Travel surveillance uncovers dengue virus dynamics and introductions in the Caribbean.

Dengue is the most prevalent mosquito-borne viral disease in humans, and cases are continuing to rise globally. In particular, islands in the Caribbean have experienced more frequent outbreaks, and all four dengue virus (DENV) serotypes have been reported in the region, leading to hyperendemicity and increased rates of severe disease. However, there is significant variability regarding virus surveillance and reporting between islands, making it difficult to obtain an accurate understanding of the epidemiological patterns in the Caribbean. To investigate this, we used travel surveillance and genomic epidemiology to reconstruct outbreak dynamics, DENV serotype turnover, and patterns of spread within the region from 2009-2022. We uncovered two recent DENV-3 introductions from Asia, one of which resulted in a large outbreak in Cuba, which was previously under-reported. We also show that while outbreaks can be synchronized between islands, they are often caused by different serotypes. Our study highlights the importance of surveillance of infected travelers to provide a snapshot of local introductions and transmission in areas with limited local surveillance and suggests that the recent DENV-3 introductions may pose a major public health threat in the region.

Design, development, and validation of multi-epitope proteins for serological diagnosis of Zika virus infections and discrimination from dengue virus seropositivity.

Zika virus (ZIKV), an arbovirus from the Flaviviridae family, is the causative agent of Zika fever, a mild and frequent oligosymptomatic disease in humans. Nonetheless, on rare occasions, ZIKV infection can be associated with Guillain-Barré Syndrome (GBS), and severe congenital complications, such as microcephaly. The oligosymptomatic disease, however, presents symptoms that are quite similar to those observed in infections caused by other frequent co-circulating arboviruses, including dengue virus (DENV). Moreover, the antigenic similarity between ZIKV and DENV, and even with other members of the Flaviviridae family, complicates serological testing due to the high cross-reactivity of antibodies. Here, we designed, produced in a prokaryotic expression system, and purified three multiepitope proteins (ZIKV-1, ZIKV-2, and ZIKV-3) for differential diagnosis of Zika. The proteins were evaluated as antigens in ELISA tests for the detection of anti-ZIKV IgG using ZIKV- and DENV-positive human sera. The recombinant proteins were able to bind and detect anti-ZIKV antibodies without cross-reactivity with DENV-positive sera and showed no reactivity with Chikungunya virus (CHIKV)- positive sera. ZIKV-1, ZIKV-2, and ZIKV-3 proteins presented 81.6%, 95%, and 66% sensitivity and 97%, 96%, and 84% specificity, respectively. Our results demonstrate the potential of the designed and expressed antigens in the development of specific diagnostic tests for the detection of IgG antibodies against ZIKV, especially in regions with the circulation of multiple arboviruses.

Spatial analysis of dengue transmission in an endemic city in Brazil reveals high spatial structuring on local dengue transmission dynamics.

In the last decades, dengue has become one of the most widespread mosquito-borne arboviruses in the world, with an increasing incidence in tropical and temperate regions. The mosquito Aedes aegypti is the dengue primary vector and is more abundant in highly urbanized areas. Traditional vector control methods have showing limited efficacy in sustaining mosquito population at low levels to prevent dengue virus outbreaks. Considering disease transmission is not evenly distributed in the territory, one perspective to enhance vector control efficacy relies on identifying the areas that concentrate arbovirus transmission within an endemic city, i.e., the hotspots. Herein, we used a 13-month timescale during the SARS-Cov-2 pandemic and its forced reduction in human mobility and social isolation to investigate the spatiotemporal association between dengue transmission in children and entomological indexes based on adult Ae. aegypti trapping. Dengue cases and the indexes Trap Positive Index (TPI) and Adult Density Index (ADI) varied seasonally, as expected: more than 51% of cases were notified on the first 2 months of the study, and higher infestation was observed in warmer months. The Moran's Eigenvector Maps (MEM) and Generalized Linear Models (GLM) revealed a strong large-scale spatial structuring in the positive dengue cases, with an unexpected negative correlation between dengue transmission and ADI. Overall, the global model and the purely spatial model presented a better fit to data. Our results show high spatial structure and low correlation between entomological and epidemiological data in Foz do Iguaçu dengue transmission dynamics, suggesting the role of human mobility might be overestimated and that other factors not evaluated herein could be playing a significant role in governing dengue transmission.

Dengue virus pathogenesis and host molecular machineries.

Dengue viruses (DENV) are positive-stranded RNA viruses belonging to the Flaviviridae family. DENV is the causative agent of dengue, the most rapidly spreading viral disease transmitted by mosquitoes. Each year, millions of people contract the virus through bites from infected female mosquitoes of the Aedes species. In the majority of individuals, the infection is asymptomatic, and the immune system successfully manages to control virus replication within a few days. Symptomatic individuals may present with a mild fever (Dengue fever or DF) that may or may not progress to a more critical disease termed Dengue hemorrhagic fever (DHF) or the fatal Dengue shock syndrome (DSS). In the absence of a universally accepted prophylactic vaccine or therapeutic drug, treatment is mostly restricted to supportive measures. Similar to many other viruses that induce acute illness, DENV has developed several ways to modulate host metabolism to create an environment conducive to genome replication and the dissemination of viral progeny. To search for new therapeutic options, understanding the underlying host-virus regulatory system involved in various biological processes of the viral life cycle is essential. This review aims to summarize the complex interaction between DENV and the host cellular machinery, comprising regulatory mechanisms at various molecular levels such as epigenetic modulation of the host genome, transcription of host genes, translation of viral and host mRNAs, post-transcriptional regulation of the host transcriptome, post-translational regulation of viral proteins, and pathways involved in protein degradation.

Dengue vector control in high-income, city settings: A scoping review of approaches and methods.

BACKGROUND: Dengue virus (DENV) is endemic to many parts of the world and has serious health and socioeconomic effects even in high-income countries, especially with rapid changes in the climate globally. We explored the literature on dengue vector control methods used in high-income, city settings and associations with dengue incidence, dengue prevalence, or mosquito vector densities. METHODS: Studies of any design or year were included if they reported effects on human DENV infection or Aedes vector indices of dengue-specific vector control interventions in high-income, city settings. RESULTS: Of 24 eligible sources, most reported research in the United States (n = 8) or Australia (n = 5). Biocontrol (n = 12) and chemical control (n = 13) were the most frequently discussed vector control methods. Only 6 sources reported data on the effectiveness of a given method in reducing human DENV incidence or prevalence, 2 described effects of larval and adult control on Aedes DENV positivity, 20 reported effectiveness in reducing vector density, using insecticide, larvicide, source reduction, auto-dissemination of pyriproxyfen and Wolbachia, and only 1 described effects on human-vector contact. CONCLUSIONS: As most studies reported reductions in vector densities, rather than any effects on human DENV incidence or prevalence, we can draw no clear conclusions on which interventions might be most effective in reducing dengue in high-income, city areas. More research is needed linking evidence on the effects of different DENV vector control methods with dengue incidence/prevalence or mosquito vector densities in high-income, city settings as this is likely to differ from low-income settings. This is a significant evidence gap as climate changes increase the global reach of DENV. The importance of community involvement was clear in several studies, although it is impossible to tease out the relative contributions of this from other control methods used.

Gut microbe blocks dengue and Zika viruses in mosquitoes.

Naturally occurring bacterium could offer an additional way to control mosquito-borne diseases.

Biased virus transmission following sequential coinfection of Aedes aegypti with dengue and Zika viruses.

BACKGROUND: Mosquito-borne arboviruses are expanding their territory and elevating their infection prevalence due to the rapid climate change, urbanization, and increased international travel and global trade. Various significant arboviruses, including the dengue virus, Zika virus, Chikungunya virus, and yellow fever virus, are all reliant on the same primary vector, Aedes aegypti. Consequently, the occurrence of arbovirus coinfection in mosquitoes is anticipated. Arbovirus coinfection in mosquitoes has two patterns: simultaneous and sequential. Numerous studies have demonstrated that simultaneous coinfection of arboviruses in mosquitoes is unlikely to exert mutual developmental influence on these viruses. However, the viruses' interplay within a mosquito after the sequential coinfection seems intricated and not well understood. METHODOLOGY/PRINCIPAL FINDINGS: We conducted experiments aimed at examining the phenomenon of arbovirus sequential coinfection in both mosquito cell line (C6/36) and A. aegypti, specifically focusing on dengue virus (DENV, serotype 2) and Zika virus (ZIKV). We firstly observed that DENV and ZIKV can sequentially infect mosquito C6/36 cell line, but the replication level of the subsequently infected ZIKV was significantly suppressed. Similarly, A. aegypti mosquitoes can be sequentially coinfected by these two arboviruses, regardless of the order of virus exposure. However, the replication, dissemination, and the transmission potential of the secondary virus were significantly inhibited. We preliminarily explored the underlying mechanisms, revealing that arbovirus-infected mosquitoes exhibited activated innate immunity, disrupted lipid metabolism, and enhanced RNAi pathway, leading to reduced susceptibility to the secondary arbovirus infections. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that, in contrast to simultaneous arbovirus coinfection in mosquitoes that can promote the transmission and co-circulation of these viruses, sequential coinfection appears to have limited influence on arbovirus transmission dynamics. However, it is important to note that more experimental investigations are needed to refine and expand upon this conclusion.

Kirigami tripod-based electrode for the development of highly stretchable dengue aptasensor.

Kirigami is one of the interesting paper art forms and the modified sub-class of origami. Kirigami paper art is widely employed in a variety of applications, and it is currently being used in biosensors because of its outstanding advantages. This is the first study on the use of a Kirigami-based aptasensor for DENV (Dengue virus)-antigen detection. In this study, the kirigami approach has been utilized to develop a stretchable, movable, and flexible sensor. The constructed stretchable-kirigami electrode helps in adjusting the connection of electrodes without disturbing the electrochemical cell zone during the experiment. To increase the sensitivity of this biosensor we have synthesized Ag-NPs (Silver nanoparticles) via chemical methods and characterized their results with the help of TEM & UV-Vis Spectroscopy. Different electrochemical approaches were used to validate the sensor response i.e., CV (Cyclic voltammetry) and LSV (Linear sweep voltammetry), which exhibited great detection capability towards dengue virus with the range of 0.1 µg/ml to 1000 µg/ml along with a detection limit of 0.1 µg/ml and showing no reactivity to the chikungunya virus antigen, making it more specific to the DENV antigen. Serum (healthy-human) was also successfully applied to validate the results of the constructed aptasensor. Integration of the Kirigami approach form with the electrochemical aptasensor that utilizes a 3-E setup (three-electrode setup) which is referred to as a tripod and collectively called Kirigami-tripod-based aptasensor. Thus, the developed integrated platform improves the sensors capabilities in terms of cost efficiency, high stretchability, and sensitivity.

Molecular identification and phylogenetic analysis of chikungunya virus among dengue-negative patients in Kolkata, India.

Dengue and chikungunya are co-circulating vector-borne diseases that share a significant number of clinical symptoms. To identify variables to aid physicians in making rapid and effective diagnostic decisions, we performed molecular diagnosis of the chikungunya virus and examined the clinical manifestations of chikungunya cases to identify the prevalence among dengue-negative individuals in Kolkata. Dengue suspected patients' samples were collected during January 2020-December 2021 and Enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) methods have been performed to confirm the prevalence of chikungunya infection among dengue-negative patients. By performing phylogenetic analysis, comparing clinical classifications, identifying disease aetiology using clinical and laboratory factors, and evaluating the time course of several clinical variables, we have evaluated the clinical manifestations linked to dengue and chikungunya virus infections. Chikungunya infection was found in 15.1% and 6.3% of the 635 dengue-negative patients, as determined by ELISA and RT-PCR, respectively. Arthritis and myalgia were more common in chikungunya-infected patients at the time of hospital admission while conjunctivitis, photosensitivity, arthralgia, Anorexia, fatigue, retro-orbital pain, vomiting, dermatitis, or swollen glands were significantly presented as an overlapping symptom. Although dengue and chikungunya infections have significant clinical overlap, basic clinical and laboratory criteria can predict these diseases at presentation for proper management. Effective management enables doctors to treat and care for patients properly and contributes to the development of control measures for these infections in a medical setting.

Antigenic distance between primary and secondary dengue infections correlates with disease risk.

Many pathogens continuously change their protein structure in response to immune-driven selection, resulting in weakened protection even in previously exposed individuals. In addition, for some pathogens, such as dengue virus, poorly targeted immunity is associated with increased risk of severe disease through a mechanism known as antibody-dependent enhancement. However, it remains unclear whether the antigenic distances between an individual's first infection and subsequent exposures dictate disease risk, explaining the observed large-scale differences in dengue hospitalizations across years. Here, we develop a framework that combines detailed antigenic and genetic characterization of viruses with details on hospitalized cases from 21 years of dengue surveillance in Bangkok, Thailand, to identify the role of the antigenic profile of circulating viruses in determining disease risk. We found that the risk of hospitalization depended on both the specific order of infecting serotypes and the antigenic distance between an individual's primary and secondary infections, with risk maximized at intermediate antigenic distances. These findings suggest that immune imprinting helps determine dengue disease risk and provide a pathway to monitor the changing risk profile of populations and to quantifying risk profiles of candidate vaccines.

Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics.

Arboviruses can emerge rapidly and cause explosive epidemics of severe disease. Some of the most epidemiologically important arboviruses, including dengue virus (DENV), Zika virus (ZIKV), Chikungunya (CHIKV) and yellow fever virus (YFV), are transmitted by Aedes mosquitoes, most notably Aedes aegypti and Aedes albopictus. After a mosquito blood feeds on an infected host, virus enters the midgut and infects the midgut epithelium. The virus must then overcome a series of barriers before reaching the mosquito saliva and being transmitted to a new host. The virus must escape from the midgut (known as the midgut escape barrier; MEB), which is thought to be mediated by transient changes in the permeability of the midgut-surrounding basal lamina layer (BL) following blood feeding. Here, we present a mathematical model of the within-mosquito population dynamics of DENV (as a model system for mosquito-borne viruses more generally) that includes the interaction of the midgut and BL which can account for the MEB. Our results indicate a dose-dependency of midgut establishment of infection as well as rate of escape from the midgut: collectively, these suggest that the extrinsic incubation period (EIP)-the time taken for DENV virus to be transmissible after infection-is shortened when mosquitoes imbibe more virus. Additionally, our experimental data indicate that multiple blood feeding events, which more closely mimic mosquito-feeding behavior in the wild, can hasten the course of infections, and our model predicts that this effect is sensitive to the amount of virus imbibed. Our model indicates that mutations to the virus which impact its replication rate in the midgut could lead to even shorter EIPs when double-feeding occurs. Mechanistic models of within-vector viral infection dynamics provide a quantitative understanding of infection dynamics and could be used to evaluate novel interventions that target the mosquito stages of the infection.

Ophthalmic manifestations as the first presenting feature in dengue fever: a 10-year study.

Purpose: To report patients who first presented with various ocular manifestations and eventually ascertained to have underlying dengue. Methods: A prospective study was conducted at multiple tertiary eye-care centers in India from 2012 to 2022. Cases reporting initially with ocular features along with fever/past history of fever over the last two weeks or with clinical features of dengue were selected. After an ophthalmological examination, patients underwent complete serological and biochemical analysis and those with reduced platelet counts were evaluated for dengue. Results: Out of 564 cases, 15 patients were verified to be afflicted with dengue eventually. A rising trend of cases was seen every year and out of 15 cases, eight cases were reported during the Covid-19 pandemic (from 2020 to 2022), but were COVID-negative. 9 cases presented with acute redness followed by diminished vision. Seven cases presented a history of fever over the last few days and one had traveled from dengue endemic area. The various ocular presentations included subconjunctival hemorrhage, viral keratitis, anterior uveitis, sixth-nerve palsy, and vitreous hemorrhage. On serological examination, all 15 patients were detected to have low platelets. All cases responded well with supportive treatment and the ocular features subsided in all within a couple of weeks with good visual recovery. Conclusion: In a tropical nation, such as India, with endemic dengue zones and increasing figures of dengue lately, ophthalmologists must include dengue fever among the differential diagnoses in various ocular presentations like subconjunctival hemorrhage, viral keratitis, anterior uveitis, sixth nerve palsy, and vitreous hemorrhage. Abbreviations: DHF = dengue hemorrhagic fever, PCR = polymerase chain reaction, RT-PCR = real-time automated reverse transcriptase (RT-PCR), SD = standard deviation, MAC-ELIS = IgM antibodies capture enzyme-linked immunosorbent assay, RE = right eye, LE = left eye, CECT = Contrast-enhanced computed tomography.

A systematic review on malaria and dengue vaccines for the effective management of these mosquito borne diseases: Improving public health.

Insect vector-borne diseases (VBDs) pose significant global health challenges, particularly in tropical and subtropical regions. The WHO has launched the "Global Vector Control Response (GVCR) 2017-2030" to address these diseases, emphasizing a comprehensive approach to vector control. This systematic review investigates the potential of malaria and dengue vaccines in controlling mosquito-borne VBDs, aiming to alleviate disease burdens and enhance public health. Following PRISMA 2020 guidelines, the review incorporated 39 new studies out of 934 identified records. It encompasses various studies assessing malaria and dengue vaccines, emphasizing the significance of vaccination as a preventive measure. The findings indicate variations in vaccine efficacy, duration of protection, and safety considerations for each disease, influencing public health strategies. The review underscores the urgent need for vaccines to combat the increasing burden of VBDs like malaria and dengue, advocating for ongoing research and investment in vaccine development.

Epidemiological investigation of dengue fever outbreak and its socioeconomic determinants in Banadir region, Somalia.

BACKGROUND: Dengue has become an alarming global problem and is endemic in many countries, particularly in tropical and subtropical countries. The aim of this study was to investigate dengue fever outbreak in Banadir Region, Somalia, to understand the risk factors (time, place, personal characteristics). METHODS: A descriptive cross-sectional study was undertaken to determine the levels of circulating anti-dengue virus antibodies and DENV NS1 antigen among Banadir Region residents, while a questionnaire survey was conducted to understand the clinical and demographic characteristics of the patients. RESULTS: A total of 735 febrile patients were studied, with 55.6% men and 44.3% women. The majority of the participants were children aged 14 years and younger. Among them, 10.8% tested positive for IgM antibodies against dengue virus (DENV), while the prevalence of DENV NS1 antigen was 11.8%. Fever and myalgia were the most common symptoms observed in the DENV-positive patients. CONCLUSIONS: A dengue fever outbreak has been confirmed in Banadir region, Somalia. This study provides information on the most affected districts and identifies risk factors contributing to DF outbreaks. The study recommends improving outbreak readiness and response, particularly in surveillance and laboratory diagnostics, by fostering intersectoral collaboration and establishing regulatory frameworks for financial and operational participation.

A 2D-proteomic analysis identifies proteins differentially regulated by two different dengue virus serotypes.

The mosquito transmitted dengue virus (DENV) is a major public health problem in many tropical and sub-tropical countries around the world. Both vaccine development and drug development are complex as the species Dengue virus consist of four distinct viruses (DENV 1 to DENV 4) each of which is composed of multiple lineages and strains. To understand the interaction of DENV with the host cell machinery, several studies have undertaken in vitro proteomic analysis of different cell lines infected with DENV. Invariably, these studies have utilized DENV 2. In this study we sought to define proteins that are differentially regulated by two different DENVs, DENV 2 and DENV 4. A 2-dimensional proteomic analysis identified some 300 protein spots, of which only 11 showed differential expression by both DENVs. Of these, only six were coordinately regulated. One protein, prohibitin 1 (PHB1) was downregulated by infection with both DENVs. Overexpression of PHB1 increased DENV protein expression, level of infection and genome copy number. DENV E protein colocalized with PHB, and there was a direct interaction between DENV 2 E protein and PHB1, but not between DENV 4 E protein and PHB1. The low number of proteins showing coordinate regulation after infection by different DENVs is a cause for concern, particularly in determining new druggable targets, and suggests that studies should routinely investigate multiple DENVs.

Glycolysis is reduced in dengue virus 2 infected liver cells.

Infections with dengue virus (DENV) remain a worldwide public health problem. A number of bona fide cellular targets of DENV have been identified including liver cells. Despite the many lines of evidence confirming the involvement of hepatocytes during DENV infection, only a few studies have used proteomic analysis to understand the modulation of the cellular proteome occurring upon DENV infection. We utilized a 2D-gel electrophoresis analysis to identify proteins that were differentially regulated by DENV 2 infection of liver (Hep3B) cells at 12 h post infection (hpi) and at 48 hpi. The analysis identifies 4 proteins differentially expressed at 12 hpi, and 14 differentially regulated at 48 hpi. One candidate protein identified as downregulated at 48 hpi in the proteomic analysis (GAPDH) was validated in western blotting in Hep3B cells, and subsequently in induced pluripotent stem cell (iPSC) derived human hepatocytes. The reduced expression of GAPDH was coupled with an increase in NADH, and a significantly reduced NAD + /NADH ratio, strongly suggesting that glycolysis is down regulated in response to DENV 2 infection. Metformin, a well characterized drug used in the treatment of diabetes mellitus, is an inhibitor of hepatic gluconeogenesis was shown to reduce the level of DENV 2 infection and new virus production. Collectively these results show that although glycolysis is reduced, glucose is still required, possibly for use by the pentose phosphate pathway to generate nucleosides required for viral replication.

Wolbachia-carrying Aedes mosquitoes for preventing dengue infection.

BACKGROUND: Dengue is a global health problem of high significance, with 3.9 billion people at risk of infection. The geographic expansion of dengue virus (DENV) infection has resulted in increased frequency and severity of the disease, and the number of deaths has increased in recent years. Wolbachia,an intracellular bacterial endosymbiont, has been under investigation for several years as a novel dengue-control strategy. Some dengue vectors (Aedes mosquitoes) can be transinfected with specific strains of Wolbachia, which decreases their fitness (ability to survive and mate) and their ability to reproduce, inhibiting the replication of dengue. Both laboratory and field studies have demonstrated the potential effect of Wolbachia deployments on reducing dengue transmission, and modelling studies have suggested that this may be a self-sustaining strategy for dengue prevention, although long-term effects are yet to be elucidated. OBJECTIVES: To assess the efficacy of Wolbachia-carrying Aedes speciesdeployments (specifically wMel-, wMelPop-, and wAlbB- strains of Wolbachia) for preventing dengue virus infection. SEARCH METHODS: We searched CENTRAL, MEDLINE, Embase, four other databases, and two trial registries up to 24 January 2024. SELECTION CRITERIA: Randomized controlled trials (RCTs), including cluster-randomized controlled trials (cRCTs), conducted in dengue endemic or epidemic-prone settings were eligible. We sought studies that investigated the impact of Wolbachia-carrying Aedes deployments on epidemiological or entomological dengue-related outcomes, utilizing either the population replacement or population suppression strategy. DATA COLLECTION AND ANALYSIS: Two review authors independently selected eligible studies, extracted data, and assessed the risk of bias using the Cochrane RoB 2 tool. We used odds ratios (OR) with the corresponding 95% confidence intervals (CI) as the effect measure for dichotomous outcomes. For count/rate outcomes, we planned to use the rate ratio with 95% CI as the effect measure. We used adjusted measures of effect for cRCTs. We assessed the certainty of evidence using GRADE. MAIN RESULTS: One completed cRCT met our inclusion criteria, and we identified two further ongoing cRCTs. The included trial was conducted in an urban setting in Yogyakarta, Indonesia. It utilized a nested test-negative study design, whereby all participants aged three to 45 years who presented at healthcare centres with a fever were enrolled in the study provided they had resided in the study area for the previous 10 nights. The trial showed that wMel-Wolbachia infected Ae aegypti deployments probably reduce the odds of contracting virologically confirmed dengue by 77% (OR 0.23, 95% CI 0.15 to 0.35; 1 trial, 6306 participants; moderate-certainty evidence). The cluster-level prevalence of wMel Wolbachia-carrying mosquitoes remained high over two years in the intervention arm of the trial, reported as 95.8% (interquartile range 91.5 to 97.8) across 27 months in clusters receiving wMel-Wolbachia Ae aegypti deployments, but there were no reliable comparative data for this outcome. Other primary outcomes were the incidence of virologically confirmed dengue, the prevalence of dengue ribonucleic acid in the mosquito population, and mosquito density, but there were no data for these outcomes. Additionally, there were no data on adverse events. AUTHORS' CONCLUSIONS: The included trial demonstrates the potential significant impact of wMel-Wolbachia-carrying Ae aegypti mosquitoes on preventing dengue infection in an endemic setting, and supports evidence reported in non-randomized and uncontrolled studies. Further trials across a greater diversity of settings are required to confirm whether these findings apply to other locations and country settings, and greater reporting of acceptability and cost are important.