most interesting finding was the high awareness of dengue in the population combined with a high proportion of appropriate community based practices that resulted in low vector indices- this was in stark contrast to our findings in public buildings such as construction sites, factories and in particular also schools. Given that schools are the sites where the most vulnerable age group for dengue spends most of the day, these findings need to be translated into new policies on where to prioritize vector control measures and public health education.
Our results suggest that the RT-Recombinase Polymerase Amplification technology (RPA) is superior over LAMP. RPA is a rapid diagnostic method for detection of dengue viruses with diagnostic sensitivity similar or comparable to the reference qRT-PCR assay, however, it has several advantages over PCR, namely user-friendliness, speed of test performance, affordability, independence of outdoor temperatures (of particular importance in tropical countries). However, in comparison to the rapid diagnostic tests such as NS1, RPA – although it can be used as a point of care test—requires an equipment to read the results, the Twistdx equipment. In other words, although the assay itself is very cheap (< 1USD), the equipment still needs to be purchased. The easiness of performing the RT-RPA assay was evaluated among new users with the user usability test and RPA was found to be highly user-friendly.
The performance of the RT-RPA test in actual diagnostic setting was evaluated in the arbovirus surveillance laboratory of the WHO Collaborating Centre for Arbovirus Reference & Research (Dengue/Severe Dengue) at the University of Malaya. The diagnostic utility of the RT-RPA assay was assessed so favorably that the WHO CC for Arbovirus in Malaysia decided to role out RPA for all their screening of samples from blood donors, as RPA is so highly sensitive, rapid and cheap (if the Twistdx reader is in place).
Societal and policy impact:
With the EU funded laboratory-enhanced surveillance project a dedicated government laboratory was set up in Sri Lanka to perform routine molecular testing for dengue. As a result, the capacity to do PCR and serotyping is now well established. Quality assurance with a subset of samples sent to the Duke-NUS Laboratory “Emerging Infectious Diseases Program” showed a high agreement, underpinning successful local capacity building. Technology transfer was achieved. Although costs for PCR are higher than for IgM and NS1, PCR would still need to be done in a randomly selected sub-group to allow for serotyping—a recommendation that we gave to the Ministry of Health. In terms of cost effectiveness, the most appropriate test in a clinical setting is NS1—but for sentinel surveillance sites, the combination of PCR with serotyping remains a priority. Sequencing should only be done in research settings, as the cost is prohibitive, and the predictive value of genotypic changes remains unresolved. Based on our findings we shared our recommendations with the Ministry of Health in Sri Lanka to continue enhancing national reference laboratories throughout the country, transfer technologies to peripheral cities for PCR and serotyping, apply lessons learned through our sentinel surveillance, and implement random selection of febrile patients both from in-and outpatient settings. The sentinel sites in Sri Lanka will continue to be used for laboratory enhanced dengue surveillance even after the funding period of DengueTools, underpinning that we set up a sustainable new system.
The high amount of public health staff in dengue control activities year-round combined with free medical care to dengue patients at secondary care hospitals place a formidable financial burden on the public health sector. We calculated the total cost of dengue control and reported hospitalizations to be USD 3.45 million (US$1.50 per capita) in Colombo district in 2012. This analysis was a first attempt to assess the economic burden of dengue response in the public health sector in Sri Lanka. Country-specific evidence is needed for setting public health priorities and deciding about the deployment of existing or new technologies. Such data will help future cost-effectiveness studies when the time comes for dengue vaccine introduction into Sri Lanka.
Strategic impact:
We did biobanking of more than 5,000 blood samples obtained during the enhanced sentinel surveillance in 6 different sites in Colombo District which has already led to various spin-off projects.
First of all, we secured an additional grant from the National Medical Research Council in Singapore to do further detailed sequencing. This information will help us to understand the potential origins of this virus and, combined with current international travel data, where the virus is most likely to go next. Recently, we have also identified regions in non-protein coding regions of the genome that are responsible for increases in epidemic potential. With full genome sequencing, we will specifically look for these genetic signatures as well as other areas of the genome that may be responsible for this epidemic.
Second, we secured more funding to benefit from a subset of 100 patients with more detailed daily clinical information, using a similar questionnaire as used by DENCO. In this subset, we took daily blood samples for biomarker testing using metabolomics, proteomic and lipidomic approaches in order to identify prognostic markers that indicate development of more severe disease.
Third, the set up of the sentinel sites and the cohort study in Ward 33 (=sub-district of Colombo) has attracted the interest of the industry. Takeda has selected this ward for a large Phase 3 trial for their dengue vaccine, leveraging on the relationships built with the community, the scientific community and the Ministry of Health.
Fourth, the promising results of RT-RPA motivated us to develop primers for Zika diagnostics. RT-RPA for Zika was highly specific, in other words we did not find cross-reactivity between the primers/probes to the six arboviruses tested. These results will lead to further work on optimizing the primers and evaluating the assay against a panel of more flaviviruses, and for the duration to positivity compared to PCR.
Fifth, our stored dengue-negative samples can be used to describe the cause of non-dengue fever.
Research area 2:
Scientific Impact
There is an urgent need for integrated and complementary population-based strategies to protect vulnerable children. Insecticide-treated bednets were a breakthrough in community based, scalable measures to reduce malaria infections that are mainly acquired during dawn and dusk and evening/night hours. Insecticide-treated clothing is an analogue idea that could be used during the day-time for day-time biting Aedes mosquitoes that transmit dengue viruses. Impregnated school uniforms to reduce dengue infections in children would be a simple, safe and cost-effective intervention, that is sustainable through community-based programmes; and our laboratory-based studies confirm that this approach is promising. Knockdown rates and mortality of mosquitoes landing on impregnated clothing was very high. Even partial body coverage (short sleeves, shorts) had an impact. However, our studies also showed rapid waning of initial close to 100% efficacy to below 20% after just 20 washes. UV light and ironing also had deleterious effects on the longevity of the impregnation. The results of entomological assessments showed that the intervention had an impact on the number of Aedes mosquitoes inside intervention schools before insecticidal activity declined. This is an important finding that encourages continued research on the use of insecticide-treated clothing as a potential strategy for dengue prevention in school children.
Long-lasting insecticide-treated bednets were a major breakthrough in the control of malaria. However, bednets do not get washed so frequently. For insecticide-treated clothing to be a viable public health intervention it should withstand regular washing. If the rapid washing-out of permethrin can be overcome by novel technological approaches, insecticide-treated clothes would deserve to be re-evaluated as a potentially cost-effective and scalable intervention.
Policy impact:
A very recent study on laundering resistance of five commercially available, factory-treated permethrin-impregnated fabrics also found that permethrin content fell by 58.1 to 98.5 % after 100 defined machine launderings, with InsectShield showing the fastest loss. InsectShield itself claims efficacy for up to 70 washes. Currently InsectShield and other companies that produce insecticide treated clothing are advertising their products in the context of protective measures against Zika. There is an urgent need for a standardised testing and licensing procedure for insecticide-impregnated commercial clothing to avoid misleading information.
Strategic impact:
Despite the urgency of the current Zika outbreak associated with serious pregnancy outcomes, we should not rush into recommending factory-impregnated clothing to pregnant women in Zika affected areas, until standardised testing and licensing procedures for insecticide-treated materials are implemented, with defined cut-off values for initial maximum and post-laundering minimum concentrations of permethrin as well as data on toxicity, homogeneity on fabrics, residual activity, and laundering resistance. Failing to do this could generate a dangerous sense of security among Zika-exposed pregnant women using impregnated clothing, since the wearer has no means of judging insecticidal efficacy. Given the increasingly epidemic proportions of Aedes-transmitted viral infections, we hope that the findings from this trial will provide strong impetus to fund research to develop appropriate and safe technologies for long-lasting insecticide-treated clothing materials that can be used for school uniforms, work place uniforms and maternity clothing alike.
The rapid wash-out effect of permethrin-impregnated uniforms have also motivated the Ministry of Science, Thailand, together with Mahidol University, to develop alternative approaches. School vests (light cotton covers over the school uniforms) could potentially also be impregnated and would have the following advantages over school uniforms: they do not get washed so frequently, they are not directly on the skin (to avoid any direct skin contact with permethrin), and there would be only one type (whereas for school uniforms, we found that there were 4 types that all needed to be impregnated: regular uniforms, sport uniforms, scout and cultural uniforms). We added acceptability studies for such vests and found the acceptability to be surprisingly high. We also added semi-field trials to evaluate the repellency effect of impregnated vests. This work investigated the ability for insecticide treated clothing to provide a "protective" or “diversion” effect to individuals not wearing the protective clothing. The repellent effect of the treated vests was low but mortality of the mosquitoes which came into contact with the treated vests was high. The use of a compound with better repellent properties may increase the protection provide to the individual as well as provide a protective effect to others in close proximity.
Another spin-off from this project was to investigate further on technologies that would overcome the rapid-wash out effect. We are pleased that our application under Horizon 2020 was successful—this is a true continuation and expansion of what we did in DengueTools to develop wearable protective strategies. One idea is to develop novel detergents containing repellents that can be used during laundry to allow active repellents to be applied to every day clothing during each wash for protection against Aedes mosquitoes. Furthermore, this project will be expanded to investigate new wash-resistant technologies, including novel silica-shell, polymer fibres and microencapsulated formulations to determine whether repellent active ingredients can be retained in fabrics for multiple washes.
Socio-economic relevance
At an average dengue incidence rate of 5.8% per year in school-aged children in Thailand, we calculated that any intervention would be cost-effective (ICER≤$16,440) if the intervention cost per child was $5.3 or less and the intervention effectiveness was 50% or higher. In fact, intervention was cost saving (ICER<0) in all scenarios in which the intervention cost per child was $2.9 or less per year and the intervention effectiveness was 50% or higher. The results suggested that the impregnated school uniform intervention would be of no interest to Thai policy makers when the intervention cost per child was $10.6 or higher per year regardless of intervention effectiveness (ICER>$16,440). Our results present the potential economic value of the use of insecticide-treated uniforms for prevention of dengue in schoolchildren in a typical dengue endemic setting.
Exploitation
With the extension of DengueTools, we investigated compounds beyond permethrin and we found a number of candidate repellents which can be incorporated into fabrics to provide protection from mosquito bites with protection of up to 45%. The London school also successfully approached commercial companies with these compounds and have demonstrated that such compounds can be successfully incorporated into fabrics and garments.
Research area 3:
Here we explored the risk of geographic spread of dengue and the risk of introduction of dengue virus into Europe. We took into account mobility data and climate change, analysed the Madeira outbreak, and studied various parameters related to Aedes albopictus in Europe with a particular focus on vector control measures for Aedes albopictus in temperate France.
Scientific impact:
We documented a convergence of various factors that put Europe increasingly at risk of dengue introduction AND establishment. We found an increasing trend of dengue virus infections imported into Europe via air passengers. The observed increasing annual trends of dengue importation and the consistent peaks in late summer underpin the urgency in determining the threshold levels for the vector and infected human populations that could facilitate novel autochthonous transmission of dengue in Europe. We calculated temperature-dependent vectorial capacity (VC) for Europe, highlighting 10 European cities and three non-European reference cities. Compared with the tropics, Europe shows pronounced seasonality and geographical heterogeneity. Although low, VC during summer is currently sufficient for dengue outbreaks in Southern Europe to commence-if sufficient vector populations (either Ae. aegypti and Ae. albopictus) were active and virus were introduced. Under various climate change scenarios, the seasonal peak and time window for dengue epidemic potential increases during the 21st century. Our study mapped dengue epidemic potential in Europe and identified seasonal time windows when major cities are most conducive for dengue transmission from 1901 to 2099. Our findings illustrate, that besides vector control, mitigating greenhouse gas emissions crucially reduces the future epidemic potential of dengue in Europe. A spin-off of these models was to remodel the risk for Zika introduction and establishment.
We developed a novel importation index, and a de-novo mathematical model, the first of its kind, to estimate the risk of importation and exportation of dengue. In the absence of data, mathematical models are important to estimate the extent of geographic spread, and the risk of importation. Such models also help the travel medicine provider give better evidence-based advice for travellers to dengue-endemic countries, and we calculated the risk for dengue in travellers to Thailand, as well as attack rates in European travellers to dengue endemic countries—such data are important for future dengue vaccine introduction, also in the travel medicine context. The scientific impact of these models went beyond dengue, and we applied them for modeling the spread of poliomyelitis in travellers and for the risk of dengue in visitors to the FIFA World Cup.
Political Impact
Our mathematical models that we developed for dengue were sought after during the Zika outbreak. We were consulted to model the risk of Zika infections in visitors to the Summer Olympics in Rio de Janeiro in August 2016, as many demanded the cancellation or postponement of these Games. A Wilder-Smith was invited as Advisor to the WHO IHR Emergency Committee for Zika led by David Heymann to advise exactly on this issue. For the WHO IHR Emergency Committee on Polio, A Wilder-Smith was also invited to be member and shared the results on polio exportation risk using the models developed under DengueTools.
Strategic and policy impact:
For dengue endemic countries we explored different drivers to tease out the main ones. We found that urbanization with population growth and increased population densities were the main drivers for dengue over the past 40 years. Our findings have significant implications for predicting future trends of the dengue epidemics given the rapid urbanization with population growth in many dengue endemic countries. It is time for policy-makers and the scientific community alike to pay more attention to the negative impact of urbanization and urban climate on diseases such as dengue.
Scientific and policy impact of our studies on Aedes albopictus in Europe
Aedes albopictus is the main vector for dengue in Europe. It differs from Ae. aegypti (considered the most important urban vector of dengue) in that it is primarily exophilic; the association with vegetation cover is an important factor in the efficacy of space sprays for adult control. Control measures taken for Aedes aegypti in tropical countries may hence not necessarily be the same as for Aedes albopictus in temperate countries. Aedes albopictus is an invasive species that is present—often abundant—in 27 European countries from Spain to Romania and is spreading rapidly northwards. Given its cold-hardiness and marked winter diapause, it could conceivably become established as far north as Scandinavia.
In our field studies, in contrast to previous evaluations of space-sprays—nearly all of which have relied on the mortality of caged mosquitoes—we assessed the impact of such treatments by monitoring the wild mosquito population on a daily (24-hour) basis. In four large-scale trials in a residential area of Nice we did not detect any change in the oviposition rate, nor the catch of adult female mosquitoes, nor any change in the parous rate. We believe that this lack of efficacy is due to lack of interaction between the target mosquitoes and the aerosol. By contrast, we observed a >90% reduction of oviposition rate and adult catch in small-scale treatments with hand-held thermal fog. We conclude that in the event of autochthonous transmission, thermal fogging may be the control method of choice despite being highly labor-intensive. This is an absolutely important finding with immediate implications for environmental agencies and policy makers.
We had observed that male Ae. albopictus were significantly more susceptible to deltamethrin than females, both by the standard WHO tube test and by topical application. When dosages were corrected for weight of the insects the difference was eliminated, i.e. the difference in susceptibility was entirely due to the smaller size of the males.
Transmission studies: We found that titres of DENV in saliva compatible with high transmission rates occurred in all strains at 28°C but none at 20°C. By contrast, infective titres
of chikungunya virus (a virus that caused its first outbreak in Europe, in Italy, in 2008) was much higher. Mean temperatures of 20°C and above are normal for several months in much of Europe. We conclude that if temperature is the key environmental factor limiting transmission, then autochthonous chikungunya virus transmission, but not so much dengue virus, is feasible in much of Europe.
We tested several different paints marketed as slow-release formulations for residual application. An organophosphate-based formulation proved the most effective. Paint applied directly to cement had poor persistence but when the surface was pre-treated with a primer there was still 100% kill after 2 years. These results can be exploited for further commercialization.
Dissemination
Key stakeholders:
Engagement with stakeholders and policy makers started early in the project to ensure the potential for scaling up our recommendations. This was particularly the case for Sri Lanka where the Ministry of Health showed great interest in the laboratory training, the setting up of a separate room for PCR testing, and the sentinel surveillance results as a result of increased PCR testing. It was then also no surprise that the President of Sri Lanka honoured our dissemination in conference in February 2016 with his presence during the inauguration ceremony. We had almost annual meetings with policy makers at the highest level in Sri Lanka including the Director of Medical Services to share about lessons learnt during the set-up of the sentinel sites. The Director of Medical Services assured us that the Sri Lankan Ministry of Health will continue funding the laboratory enhanced surveillance in Sri Lanka beyond the EU DengueTools funding period.
The key stakeholders of the dissemination conference included international organizations such as the World Health Organization (WHO), the Centre for Global Health Research, University of Umeå, Sweden, Partnership for Dengue Control (PDC), France, and the Epidemiology Unit, Ministry of Health Sri Lanka among others. We had several invited speakers from US universities. All industry partners participated in the meeting, in particular Takeda, Sanofi Pasteur and GSK.
Scientific dissemination:
48 scientific publications have been the result so far, with 5 more currently under review, and 6 in preparation for submission.
As a result of DengueTools, we had 15 PhD Students and 7 Msc students.
The scientific dissemination meeting was at the 9th European Congress on Tropical Medicine and International Health (ECTMIH) 6-10 September. The Organizing Committee had invited Prof Wilder-Smith to be the Convener of one 90 minute symposium on dengue, and we invited the other two EU funded dengue consortia (Denfree and IDAMS) to join under the topic of “Dengue Control”. During ECTMIH, we were invited to give 8 oral presentations, and 9 posters. There were several conferences where DengueTools funded research was presented:
No Conference Abbreviated Name Venue Date Oral/Poster presentation
1 Singapore International Conference on Dengue and Emerging Infections - Singapore 21-23 Nov 2012 1 poster WP 2
2 Research Colloquium: Building on Heritage - University of Malaya, Malaysia 21-23 Jan 2013 1 poster WP 2
3 8th European Congress on Tropical Medicine and International Health 2013 ECTMIH Copenhagen, Denmark 10-13 Sept 2013 6 posters & 5 oral WP 2, WP 4, WP 8 & WP 10
4 The 2nd International Society for Neglected Tropical Diseases ISNTD Bites London, UK 15 October 2013 1 poster WP 2
5 62nd Annual Meeting of the American Society of Tropical Medicine and Hygiene ASTMH Washington, DC 13-17 Nov 2013 2 posters & 1 oral WP 2, WP 4 & WP 6
6 MOSTI Commercialization Conference and Exhibition 2014 MCCE Selangor, Malaysia 13-15 August 2014 1 poster WP 2
7 Fifth European Congress of Virology ECV Lyon, France 11-14 Sept 2013 1 poster WP 6
8 Annual Consortium of DengueTools Project - London 01-02 Sept 2014 1 oral WP 7
9 (EDENext), Genes, Ecosystems and risk of infection, congress 2015 GERI Heraklion, Crete, Greece 21-23 April 2015 1 oral
WP 7
10 Impact of Environmental Changes on Infectious Diseases 2015 IECID Sitges, Spain 23-25 Mar 2015 2 posters WP 8
11 International Symposium on Mathematical and Computational Biology BIOMAT Toronto, Canada 04-08 Nov 2013 1 oral WP 8
12 International Forum on Climate Change and Health - Beijing, China 26-28 Nov 2013 1 oral WP 8
13 Dengue: The Way Forward 2013 - Colombo, Sri Lanka 16 Oct 2013 7 oral -
14 Dengue – to stem the tide 2016 - Colombo, Sri Lanka 24-26 Feb 2016 7 oral -
Total Number of oral presentations: 24
Total Number of poster presentations: 15
Collaboration with other two EU funded dengue consortia
We had a planning meeting with the leaders of the other 2 EU funded dengue consortia in London in 2011, followed by a meeting with members of all three consortia on dengue mapping in France. DengueTools organized a working group meeting with all the mathematical modellers from the three EU funded dengue consortia. Furthermore, all three consortia presented their scientific findings at the ECTMIH conference in Basel where also the EU scientific officer attended, organized by DengueTools as convener. At the DengueTools final dissemination event in Sri Lanka, representatives from the other two consortia were invited and shared their work.
List of Websites:
www.denguetools.net
contact: Prof Annelies Wilder-Smith, annelies.wilder-smith@umu.se
Dr. Raman Preet, raman.preet@umu.se
Dr. Karl-Erik Renhorn, karl-erik.renhorn@umu.se