Correlation of serotype-specific dengue virus infection with clinical manifestations

Background

Disease caused by the dengue virus (DENV) is a significant cause of morbidity throughout the world. Although prior research has focused on the association of specific DENV serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) with the development of severe outcomes such as dengue hemorrhagic fever and dengue shock syndrome, relatively little work has correlated other clinical manifestations with a particular DENV serotype. The goal of this study was to estimate and compare the prevalence of non-hemorrhagic clinical manifestations of DENV infection by serotype.

Methodology and Principal Findings

Between the years 2005–2010, individuals with febrile disease from Peru, Bolivia, Ecuador, and Paraguay were enrolled in an outpatient passive surveillance study. Detailed information regarding clinical signs and symptoms, as well as demographic information, was collected. DENV infection was confirmed in patient sera with polyclonal antibodies in a culture-based immunofluorescence assay, and the infecting serotype was determined by serotype-specific monoclonal antibodies. Differences in the prevalence of individual and organ-system manifestations were compared across DENV serotypes. One thousand seven hundred and sixteen individuals were identified as being infected with DENV-1 (39.8%), DENV-2 (4.3%), DENV-3 (41.5%), or DENV-4 (14.4%). When all four DENV serotypes were compared with each other, individuals infected with DENV-3 had a higher prevalence of musculoskeletal and gastrointestinal manifestations, and individuals infected with DENV-4 had a higher prevalence of respiratory and cutaneous manifestations.

Conclusions/Significance

Specific clinical manifestations, as well as groups of clinical manifestations, are often overrepresented by an individual DENV serotype.     

Recent emergence of dengue virus serotype 4 in French Polynesia results from multiple introductions from other South Pacific Islands

Background

Infection by dengue virus (DENV) is a major public health concern in hundreds of tropical and subtropical countries. French Polynesia (FP) regularly experiences epidemics that initiate, or are consecutive to, DENV circulation in other South Pacific Island Countries (SPICs). In January 2009, after a decade of serotype 1 (DENV-1) circulation, the first cases of DENV-4 infection were reported in FP. Two months later a new epidemic emerged, occurring about 20 years after the previous circulation of DENV-4 in FP. In this study, we investigated the epidemiological and molecular characteristics of the introduction, spread and genetic microevolution of DENV-4 in FP.

Methodology/Principal Findings

Epidemiological data suggested that recent transmission of DENV-4 in FP started in the Leeward Islands and this serotype quickly displaced DENV-1 throughout FP. Phylogenetic analyses of the nucleotide sequences of the envelope (E) gene of 64 DENV-4 strains collected in FP in the 1980s and in 2009–2010, and some additional strains from other SPICs showed that DENV-4 strains from the SPICs were distributed into genotypes IIa and IIb. Recent FP strains were distributed into two clusters, each comprising viruses from other but distinct SPICs, suggesting that emergence of DENV-4 in FP in 2009 resulted from multiple introductions. Otherwise, we observed that almost all strains collected in the SPICs in the 1980s exhibit an amino acid (aa) substitution V287I within domain I of the E protein, and all recent South Pacific strains exhibit a T365I substitution within domain III.

Conclusions/Significance

This study confirmed the cyclic re-emergence and displacement of DENV serotypes in FP. Otherwise, our results showed that specific aa substitutions on the E protein were present on all DENV-4 strains circulating in SPICs. These substitutions probably acquired and subsequently conserved could reflect a founder effect to be associated with epidemiological, geographical, eco-biological and social specificities in SPICs.     

Spatial and temporal clustering of dengue virus transmission in Thai villages

Background

Transmission of dengue viruses (DENV), the leading cause of arboviral disease worldwide, is known to vary through time and space, likely owing to a combination of factors related to the human host, virus, mosquito vector, and environment. An improved understanding of variation in transmission patterns is fundamental to conducting surveillance and implementing disease prevention strategies. To test the hypothesis that DENV transmission is spatially and temporally focal, we compared geographic and temporal characteristics within Thai villages where DENV are and are not being actively transmitted.

Methods and Findings

Cluster investigations were conducted within 100 m of homes where febrile index children with (positive clusters) and without (negative clusters) acute dengue lived during two seasons of peak DENV transmission. Data on human infection and mosquito infection/density were examined to precisely (1) define the spatial and temporal dimensions of DENV transmission, (2) correlate these factors with variation in DENV transmission, and (3) determine the burden of inapparent and symptomatic infections. Among 556 village children enrolled as neighbors of 12 dengue-positive and 22 dengue-negative index cases, all 27 DENV infections (4.9% of enrollees) occurred in positive clusters (p < 0.01; attributable risk [AR] = 10.4 per 100; 95% confidence interval 1–19.8 per 100]. In positive clusters, 12.4% of enrollees became infected in a 15-d period and DENV infections were aggregated centrally near homes of index cases. As only 1 of 217 pairs of serologic specimens tested in positive clusters revealed a recent DENV infection that occurred prior to cluster initiation, we attribute the observed DENV transmission subsequent to cluster investigation to recent DENV transmission activity. Of the 1,022 female adult Ae. aegypti collected, all eight (0.8%) dengue-infected mosquitoes came from houses in positive clusters; none from control clusters or schools. Distinguishing features between positive and negative clusters were greater availability of piped water in negative clusters (p < 0.01) and greater number of Ae. aegypti pupae per person in positive clusters (p = 0.04). During primarily DENV-4 transmission seasons, the ratio of inapparent to symptomatic infections was nearly 1:1 among child enrollees. Study limitations included inability to sample all children and mosquitoes within each cluster and our reliance on serologic rather than virologic evidence of interval infections in enrollees given restrictions on the frequency of blood collections in children.

Conclusions

Our data reveal the remarkably focal nature of DENV transmission within a hyperendemic rural area of Thailand. These data suggest that active school-based dengue case detection prompting local spraying could contain recent virus introductions and reduce the longitudinal risk of virus spread within rural areas. Our results should prompt future cluster studies to explore how host immune and behavioral aspects may impact DENV transmission and prevention strategies. Cluster methodology could serve as a useful research tool for investigation of other temporally and spatially clustered infectious diseases.     

Kinetics of viremia and NS1 antigenemia are shaped by immune status and virus serotype in adults with dengue

Background

Dengue is a major public health problem in tropical and subtropical countries. Exploring the relationships between virological features of infection with patient immune status and outcome may help to identify predictors of disease severity and enable rational therapeutic strategies.

Methods

Clinical features, antibody responses and virological markers were characterized in Vietnamese adults participating in a randomised controlled treatment trial of chloroquine.

Results

Of the 248 patients with laboratory-confirmed dengue and defined serological and clinical classifications 29 (11.7%) had primary DF, 150 (60.5%) had secondary DF, 4 (1.6%) had primary DHF and 65 (26.2%) had secondary DHF. DENV-1 was the commonest serotype (57.3%), then DENV-2 (20.6%), DENV-3 (15.7%) and DENV-4 (2.8%). DHF was associated with secondary infection (Odds ratio = 3.13, 95% CI 1.04–12.75). DENV-1 infections resulted in significantly higher viremia levels than DENV-2 infections. Early viremia levels were higher in DENV-1 patients with DHF than with DF, even if the peak viremia level was often not observed because it occurred prior to enrolment. Peak viremias were significantly less often observed during secondary infections than primary for all disease severity grades (P = 0.001). The clearance of DENV viremia and NS1 antigenemia occurs earlier and faster in patients with secondary dengue (P<0.0001). The maximum daily rate of viremia clearance was significantly higher in patients with secondary infections than primary (P<0.00001).

Conclusions

Collectively, our findings suggest that the early magnitude of viremia is positively associated with disease severity. The clearance of DENV is associated with immune status, and there are serotype dependent differences in infection kinetics. These findings are relevant for the rational design of randomized controlled trials of therapeutic interventions, especially antivirals.     

Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages

Background

Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages.

Methodology/Principal Findings

Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile “index” cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (p<0.001). Significantly more Ae. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (p<0.001). The DENV infection rate in contacts was 47.4% in houses with infectious mosquitoes, 28.7% in other houses in the same cluster, and 10.8% in positive clusters without infectious mosquitoes (p<0.001). Ae. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes.

Conclusions/Significance

Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission).     

Incomplete Protection against Dengue Virus Type 2 Re-infection in Peru

Background

Nearly half of the world’s population is at risk for dengue, yet no licensed vaccine or anti-viral drug is currently available. Dengue is caused by any of four dengue virus serotypes (DENV-1 through DENV-4), and infection by a DENV serotype is assumed to provide life-long protection against re-infection by that serotype. We investigated the validity of this fundamental assumption during a large dengue epidemic caused by DENV-2 in Iquitos, Peru, in 2010–2011, 15 years after the first outbreak of DENV-2 in the region.

Methodology/Principal Findings

We estimated the age-dependent prevalence of serotype-specific DENV antibodies from longitudinal cohort studies conducted between 1993 and 2010. During the 2010–2011 epidemic, active dengue cases were identified through active community- and clinic-based febrile surveillance studies, and acute inapparent DENV infections were identified through contact tracing studies. Based on the age-specific prevalence of DENV-2 neutralizing antibodies, the age distribution of DENV-2 cases was markedly older than expected. Homologous protection was estimated at 35.1% (95% confidence interval: 0%–65.2%). At the individual level, pre-existing DENV-2 antibodies were associated with an incomplete reduction in the frequency of symptoms. Among dengue cases, 43% (26/66) exhibited elevated DENV-2 neutralizing antibody titers for years prior to infection, compared with 76% (13/17) of inapparent infections (age-adjusted odds ratio: 4.2; 95% confidence interval: 1.1–17.7).

Conclusions/Significance

Our data indicate that protection from homologous DENV re-infection may be incomplete in some circumstances, which provides context for the limited vaccine efficacy against DENV-2 in recent trials. Further studies are warranted to confirm this phenomenon and to evaluate the potential role of incomplete homologous protection in DENV transmission dynamics.     

Dengue Virus Serotype 2 Blocks Extracellular Signal-Regulated Kinase and Nuclear Factor-κB Activation to Downregulate Cytokine Production

Background

Dengue virus (DENV) infection is the most common mosquito-borne viral disease threatening human health around the world. Type I interferon (IFN) and cytokine production are crucial in the innate immune system. We previously reported that DENV serotype 2 (DENV-2) induced low levels of interferon regulatory factor 3 and NF-κB activation, thus leading to reduced production of IFN-β in the early phase of infection. Here, we determined whether DENV infection not only hampers type I IFN activation but also cytokine production triggered by Toll-like receptor (TLR) signaling.

Methodology/Principal Findings

We used quantitative RT-PCR and found that only low levels of IFN-β and inflammatory cytokines such as interleukin 10 (IL-10), IL-12 and tumor necrosis factor α (TNFα) mRNA were detected in DENV-2–infected bone-marrow–derived dendritic cells. Furthermore, DENV-2 infection repressed cytokine production triggered by TLR signaling. To elucidate the molecular mechanisms underlying this suppression event, we measured NF-κB activation by p65 nuclear translocation and luciferase reporter assay and found that NF-κB activation triggered by TLR ligands was blocked by DENV-2 infection. As well, extracellular signal-regulated kinase (ERK) activity was suppressed by DENV-2 infection.

Conclusions/Significance

To downregulate the host innate immunity, DENV-2 by itself is a weak inducer of type I IFN and cytokines, furthermore DENV-2 can also block the TLR-triggered ERK–NF-κB activation and cytokine production.     

Dengue Virus Neutralizing Antibody Levels Associated with Protection from Infection in Thai Cluster Studies

Background

Long-term homologous and temporary heterologous protection from dengue virus (DENV) infection may be mediated by neutralizing antibodies. However, neutralizing antibody titers (NTs) have not been clearly associated with protection from infection.

Methodology/Principal Findings

Data from two geographic cluster studies conducted in Kamphaeng Phet, Thailand were used for this analysis. In the first study (2004–2007), cluster investigations of 100-meter radius were triggered by DENV-infected index cases from a concurrent prospective cohort. Subjects between 6 months and 15 years old were evaluated for DENV infection at days 0 and 15 by DENV PCR and IgM ELISA. In the second study (2009–2012), clusters of 200-meter radius were triggered by DENV-infected index cases admitted to the provincial hospital. Subjects of any age ≥6 months were evaluated for DENV infection at days 0 and 14. In both studies, subjects who were DENV PCR positive at day 14/15 were considered to have been “susceptible” on day 0. Comparison subjects from houses in which someone had documented DENV infection, but the subject remained DENV negative at days 0 and 14/15, were considered “non-susceptible.” Day 0 samples were presumed to be from just before virus exposure, and underwent plaque reduction neutralization testing (PRNT). Seventeen “susceptible” (six DENV-1, five DENV-2, and six DENV-4), and 32 “non-susceptible” (13 exposed to DENV-1, 10 DENV-2, and 9 DENV-4) subjects were evaluated. Comparing subjects exposed to the same serotype, receiver operating characteristic (ROC) curves identified homotypic PRNT titers of 11, 323 and 16 for DENV-1, -2 and -4, respectively, to differentiate “susceptible” from “non-susceptible” subjects.

Conclusions/Significance

PRNT titers were associated with protection from infection by DENV-1, -2 and -4. Protective NTs appeared to be serotype-dependent and may be higher for DENV-2 than other serotypes. These findings are relevant for both dengue epidemiology studies and vaccine development efforts.     

Age-specificity of clinical dengue during primary and secondary infections

Background

This study aims to estimate the age-specific risks of clinical dengue attack (i.e., the risk of symptomatic dengue among the total number of dengue virus (DENV) infections) during primary and secondary infections.

Methods

We analyzed two pieces of epidemiological information in Binh Thuan province, southern Vietnam, i.e., age-specific seroprevalence and a community-wide longitudinal study of clinical dengue attack. The latter data set stratified febrile patients with DENV infection by age as well as infection parity. A simple modeling approach was employed to estimate the age-specific risks of clinical dengue attack during primary and secondary infections.

Results

Using the seroprevalence data, the force of infection was estimated to be 11.7% (95% confidence intervals (CI): 10.8–12.7) per year. Median age (and the 25–75 percentiles) of dengue fever patients during primary and secondary infections were 12 (9–20) and 20 (14–31) years, respectively. The estimated age-specific risk of clinical dengue increases as a function of age for both primary and secondary infections; the estimated proportion of symptomatic patients among the total number of infected individuals was estimated to be <7% for those aged <10 years for both primary and secondary infections, but increased as patients become older, reaching to 8–11% by the age of 20 years.

Conclusions/Significance

For both primary and secondary infections, higher age at DENV infection was shown to result in higher risk of clinical attack. Age as an important modulator of clinical dengue explains recent increase in dengue notifications in ageing countries in Southeast Asia, and moreover, poses a paradoxical problem of an increase in adult patients resulting from a decline in the force of infection, which may be caused by various factors including time-dependent variations in epidemiological, ecological and demographic dynamics.     

Detection of Serotype-Specific Antibodies to the Four Dengue Viruses Using an Immune Complex Binding (ICB) ELISA

Background

Dengue virus (DENV) infections are preferentially diagnosed by detection of specific IgM antibodies, DENV NS1 antigen assays or by amplification of viral RNA in serum samples of the patients. The type-specific immunity to the four worldwide circulating DENV serotypes can be determined by neutralization assays. An alternative to the complicated neutralization assays would be helpful to study the serotype-specific immune response in people in DENV hyperendemic areas but also in subjects upon DENV vaccination.

Methods

In consecutive samples of patients with DENV-1- 4 infection type-specific antibodies were detected using an immune complex binding (ICB) ELISA. During incubation of serum samples and enzyme- labeled recombinant envelope domain III (EDIII) antigens immune complexes (ICs) are formed, which are simultaneously bound to a solid phase coated with an Fc–receptor (CD32). After a single washing procedure the bound labeled ICs can be determined. To further improve type-specific reactions high concentrations of competing heterologous unlabeled ED III proteins were added to the labeled antigens.

Results

Follow-up serum samples of 64 patients with RT-PCR confirmed primary DENV-1, -2, -3 or -4 infections were tested against four enzyme-labeled recombinant DENV EDIII antigens. Antibodies to the EDIII antigens were found in 55 patients (sensitivity 86%). A complete agreement between the serotype detected by PCR in early samples and the serotype-specific antibody in later samples was found. Type-specific anti-EDIII antibodies were first detected 9–20 days after onset of the disease. In 21% of the samples collected from people in Vietnam secondary infections with antibodies to two serotypes could be identified.

Conclusions

The data obtained with the ICB-ELISA show that after primary DENV infection the corresponding type-specific antibodies are detected in almost all samples collected at least two weeks after onset of the disease. The method will be of value to determine the distribution of the various type-specific anti–DENV antibodies in DENV endemic areas.     

Seroepidemiology of Dengue virus in Mayotte, Indian Ocean, 2006

Background

Although Dengue virus (DENV) circulation had been documented in neighbouring South-western Indian Ocean Islands, its presence in Mayotte is poorly characterised. To address this issue, we aimed to assess the seroprevalence of dengue IgG antibodies (DENV-IgG Ab) among the population and to investigate potential associations with individual and household characteristics.

Methods/Principal Findings

In November–December 2006 we conducted a cross-sectional serologic survey in Mayotte among 1,154 inhabitants aged ≥2 years by using a multistage cluster random sampling method. The overall prevalence of DENV-specific IgG antibodies (ELISA) was 22.73% (95% CI, 18.16–27.31). The age-specific seroprevalence increased with age (χ² for trend = 11.86, P<0.0006), and was linked with previous known outbreaks in this region. In multivariate analysis, older age, being born in the Comoros and living in a household with a low socioeconomic index were positively associated with DENV IgG antibody positivity.

Conclusions

These findings document substantial prior exposure of the population of Mayotte to DENV and highlight the risk of severe illness due to the possibility of sequential DENV infections. Further investigations characterizing current DENV circulation patterns and associated serotypes are needed.     

The Prevalence and Endemic Nature of Dengue Infections in Guangdong,South China: An Epidemiological,Serological, and Etiological Study from 2005–2011

Objectives

Frequent outbreaks of dengue are considered to be associated with an increased risk for endemicity of the disease. The occurrence of a large number of indigenous dengue cases in consecutive years indicates the possibility of a changing dengue epidemic pattern in Guangdong, China.

Methods

To have a clear understanding of the current dengue epidemic, a retrospective study of epidemiological profile, serological response, and virological features of dengue infections from 2005–2011 was conducted. Case data were collected from the National Notifiable Infectious Diseases Reporting Network. Serum samples were collected and prepared for serological verification and etiological confirmation. Incidence, temporal and spatial distribution, and the clinical manifestation of dengue infections were analyzed. Pearson''s Chi-Square test was used to compare incidences between different age groups. A seroprevalence survey was implemented in local healthy inhabitants to obtain the overall positive rate for the specific immunoglobulin (Ig) G antibody against dengue virus (DENV).

Results

The overall annual incidence rate was 1.87/100000. A significant difference was found in age-specific incidence (Pearson''s Chi-Square value 498.008, P<0.001). Children under 5 years of age had the lowest incidence of 0.28/100000. The vast majority of cases presented with a mild manifestation typical to dengue fever. The overall seroprevalence of dengue IgG antibody in local populations was 2.43% (range 0.28%–5.42%). DENV-1 was the predominant serotype in circulation through the years, while all 4 serotypes were identified in indigenous patients from different outbreak localities since 2009.

Conclusions

A gradual change in the epidemic pattern of dengue infection has been observed in recent years in Guangdong. With the endemic nature of dengue infections, the transition from a monotypic to a multitypic circulation of dengue virus in the last several years will have an important bearing on the prevention and control of dengue in the province and in the neighboring districts.     

Immunological and viral determinants of dengue severity in hospitalized adults in Ha Noi, Viet Nam

Background

The relationships between the infecting dengue serotype, primary and secondary infection, viremia and dengue severity remain unclear. This cross-sectional study examined these interactions in adult patients hospitalized with dengue in Ha Noi.

Methods and Findings

158 patients were enrolled between September 16 and November 11, 2008. Quantitative RT-PCR, serology and NS1 detection were used to confirm dengue infection, determine the serotype and plasma viral RNA concentration, and categorize infections as primary or secondary. 130 (82%) were laboratory confirmed. Serology was consistent with primary and secondary infection in 34% and 61%, respectively. The infecting serotype was DENV-1 in 42 (32%), DENV-2 in 39 (30%) and unknown in 49 (38%). Secondary infection was more common in DENV-2 infections (79%) compared to DENV-1 (36%, p<0.001). The proportion that developed dengue haemorrhagic fever (DHF) was 32% for secondary infection compared to 18% for primary infection (p = 0.14), and 26% for DENV-1 compared to 28% for DENV-2. The time until NS1 and plasma viral RNA were undetectable was shorter for DENV-2 compared to DENV-1 (p≤0.001) and plasma viral RNA concentration on day 5 was higher for DENV-1 (p = 0.03). Plasma viral RNA concentration was higher in secondary infection on day 5 of illness (p = 0.046). We didn''t find an association between plasma viral RNA concentration and clinical severity.

Conclusion

Dengue is emerging as a major public health problem in Ha Noi. DENV-1 and DENV-2 were the prevalent serotypes with similar numbers and clinical presentation. Secondary infection may be more common amongst DENV-2 than DENV-1 infections because DENV-2 infections resulted in lower plasma viral RNA concentrations and viral RNA concentrations were higher in secondary infection. The drivers of dengue emergence in northern Viet Nam need to be elucidated and public health measures instituted.     

Quantifying the spatial dimension of dengue virus epidemic spread within a tropical urban environment

Background

Dengue infection spread in naive populations occurs in an explosive and widespread fashion primarily due to the absence of population herd immunity, the population dynamics and dispersal of Ae. aegypti, and the movement of individuals within the urban space. Knowledge on the relative contribution of such factors to the spatial dimension of dengue virus spread has been limited. In the present study we analyzed the spatio-temporal pattern of a large dengue virus-2 (DENV-2) outbreak that affected the Australian city of Cairns (north Queensland) in 2003, quantified the relationship between dengue transmission and distance to the epidemic''s index case (IC), evaluated the effects of indoor residual spraying (IRS) on the odds of dengue infection, and generated recommendations for city-wide dengue surveillance and control.

Methods and Findings

We retrospectively analyzed data from 383 DENV-2 confirmed cases and 1,163 IRS applications performed during the 25-week epidemic period. Spatial (local k-function, angular wavelets) and space-time (Knox test) analyses quantified the intensity and directionality of clustering of dengue cases, whereas a semi-parametric Bayesian space-time regression assessed the impact of IRS and spatial autocorrelation in the odds of weekly dengue infection. About 63% of the cases clustered up to 800 m around the IC''s house. Most cases were distributed in the NW-SE axis as a consequence of the spatial arrangement of blocks within the city and, possibly, the prevailing winds. Space-time analysis showed that DENV-2 infection spread rapidly, generating 18 clusters (comprising 65% of all cases), and that these clusters varied in extent as a function of their distance to the IC''s residence. IRS applications had a significant protective effect in the further occurrence of dengue cases, but only when they reached coverage of 60% or more of the neighboring premises of a house.

Conclusion

By applying sound statistical analysis to a very detailed dataset from one of the largest outbreaks that affected the city of Cairns in recent times, we not only described the spread of dengue virus with high detail but also quantified the spatio-temporal dimension of dengue virus transmission within this complex urban environment. In areas susceptible to non-periodic dengue epidemics, effective disease prevention and control would depend on the prompt response to introduced cases. We foresee that some of the results and recommendations derived from our study may also be applicable to other areas currently affected or potentially subject to dengue epidemics.     

Households as Foci for Dengue Transmission in Highly Urban Vietnam

Background

Dengue control programs commonly employ reactive insecticide spraying around houses of reported cases, with the assumption that most dengue virus (DENV) transmission occurs in the home. Focal household transmission has been demonstrated in rural settings, but it is unclear whether this holds true in dense and mobile urban populations. We conducted a prospective study of dengue clustering around households in highly urban Ho Chi Minh City, Vietnam.

Methods

We enrolled 71 index cases with suspected dengue (subsequently classified as 52 dengue cases and 19 non-dengue controls); each initiated the enrollment of a cluster of 25–35 household members and neighbors who were followed up over 14 days. Incident DENV infections in cluster participants were identified by RT-PCR, NS1-ELISA, and/or DENV-IgM/-IgG seroconversion, and recent infections by DENV-IgM positivity at baseline.

Principal Findings/Conclusions

There was no excess risk of DENV infection within dengue case clusters during the two-week follow-up, compared to control clusters, but the prevalence of recent DENV infection at baseline was two-fold higher in case clusters than controls (OR 2.3, 95%CI 1.0–5.1, p = 0.05). Prevalence of DENV infection in Aedes aegypti was similar in case and control houses, and low overall (1%). Our findings are broadly consistent with household clustering of dengue risk, but indicate that any clustering is at a short temporal scale rather than sustained chains of localized transmission. This suggests that reactive perifocal insecticide spraying may have a limited impact in this setting.     

Insights into the internalization and retrograde trafficking of Dengue 2 virus in BHK-21 cells

Background

Dengue virus (DENV) enters cells via endocytosis, traffics to perinuclear (PN) region, the site of morphogenesis and exits by exocytosis. This study aims to understand the role of dynamin II, endosomes, microtubules (MT) and dynein in the early events of DENV replication.

Findings

Using double immunoflourescence labelling of DENV-2 infected BHK-21 cells it was observed that the surface envelope (E) protein of the virion associated with dynamin II from 0–30 min post infection (p.i.). The sphincter like array of dynamin II supported its pinchase-like activity. The association with endosomes was observed from 0 min at cell periphery to 30 min in the perinuclear (PN) region, suggesting that internalization continued for 30 min. Association of E protein with alpha-tubulin was observed from 8 h indicating that it was the newly translated protein that trafficked on the MT. Dynein was found to associate with the E protein from 4 h in the cytoplasm to 48 h in the PN region and dissociate at 72 h. Association of E protein with dynein was confirmed by immunoprecipitation. Overexpression of dynamitin, which disrupts the dynein complex, resulted in loss of trafficking of viral E and core proteins. The findings corroborated with the growth kinetics assessed by quantitation of viral RNA in infected BHK-21 cells. The detection of E protein at 4 h–8 h correlated with detectable increase in viral RNA from 8 h. The detection of high concentrations of E protein in the PN region at 24–48 h coincided with release of virus into the supernatant starting from 36 h p.i. The dissociation of dynein from E protein by 72 h was coincident with maximum release of virus, hinting at a possible negative feedback for viral protein translation.

Conclusion

The study shows for the first time the association of dynamin II with DENV-2 during entry and dynein dependent retrograde trafficking of DENV proteins on microtubules.     

Preexisting Japanese encephalitis virus neutralizing antibodies and increased symptomatic dengue illness in a school-based cohort in Thailand

Background

Dengue viruses (DENVs) and Japanese encephalitis virus (JEV) have significant cross-reactivity in serological assays; the clinical implications of this remain undefined. An improved understanding of whether and how JEV immunity modulates the clinical outcome of DENV infection is important as large-scale DENV vaccine trials will commence in areas where JEV is co-endemic and/or JEV immunization is routine.

Methods and Findings

The association between preexisting JEV neutralizing antibodies (NAbs) and the clinical severity of DENV infection was evaluated in a prospective school-based cohort in Thailand that captured asymptomatic, non-hospitalized, and hospitalized DENV infections. Covariates considered included age, baseline DENV antibody status, school of attendance, epidemic year, and infecting DENV serotype. 942 children experienced at least one DENV infection between 1998 and 2002, out of 3,687 children who were enrolled for at least one full year. In crude analysis, the presence of JEV NAbs was associated with an increased occurrence of symptomatic versus asymptomatic infection (odds ratio [OR] = 1.55, 95% CI: 1.08–2.23) but not hospitalized illness or dengue hemorrhagic fever (DHF). The association was strongest in children with negative DENV serology (DENV-naive) (OR = 2.75, 95% CI: 1.12–6.72), for whom the presence of JEV NAbs was also associated with a symptomatic illness of longer duration (5.4 days for JEV NAb+ versus 2.6 days for JEV NAb-, p = 0.048). JEV NAbs were associated with increased DHF in younger children with multitypic DENV NAb profiles (OR = 4.05, 95% CI: 1.18 to 13.87). Among those with JEV NAbs, the association with symptomatic illness did not vary by antibody titer.

Interpretation

The prior existence of JEV NAbs was associated with an increased probability of symptomatic as compared to asymptomatic DENV illness. These findings are in contrast to previous studies suggesting an attenuating effect of heterologous flavivirus immunity on DENV disease severity.     

Identifying residual foci of Plasmodium falciparum infections for malaria elimination: the urban context of Khartoum, Sudan

Background

Identifying the location and size of residual foci of infections is critical where malaria elimination is the primary goal. Here the spatial heterogeneity of Plasmodium falciparum infections within the urban extent of Khartoum state in Sudan is investigated using data from cross-sectional surveys undertaken from 1999 to 2008 to inform the Khartoum Malaria Free Initiative (KMFI).

Methods

From 1999–2008 the KMFI undertook cross-sectional surveys of 256 clusters across 203 random samples of residential blocks in the urban Khartoum state in September of each year. Within sampled blocks, at least five persons, including at least one child under the age of five years, were selected from each household. Blood smears were collected from the sampled individuals to examine the presence of P. falciparum parasites. Residential blocks were mapped. Data were analysed for spatial clustering using the Bernoulli model and the significance of clusters were tested using the Kulldorff scan statistic.

Results

A total of 128,510 malaria slide examinations were undertaken during the study period. In 1999, overall prevalence was 2.5%, rising to 3.2% in 2000 and consistently staying below 1% in subsequent years. From 2006, over 90% of all surveyed clusters reported no infections. Spatial clustering of infections was present in each year but not statistically significant in the years 2001, 2002, 2004 and 2008. Spatial clusters of high infection were often located at the junction of the Blue and White Niles.

Conclusion

Persisting foci of malaria infection in Khartoum are likely to distort wide area assessments and disproportionately affect future transmission within the city limits. Improved investments in surveillance that combines both passive and active case detection linked to a geographic information system and a more detailed analysis of the location and stability of foci should be undertaken to facilitate and track malaria elimination in the state of Khartoum.     

Dengue incidence in urban and rural Cambodia: results from population-based active fever surveillance, 2006-2008

Background

Dengue vaccines are now in late-stage development, and evaluation and robust estimates of dengue disease burden are needed to facilitate further development and introduction. In Cambodia, the national dengue case-definition only allows reporting of children less than 16 years of age, and little is known about dengue burden in rural areas and among older persons. To estimate the true burden of dengue in the largest province of Cambodia, Kampong Cham, we conducted community-based active dengue fever surveillance among the 0-to-19–year age group in rural villages and urban areas during 2006–2008.

Methods and Findings

Active surveillance for febrile illness was conducted in 32 villages and 10 urban areas by mothers trained to use digital thermometers combined with weekly home visits to identify persons with fever. An investigation team visited families with febrile persons to obtain informed consent for participation in the follow-up study, which included collection of personal data and blood specimens. Dengue-related febrile illness was defined using molecular and serological testing of paired acute and convalescent blood samples. Over the three years of surveillance, 6,121 fever episodes were identified with 736 laboratory-confirmed dengue virus (DENV) infections for incidences of 13.4–57.8/1,000 person-seasons. Average incidence was highest among children less than 7 years of age (41.1/1,000 person-seasons) and lowest among the 16-to-19–year age group (11.3/1,000 person-seasons). The distribution of dengue was highly focal, with incidence rates in villages and urban areas ranging from 1.5–211.5/1,000 person-seasons (median 36.5). During a DENV-3 outbreak in 2007, rural areas were affected more than urban areas (incidence 71 vs. 17/1,000 person-seasons, p<0.001).

Conclusion

The large-scale active surveillance study for dengue fever in Cambodia found a higher disease incidence than reported to the national surveillance system, particularly in preschool children and that disease incidence was high in both rural and urban areas. It also confirmed the previously observed focal nature of dengue virus transmission.