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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).

Six assays were evaluated in this study to determine their suitability for the diagnosis of acute dengue infection using samples from 259 Sri Lankan patients with acute fevers (99 confirmed dengue cases and 160 patients with other confirmed acute febrile illnesses): (i) the Merlin dengue fever IgG & IgM combo device (Merlin), (ii) the Standard Diagnostics Dengue Duo nonstructural 1 (NS1) antigen and IgG/IgM combo device (Standard Diagnostics, South Korea), (iii) the Biosynex Immunoquick dengue fever IgG and IgM (Biosynex, France) assay, (iv) the Bio-Rad NS1 antigen strip (Bio-Rad, France), (v) the Panbio Dengue Duo IgG/IgM Cassette (Inverness, Australia), and (vi) the Panbio dengue NS1 antigen strip (Inverness, Australia). The median number of days of fever prior to admission sample collection was 5 days (interquartile range, 3 to 7 days). Sensitivity and specificity of the NS1 antigen tests ranged from 49 to 59% and from 93 to 99%, respectively, and sensitivity and sensitivity of the IgM antibody test ranged from 71 to 80% and from 46 to 90%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics Dengue Duo test gave the best compromise of sensitivity and specificity (93% and 89%, respectively) and provided the best sensitivity in patients presenting at different times after fever onset. The Merlin IgM/IgG antibody tests correctly classified 64% and 86% of the primary and secondary dengue infection cases, respectively, and the Standard Diagnostics IgM/IgG antibody tests correctly classified 71% and 83% of the primary and secondary dengue infection cases, respectively. This study provides strong evidence of the value of combining dengue antigen- and antibody-based test results in the rapid diagnostic test (RDT) format for the acute diagnosis of dengue.

A Sri Lankan fever cohort (n = 292 patients; 17.8% prevalence) was used to assess two standard diagnostic Chikungunya IgM tests. The immunochromatographic test (ICT) acute sample sensitivity (SN) was 1.9 to 3.9%, and specificity (SP) was 92.5 to 95.0%. The enzyme-linked immunosorbent assay (ELISA) gave an acute sample SN of 3.9% and an SP of 92.5% and a convalescent sample SN of 84% and an SP of 91%. These assays are not suitable for the acute diagnosis of Chikungunya virus infection.

The objective of this study was to determine the prevalence and genotypic range of Hepatitis E virus (HEV) in the pig population of northern Lao People's Democratic Republic (PDR). We collected 181 faecal samples from indigenous-breed pigs ≤ 6 months of age and the faeces was stored in RNA stabilisation buffer due to cold-chain and transport limitations. Twenty-one (11.6%) pigs had detectable HEV RNA and 43.5% of village pig herds were infected. Based on a 240 base pair-nucleotide sequence flanking the junction of open reading frames 1, 2 and 3 (ORF1, ORF2 and ORF3) the isolates were phylogenetically classified within genotype 4. Phylogenetic analyses revealed distinct genetic groupings of the Lao HEV isolates and two groups clustered with human and pig HEV isolates from China. This was the first study to demonstrate genotype 4 HEV in Lao PDR and indicates pigs are a potential reservoir for human HEV infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A Phase I/II observer-blind, randomized, controlled trial evaluated the safety and immunogenicity of a dengue virus (DENV) vaccine candidate in healthy Thai infants (aged 12-15 months) without measurable pre-vaccination neutralizing antibodies to DENV and Japanese encephalitis virus. Fifty-one subjects received two doses of either DENV (N = 34; four received 1/10th dose) or control vaccine (N = 17; dose 1, live varicella; dose 2, Haemophilus influenzae type b). After each vaccine dose, adverse events (AEs) were solicited for 21 days, and non-serious AEs were solicited for 30 days; serious AEs (SAEs) were recorded throughout the study. Laboratory safety assessments were performed at 10 and 30 days; neutralizing antibodies were measured at 30 days. The DENV vaccine was well-tolerated without any related SAEs. After the second dose, 85.7% of full-dose DENV vaccinees developed at least trivalent and 53.6% developed tetravalent neutralizing antibodies ≥ 1:10 to DENV (control group = 0%). This vaccine candidate, therefore, warrants continued development in this age group (NCT00322049; clinicaltrials.gov).

Background: Evidence is needed on the effectiveness of non-pharmaceutical interventions (NPIs) to reduce influenza transmission., Methodology: We studied NPIs in households with a febrile, influenza-positive child. Households were randomized to control, hand washing (HW), or hand washing plus paper surgical face masks (HW + FM) arms. Study nurses conducted home visits within 24 hours of enrollment and on days 3, 7, and 21. Respiratory swabs and serum were collected from all household members and tested for influenza by RT-PCR or serology., Principal Findings: Between April 2008 and August 2009, 991 (16·5%) of 5995 pediatric influenza-like illness patients tested influenza positive. Four hundred and forty-two index children with 1147 household members were enrolled, and 221 (50·0%) were aged <6 years. Three hundred and ninety-seven (89·8%) households reported that the index patient slept in the parents' bedroom. The secondary attack rate was 21·5%, and 56/345 (16·3%; 95% CI 12·4-20·2%) secondary cases were asymptomatic. Hand-washing subjects reported 4·7 washing episodes/day, compared to 4·9 times/day in the HW + FM arm and 3·9 times/day in controls (P = 0·001). The odds ratios (ORs) for secondary influenza infection were not significantly different in the HW arm (OR = 1·20; 95% CI 0·76-1·88; P-0.442), or the HW + FM arm (OR = 1·16; 95% CI .0·74-1·82; P = 0.525)., Conclusions: Influenza transmission was not reduced by interventions to promote hand washing and face mask use. This may be attributable to transmission that occurred before the intervention, poor facemask compliance, little difference in hand-washing frequency between study groups, and shared sleeping arrangements. A prospective study design and a careful analysis of sociocultural factors could improve future NPI studies., (© 2011 Blackwell Publishing Ltd.)

The Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System Operations (AFHSC-GEIS) initiated a coordinated, multidisciplinary program to link data sets and information derived from eco-climatic remote sensing activities, ecologic niche modeling, arthropod vector, animal disease-host/reservoir, and human disease surveillance for febrile illnesses, into a predictive surveillance program that generates advisories and alerts on emerging infectious disease outbreaks. The program's ultimate goal is pro-active public health practice through pre-event preparedness, prevention and control, and response decision-making and prioritization. This multidisciplinary program is rooted in over 10 years experience in predictive surveillance for Rift Valley fever outbreaks in Eastern Africa. The AFHSC-GEIS Rift Valley fever project is based on the identification and use of disease-emergence critical detection points as reliable signals for increased outbreak risk. The AFHSC-GEIS predictive surveillance program has formalized the Rift Valley fever project into a structured template for extending predictive surveillance capability to other Department of Defense (DoD)-priority vector- and water-borne, and zoonotic diseases and geographic areas. These include leishmaniasis, malaria, and Crimea-Congo and other viral hemorrhagic fevers in Central Asia and Africa, dengue fever in Asia and the Americas, Japanese encephalitis (JE) and chikungunya fever in Asia, and rickettsial and other tick-borne infections in the U.S., Africa and Asia.

Capacity-building initiatives related to public health are defined as developing laboratory infrastructure, strengthening host-country disease surveillance initiatives, transferring technical expertise and training personnel. These initiatives represented a major piece of the Armed Forces Health Surveillance Center, Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) contributions to worldwide emerging infectious disease (EID) surveillance and response. Capacity-building initiatives were undertaken with over 80 local and regional Ministries of Health, Agriculture and Defense, as well as other government entities and institutions worldwide. The efforts supported at least 52 national influenza centers and other country-specific influenza, regional and U.S.-based EID reference laboratories (44 civilian, eight military) in 46 countries worldwide. Equally important, reference testing, laboratory infrastructure and equipment support was provided to over 500 field sites in 74 countries worldwide from October 2008 to September 2009. These activities allowed countries to better meet the milestones of implementation of the 2005 International Health Regulations and complemented many initiatives undertaken by other U.S. government agencies, such as the U.S. Department of Health and Human Services, the U.S. Agency for International Development and the U.S. Department of State.

The Armed Forces Health Surveillance Center's Division of Global Emerging Infections Surveillance and Response System (AFHSC-GEIS) supports and oversees surveillance for emerging infectious diseases, including respiratory diseases, of importance to the U.S. Department of Defense (DoD). AFHSC-GEIS accomplishes this mission by providing funding and oversight to a global network of partners for respiratory disease surveillance. This report details the system's surveillance activities during 2009, with a focus on efforts in responding to the novel H1N1 Influenza A (A/H1N1) pandemic and contributions to global public health. Active surveillance networks established by AFHSC-GEIS partners resulted in the initial detection of novel A/H1N1 influenza in the U.S. and several other countries, and viruses isolated from these activities were used as seed strains for the 2009 pandemic influenza vaccine. Partners also provided diagnostic laboratory training and capacity building to host nations to assist with the novel A/H1N1 pandemic global response, adapted a Food and Drug Administration-approved assay for use on a ruggedized polymerase chain reaction platform for diagnosing novel A/H1N1 in remote settings, and provided estimates of seasonal vaccine effectiveness against novel A/H1N1 illness. Regular reporting of the system's worldwide surveillance findings to the global public health community enabled leaders to make informed decisions on disease mitigation measures and controls for the 2009 A/H1N1 influenza pandemic. AFHSC-GEIS's support of a global network contributes to DoD's force health protection, while supporting global public health.

Background: Dengue viruses are a major cause of morbidity in tropical and subtropical regions of the world. Inapparent dengue is an important component of the overall burden of dengue infection. It provides a source of infection for mosquito transmission during the course of an epidemic, yet by definition is undetected by health care providers. Previous studies of inapparent or subclinical infection have reported varying ratios of symptomatic to inapparent dengue infection., Methodology/principal Findings: In a prospective study of school children in Northern Thailand, we describe the spatial and temporal variation of the symptomatic to inapparent (S:I) dengue illness ratio. Our findings indicate that there is a wide fluctuation in this ratio between and among schools in a given year and within schools over several dengue seasons. The most important determinants of this S:I ratio for a given school were the incidence of dengue infection in a given year and the incidence of infection in the preceding year. We found no association between the S:I ratio and age in our population., Conclusions/significance: Our findings point to an important aspect of virus-host interactions at either a population or individual level possibly due to an effect of heterotypic cross-reactive immunity to reduce dengue disease severity. These findings have important implications for future dengue vaccines.

Dengue viral isolation is necessary for definitive diagnosis, pathogenesis and evolutionary research, vaccine candidates, and diagnostic materials. Using standardized techniques, we analyzed isolation rates of 1,544 randomly selected polymerase chain reaction (PCR)-positive samples, representing all four dengue serotypes, from patients with serologically confirmed dengue infections and evaluated whether clinical and laboratory results could be predictive of isolation using standard and mosquito isolation techniques. Viruses were isolated from 62.5% of the samples by direct application to C6/36 cells and increased to 79.4% when amplifying C6/36 negative samples by intrathorasic inoculation in Toxyrhynchites splendens mosquitoes. High viremia, measured by reverse transcriptase (RT)-PCR, was a strong predictor for viral isolation by either method. Isolation was most successful in samples collected early in the disease, had low antibody levels, temperatures greater than 38°C, and had a final clinical diagnosis of dengue fever. Dengue serotypes also played a role in the success of viral isolation.

Clinically useful diagnostic tests of dengue virus infection are lacking. We prospectively evaluated the performance of real-time reverse transcriptase (rRT)-PCR, NS-1 antigen and IgM antibody tests to confirm dengue virus infection in acute blood specimens from 162 patients presenting with undifferentiated febrile illness compatible with dengue infection. rRT-PCR was the most sensitive test (89%) and potentially could be used as a single test for confirmation of dengue infection. NS-1 antigen and IgM antibody were not sufficiently sensitive to be used as a single confirmatory test with sensitivities of 54% and 17% respectively. The specificities of rRT-PCR, NS-1 antigen and IgM antibody tests were 96%, 100% and 88% respectively. Combining NS-1 and rRT-PCR or the combination of all three tests resulted in the highest sensitivity (93%) but specificities dropped to 96% and 83% respectively. We conclude that at least the combination of two tests, either agent detection (rRT-PCR) or antigen detection (NS-1) plus IgM antibody detection should be used for laboratory confirmation of dengue infection., (Copyright © 2010 Royal Society of Tropical Medicine and Hygiene. All rights reserved.)

Background: Rational infection control guidance requires an improved understanding of influenza transmission. We studied households with an influenza-infected child to measure the prevalence of influenza contamination, the effect of hand washing, and associations with humidity and temperature., Methodology: We identified children with influenza and randomly assigned their households to hand washing and control arms. Six common household surfaces and the fingertips of the index patient and symptomatic family members were swabbed. Specimens were tested by real-time reverse-transcription polymerase chain reaction (rRT-PCR), and specimens with positive results were placed on cell culture. A handheld psychrometer measured meteorological data., Results: Sixteen (17.8%) of 90 households had influenza A-positive surfaces by rRT-PCR, but no viruses could be cultured. The fingertips of 15 (16.6%) of the index patients had results positive for influenza A, and 1 virus was cultured. Index patients with seasonal influenza infections shed more virus than did patients with pandemic influenza infection. Control households had a higher prevalence of surface contamination (11 [24.4%] of 45) than did hand washing households (5 [11.1%] of 45); prevalence risk difference (PRD), 13.3%; [95% confidence interval {CI}, −2.2% to 28.9%]; P = .09). Households in which the age of the index patient was ≤8 years had a significantly higher prevalence of contamination (PRD ,19.1%; 95% CI, 5.3% -32.9%; P = .02). Within the strata of households with secondary infections, an effect of lower absolute humidity is suggested (P = .07)., Conclusions: We documented influenza virus RNA contamination on household surfaces and on the fingertips of ill children. Homes with younger children were more likely than homes of older children to have contaminated surfaces. Lower absolute humidity favors surface contamination in households with multiple infections. Increased hand washing can reduce influenza contamination in the home.

Background: Currently dengue viruses (DENV) pose an increasing threat to over 2.5 billion people in over 100 tropical and sub-tropical countries worldwide. International air travel is facilitating rapid global movement of DENV, increasing the risk of severe dengue epidemics by introducing different serotypes. Accurate diagnosis is critical for early initiation of preventive measures. Different reverse transcriptase PCR (RT-PCR) methods are available, which should be evaluated and standardized. Epidemiological and laboratory-based surveillance is required to monitor and guide dengue prevention and control programmes, i.e., by mosquito control or possible vaccination (as soon as an effective and safe vaccine becomes available)., Objective: The purpose of the external quality assurance (EQA) study described is to assess the efficiency and accuracy of dengue molecular diagnosis methods applied by expert laboratories., Study Design: A panel of 12 human plasma samples was distributed and tested for DENV-specific RNA. The panel comprised 9 samples spiked with different DENV serotypes (DENV-1 to DENV-4), including 10-fold dilution series of DENV-1 and DENV-3. Two specificity controls consisted of a sample with a pool of 4 other flaviviruses and a sample with chikungunya virus. A negative control sample was also included., Results: Thirty-seven laboratories (from Europe, Middle East Asia, Asia, the Americas/Caribbean, and Africa) participated in this EQA study, and reports including 46 sets of results were returned. Performance among laboratories varied according to methodologies used. Only 5 (10.9%) data sets met all criteria with optimal performance, and 4 (8.7%) with acceptable performance, while 37 (80.4%) reported results showed the need for improvement regarding accomplishment of dengue molecular diagnosis. Failures were mainly due to lack of sensitivity and the presence of false positives., Conclusions: The EQA provides information on each laboratory's efficacy of RT-PCR techniques for dengue diagnosis and indicates for most laboratories an urgent need to improve sensitivity and specificity.

Background: A novel influenza A/H1N1 emerged in early 2009 and by June 2009 was declared a pandemic by WHO. Rapid influenza antigen detection tests have been used to diagnose seasonal influenza but have not been adequately evaluated for the pandemic strain among all age groups. In the Philippines, pandemic influenza A/H1N1 2009 was first detected in May 2009 and by July 2009, 3207 cases and 6 deaths were reported., Objectives: Using RT-PCR as the gold standard, clinical sensitivity/specificity of Quidel QuickVue (QV) influenza A+B was estimated across all age groups for pandemic influenza A/H1N1 using nasal swabs in a hospital setting. Effect of age, viral titers (Ct values), and timing of collection on QV sensitivity to detect pandemic influenza A/H1N1 2009 was also determined., Study Design: Febrile patients with influenza-like illness (n=360) at the V. Luna General Hospital, Manila from 1 June to 31 August 2009 were included. Nasal swabs were tested using QV and RT-PCR., Results: Of 360 nasal specimens 226 (63%) were positive for pandemic influenza A/H1N1. QV sensitivity was 63% (95% confidence interval (CI): 56-69%), specificity was 96% (95% CI: 91-99%), positive predictive value was 97% (CI: 93-99%), and negative predictive value was 57% (95% CI: 49-64%). Patient's age, fever severity, presenting symptoms or number of symptoms did not significantly affect QV sensitivity, however QV sensitivity was correlated with decreasing Ct values., Conclusion: QuickVue demonstrated moderate sensitivity for pandemic influenza A/H1N1 infection. There was a significant inverse association between Ct values and QV sensitivity for pandemic influenza A/H1N1., (Copyright 2010 Elsevier B.V. All rights reserved.)

Background: In response to the 2009 H1N1 pandemic the US CDC and WHO rapidly developed and distributed a real-time RT-PCR kit to detect this strain in clinical samples. The results from the WHO swH1 primer and probe set exhibited diverse sensitivities for the 2009 influenza A/H1N1 strains in Southeast Asia (SEA)., Objective: Investigate the primer and probe-template mismatches among the 2009 influenza A/H1N1 strains in SEA that reduced the real-time RT-PCR sensitivity., Study Design: Thirty-seven swH1 positive samples categorized into sensitive and insensitive groups based on real-time RT-PCR results were selected for hemagglutinin (HA) gene sequencing. The sequence in swH1 primer and probe binding regions of the viruses was examined for mismatches. Phylogenetic analysis was performed to investigate the diversity among these viruses. Primers and probe were redesigned to match each of our sequences and tested to determine the impact on sensitivity., Results: HA sequencing of the viruses isolated from patients with high and low sensitivities revealed that a single mismatch at the 3rd base of the probe reduced sensitivity in 23/37 viruses. Homologous primers and probes increased the sensitivity (mean difference 4.66Ct P<0.0001). Phylogenetic tree revealed that the viruses in this study clustered into two groups, coinciding with RT-PCR sensitivity., Conclusion: Results obtained indicate that at least two variants of the novel H1N1 transmitting in SEA and the mutations in HA gene have a direct effect on the detection by using WHO swH1 primer and probe set., (Published by Elsevier B.V.)

We identified febrile pediatric outpatients seeking care for influenza like illness in Bangkok. Two nasal and 1 throat swab were tested using the QuickVue A+B rapid influenza kit and reverse transcription-polymerase chain reaction. Among 142 pandemic influenza A (H1N1)-positive patients, the QuickVue test identified 89 positive tests for a sensitivity of 62.7% (95% confidence interval [CI]: 54.7-70.6). Specificity was 99.2% (95% CI: 98-100). In the 0 to 2 years age group, sensitivity was 76.7% (95% CI: 61.5-91.8). Throat and nasal swabs are equally useful diagnostic specimens for reverse transcription-polymerase chain reaction diagnosis.

Background: It is unclear whether dengue serotypes differ in their propensity to cause severe disease. We analyzed differences in serotype-specific disease severity in children presenting for medical attention in Bangkok, Thailand., Methodology/principal Findings: Prospective studies were conducted from 1994 to 2006. Univariate and multivariate logistic and multinomial logistic regressions were used to determine if dengue hemorrhagic fever (DHF) and signs of severe clinical disease (pleural effusion, ascites, thrombocytopenia, hemoconcentration) were associated with serotype. Crude and adjusted odds ratios were calculated. There were 162 (36%) cases with DENV-1, 102 (23%) with DENV-2, 123 (27%) with DENV-3, and 64 (14%) with DENV-4. There was no significant difference in the rates of DHF by serotype: DENV-2 (43%), DENV-3 (39%), DENV-1 (34%), DENV-4 (31%). DENV-2 was significantly associated with increased odds of DHF grade I compared to DF (OR 2.9 95% CI 1.1, 8.0), when using DENV-1 as the reference. Though not statistically significant, DENV-2 had an increased odds of total DHF and DHF grades II, III, and IV. Secondary serologic response was significantly associated with DHF (OR 6.2) and increased when considering more severe grades of DHF. DENV-2 (9%) and -4 (3%) were significantly less often associated with primary disease than DENV-1 (28%) and -3 (33%). Restricting analysis to secondary cases, we found DENV-2 and DENV-3 to be twice as likely to result in DHF as DEN-4 (p = 0.05). Comparing study years, we found the rate of DHF to be significantly less in 1999, 2000, 2004, and 2005 than in 1994, the study year with the highest percentage of DHF cases, even when controlling for other variables., Conclusions/significance: As in other studies, we find secondary disease to be strongly associated with DHF and with more severe grades of DHF. DENV-2 appears to be marginally associated with more severe dengue disease as evidenced by a significant association with DHF grade I when compared to DENV-1. In addition, we found non-significant trends with other grades of DHF. Restricting the analysis to secondary disease we found DENV-2 and -3 to be twice as likely to result in DHF as DEN-4. Differences in severity by study year may suggest that other factors besides serotype play a role in disease severity.

Infants are a vulnerable and unique population at risk for dengue in endemic areas. This report describes the incidence and presenting clinical features of infant dengue virus (DENV) infections from a prospective community-based study performed between January 2007 and May 2009 in the Philippines. DENV3 was the predominant infecting serotype over a wide spectrum of disease severity, ranging from inapparent infection to dengue hemorrhagic fever (DHF). In 2007, the incidence of inapparent DENV infections during infancy was 103 per 1,000 persons person-years and 6-fold higher than symptomatic dengue. The age-specific incidence of infant DHF was 0.5 per 1,000 persons over the age of 3-8 months, and it disappeared by age 9 months. A febrile seizure, macular rash, petechiae, and lower platelet count were presenting clinical features associated with DENV infection among infants with acute undifferentiated febrile illnesses. Community-based studies can help to delineate the incidence rates, disease spectrum, and clinical features of DENV infections during infancy.

Eighteen dengue viruses (DENVs) representing all four serotypes, isolated from pediatric patients at children's hospital, Queen Sirikit National Institute of Child Health, Bangkok, Thailand exhibiting a diverse spectrum of disease ranging from uncomplicated dengue fever (DF) to severe dengue hemorrhagic fever (DHF), were tested for their ability to attach to host cells, replicate and interfere with the IFNalpha signaling pathway by interfering with signal transducer and activator of transcription 1 (STAT-1) function. Although most isolates suppressed IFNalpha-induced STAT-1 phosphorylation, our results showed no difference between DENV strains associated with DF and those associated with DHF. However, the DHF isolates tended replicate to higher titers in dendritic cells (DCs) than the DF isolates, but this ability was independent of their cell-binding capability. Our results suggest that the emergence early in infection of viruses with a high degree of replication fitness may play an important role in DENV pathogenesis.

Background: An increase in the average age of dengue hemorrhagic fever (DHF) cases has been reported in Thailand. The cause of this increase is not known. Possible explanations include a reduction in transmission due to declining mosquito populations, declining contact between human and mosquito, and changes in reporting. We propose that a demographic shift toward lower birth and death rates has reduced dengue transmission and lengthened the interval between large epidemics., Methods and Findings: Using data from each of the 72 provinces of Thailand, we looked for associations between force of infection (a measure of hazard, defined as the rate per capita at which susceptible individuals become infected) and demographic and climactic variables. We estimated the force of infection from the age distribution of cases from 1985 to 2005. We find that the force of infection has declined by 2% each year since a peak in the late 1970s and early 1980s. Contrary to recent findings suggesting that the incidence of DHF has increased in Thailand, we find a small but statistically significant decline in DHF incidence since 1985 in a majority of provinces. The strongest predictor of the change in force of infection and the mean force of infection is the median age of the population. Using mathematical simulations of dengue transmission we show that a reduced birth rate and a shift in the population's age structure can explain the shift in the age distribution of cases, reduction of the force of infection, and increase in the periodicity of multiannual oscillations of DHF incidence in the absence of other changes., Conclusions: Lower birth and death rates decrease the flow of susceptible individuals into the population and increase the longevity of immune individuals. The increase in the proportion of the population that is immune increases the likelihood that an infectious mosquito will feed on an immune individual, reducing the force of infection. Though the force of infection has decreased by half, we find that the critical vaccination fraction has not changed significantly, declining from an average of 85% to 80%. Clinical guidelines should consider the impact of continued increases in the age of dengue cases in Thailand. Countries in the region lagging behind Thailand in the demographic transition may experience the same increase as their population ages. The impact of demographic changes on the force of infection has been hypothesized for other diseases, but, to our knowledge, this is the first observation of this phenomenon. Please see later in the article for the Editors' Summary.

Background: Several observations support the hypothesis that vector-driven selection plays an important role in shaping dengue virus (DENV) genetic diversity. Clustering of DENV genetic diversity at a particular location may reflect underlying genetic structure of vector populations, which combined with specific vector genotype x virus genotype (G x G) interactions may promote adaptation of viral lineages to local mosquito vector genotypes. Although spatial structure of vector polymorphism at neutral genetic loci is well-documented, existence of G x G interactions between mosquito and virus genotypes has not been formally demonstrated in natural populations. Here we measure G x G interactions in a system representative of a natural situation in Thailand by challenging three isofemale families from field-derived Aedes aegypti with three contemporaneous low-passage isolates of DENV-1., Results: Among indices of vector competence examined, the proportion of mosquitoes with a midgut infection, viral RNA concentration in the body, and quantity of virus disseminated to the head/legs (but not the proportion of infected mosquitoes with a disseminated infection) strongly depended on the specific combinations of isofemale families and viral isolates, demonstrating significant G x G interactions., Conclusion: Evidence for genetic specificity of interactions in our simple experimental design indicates that vector competence of Ae. aegypti for DENV is likely governed to a large extent by G x G interactions in genetically diverse, natural populations. This result challenges the general relevance of conclusions from laboratory systems that consist of a single combination of mosquito and DENV genotypes. Combined with earlier evidence for fine-scale genetic structure of natural Ae. aegypti populations, our finding indicates that the necessary conditions for local DENV adaptation to mosquito vectors are met.

We report on a viral hepatitis serosurvey done on 381 nursing students in Bangkok; 360 (94%) were female. The mean age was 20 (+/-3.6) years (range 16-41 years); 143, 92, 86 and 59 students came from Thailand's Central, North, Northeast and South provinces, respectively. The overall prevalence of hepatitis A, B, C and E antibody in the students was 8.9%, 10.8%, 0.5% and 11.5%, respectively. The highest seroprevalence to hepatitis A was observed in cadets from the southern region of Thailand. Seroprevalence rates to hepatitis A and B were less than in other reported studies in Thailand.

An epitope-blocking enzyme-linked immunosorbent assay (b-ELISA) was evaluated for the diagnosis of West Nile virus (WNV) infections in humans. Sera from patients diagnosed with WNV infections from an outbreak in 2003 in Colorado and from patients diagnosed with dengue virus infections from Mexico and Thailand were tested with the b-ELISA. The b-ELISAs were performed using the WNV-specific monoclonal antibody (MAb) 3.1112G and the flavivirus-specific MAb 6B6C-1. Although the WNV-specific b-ELISA was effective in diagnosing WNV infections in humans from Colorado, it was not efficacious for diagnosing WNV infections in serum specimens from Mexico and Thailand. In serum specimens from patients from Colorado, the WNV b-ELISA and the WNV plaque reduction neutralization test showed an overall agreement of 91%. The sensitivity and specificity of the WNV b-ELISA were 89% and 92%, respectively, with a false-positive rate of 5%, based on receiver operating characteristic analysis. In contrast, false-positive rate results in specimens from the countries of Mexico and Thailand, where flaviviruses are endemic, were 79% and 80%, presumably due to the presence of antibodies resulting from previous dengue virus infections in Mexico and/or Japanese encephalitis virus infections or vaccination in Thailand. Thus, in regions where people have experienced previous or multiple flavivirus infections, the use of the b-ELISA for WNV diagnosis is contraindicated.

Though thrombocytopenia or dysfunction of platelets is common in dengue virus infection, the role of platelets has not been established. We enrolled 33 hospitalized children with serologically confirmed dengue virus infection. Blood specimens were collected during hospitalization. Platelets and plasma were isolated from the whole blood. Detection of dengue virus in plasma and platelets was carried out by RT-PCR with primers that can differentiate different dengue serotypes simultaneously, and by electron transmission microscopy (EM). Dengue viral RNA was detected in the platelets and plasma by conventional RT-PCR. A significantly higher percentage of dengue viral RNA was detected in platelets than in plasma (p = 0.03). Platelets isolated 5 days after onset of fever were most likely positive for viral RNA. Concurrent infection or co-circulation with multiple dengue serotypes was observed in 12% of patients. Infrequently, negative-stranded dengue viral RNA was detected in platelets and in plasma. Importantly, EM confirmed the presence of dengue viral-like particles inside platelets prepared from dengue patients. Our findings suggest the presence of dengue virus in platelets may be associated with the dysfunction of platelets observed in dengue patients.

An outbreak, characterised by fever, arthralgia and myalgia, in Malé and other islands of the Maldives began in December 2006. The illness was suspected as being due to chikungunya virus based on clinical symptoms and the prevailing chikungunya epidemic in the Indian Ocean region. The Department of Public Health initiated an investigation and collected blood samples from 67 patients; 21% were IgM-positive for chikungunya and 96% were PCR-positive. Six percent were positive for dengue by enzyme immunosorbent assay and 4% had evidence of dual infection. This is the first time that chikungunya fever has been confirmed in the Maldives. A total of 11,879 suspected and confirmed cases affecting 61% of inhabited islands (attack rate ranging from 0% to 72% on individual islands) were reported by the end of the epidemic in April 2007.

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.

In order to detect and identify dengue serotypes in serum samples, we developed a single-step quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay (referred to as Q-PCR). Sets of primers were selected from the capsid region of the viral genome. Dengue serotypes 1/3 and 2/4 were detected in two separate duplex amplification reactions using specific primers and fluorogenic TaqMan probes. Results obtained with this Q-PCR and the classical nested RT-PCR (N-PCR) assays were compared using a panel of 97 representative human sera collected from patients in Bangkok, Thailand. It is shown that the Q-PCR is a rapid, sensitive and reproducible tool for the detection and quantitation of the four dengue serotypes in clinical samples, and therefore of great interest for diagnostic use or for large cohort studies.

To determine the extent and structure of genetic variation in dengue viruses (DENV) on a restricted spatial and temporal scale, we sequenced the E (envelope) genes of DENV-1, -2, and -3 isolates collected in 2001 from children enrolled in a prospective school-based study in Kamphaeng Phet, Thailand, and diagnosed with dengue disease. Our analysis revealed substantial viral genetic variation in both time and space, with multiple viral lineages circulating within individual schools, suggesting the frequent gene flow of DENV into this microenvironment. More-detailed analyses of DENV-2 samples revealed strong clustering of viral isolates within individual schools and evidence of more-frequent viral gene flow among schools closely related in space. Conversely, we observed little evolutionary change in those viral isolates sampled over multiple time points within individual schools, indicating a low rate of mutation fixation. These results suggest that frequent viral migration into Kamphaeng Phet, coupled with population (school) subdivision, shapes the genetic diversity of DENV on a local scale, more so than in situ evolution within school catchment areas.

Global surveillance for a novel rhinovirus genotype indicated its association with community outbreaks and pediatric respiratory disease in Africa, Asia, Australia, Europe, and North America. Molecular dating indicates that these viruses have been circulating for at least 250 years.

A live-attenuated tetravalent dengue virus (DENV) vaccine candidate has been well tolerated and immunogenic in healthy, US flavivirus naive adult volunteers. We conducted a pilot, safety, and immunogenicity trial of the vaccine candidate in healthy Thai children (6-7 years of age) to prepare for its eventual evaluation in Thai infants. In an uncontrolled, open clinical trial, the investigational vaccine was administered on study Days 0 and 180 to seven volunteers residing in Bangkok without neutralizing antibodies to DENV1-4 or to Japanese encephalitis virus (JEV). Clinical and laboratory safety assessments were completed during the 30 days after each vaccine dose, and immunogenicity was determined at Day 30. In this study, the vaccine was well tolerated with no serious adverse events or alert laboratory values. One volunteer experienced fever (38.2 degrees C, < 2 days) and associated DENV4 vaccine viremia 7 days after Dose 2. One month after Dose 2, six volunteers in the per-protocol analysis exhibited a tetravalent neutralizing antibody response with DENV1-4 geometric mean titers of 55, 475, 350, and 171, respectively. Ten weeks (~75 days) after Dose 2, five of the six volunteers continued to exhibit a tetravalent neutralizing antibody profile; one volunteer's DENV4 PRNT50 titer fell below the assay cut-off (29 --> < 10); (clinicaltrials.gov NCT00384670).

We evaluated 2 commercial enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of dengue infection; one a serologic test for immunoglobulin M (IgM) antibodies, the other based on detection of dengue virus nonstructural 1 (NS1) antigen. Using gold standard reference serology on paired sera, 41% (38/92 patients) were dengue confirmed, with 4 (11%) acute primary and 33 (87%) acute secondary infections (1 was of indeterminate status). Sensitivity of the NS1-ELISA was 63% (95% confidence interval [CI], 53-73) on admission samples but was much less sensitive (5%; 95% CI, 1-10) on convalescent samples. The IgM capture ELISA had a lower but statistically equivalent sensitivity compared with the NS1-ELISA for admission samples (45%; 95% CI, 35-55) but was more sensitive on convalescent samples (58%; 95% CI, 48-68). The results of the NS1 and IgM capture ELISAs were combined using a logical OR operator, increasing the sensitivity for admission samples (79%; 95% CI, 71-87), convalescent samples (63%; 95% CI, 53-73), and all samples (71%; 95% CI, 65-78).

Immunity to a single dengue virus (DENV) infection does not provide heterologous immunity to subsequent infection. In fact, the greatest risk for dengue hemorrhagic fever (DHF) is with a second DENV serotype exposure. The risk for DHF with a third or fourth dengue infection relative to a first or second exposure is not known. An analysis of our database of children admitted to the Queen Sirikit National Institute of Child Health and Kamphaeng Phet Provincial Hospital with suspected dengue illness revealed that the number of dengue admissions caused by a third or fourth DENV infection was extremely low (0.08-0.8%). Once admitted, the risk for DHF relative to dengue fever was not different for those experiencing third or fourth DENV infections over those experiencing a second DENV infection. We document new dengue serotype infection sequences leading to DHF of 1-4, 2-3, 3-1, and 3-4.

There is an urgent need for accurate and simple dengue virus infection diagnostic assays in limited-resource settings of dengue endemicity, to assist patient management. Using a panel of reference samples (S. D. Blacksell, P. N. Newton, D. Bell, J. Kelley, M. P. Mammen, D. W. Vaughn, V. Wuthiekanun, A. Sungkakum, A. Nisalak, and N. P. Day, Clin. Infect. Dis. 42:1127-1134, 2006), we recently evaluated eihgt commercially available immunochromatographic rapid diagnostic tests (RDTs) designed to detect dengue virus-specific immunoglobulin M (IgM) and/or IgG. We found that 6/8 RDTs had sensitivities of less than 50% (range, 6 to 65%), but specificities were generally high. Here, in conjuction with dengue virus serotyping by reverse transcriptase PCR and in the limited-resource setting of Laos, where dengue virus is endemic, we evaluated the same eight RDTs against a previously validated dengue IgM/IgG enzyme-linked immunosorbent assay for diagnosis of acute dengue virus infection. Paired serum samples were collected from 87 patients, of whom 38 had confirmed dengue virus infections (4 had primary infections, 33 had secondary infections, and 1 had an infection of indeterminate status). RDT sensitivity was low, with 7/8 RDTs having admission sample sensitivities of less than 20% (range, 4 to 26%). The majority (6/8) of the RDTs, demonstrated high specificity (>95%). Kappa statistic values ranged from 6 to 54% for the RDTs, demonstrating poor to moderate variation between three operators. No RDT adequately differentiated between primary and secondary dengue virus infections. The findings of this study suggest that currently available RDTs based on the detection of IgM antibodies for the diagnosis of acute dengue virus infections are unlikely to be useful for patient management.

In response to a January 2005 report of dengue hemorrhagic fever (DHF) in Timor Leste, the World Health Organization sent a team to assist the National Hospital Guido Valadares (NHGV) in Dili with clinical case management and diagnostic support. The hospital reported 67 admissions including 8 deaths (case fatality rate approximately 12%) over the previous weeks with case histories clinically compatible with DHF. During the intervention, an additional 44 suspected dengue patients were admitted to the pediatric ward of NHGV. Among 41 patients with clinical diagnoses of dengue fever or DHF, 38 (93%) were laboratory confirmed. Although cause and effect cannot be definitely attributed, the case fatality rate decreased to 3.6% after the intervention with education about dengue management strategies.

Dengue is one of the most important diseases in the tropical and subtropical regions of the world, with an estimated 2.5 billion people being at risk. Detection of dengue virus infections has great importance for the clinical management of patients, surveillance, and clinical trial assessments. Traditionally, blood samples are collected in serum separator tubes, processed for serum, and then taken to the laboratory for analysis. The use of whole blood has the potential advantages of requiring less blood, providing quicker results, and perhaps providing better sensitivity during the acute phase of the disease. We compared the results obtained by reverse transcriptase PCR (RT-PCR) with blood drawn into tubes containing EDTA and those obtained by RT-PCR with blood samples in serum separator tubes from 108 individuals clinically suspected of being infected with dengue virus. We determined that the extraction of RNA from whole blood followed by RT-PCR resulted in a higher detection rate than the use of serum or plasma. Using a selection of these samples, we also found that our ability to detect virus by direct C6/36 cell culture and mosquito inoculation was enhanced by using whole blood but not to the same extent as that seen by the use of RT-PCR.

Why certain individuals progress to severe dengue disease is unknown. In this study, blood groups associated with dengue disease were investigated. ABO phenotypes were identified by use of serum from 399 patients with dengue-virus infection who participated in a cohort study. ABO blood-group frequencies were similar in primary versus secondary dengue-virus infections. However, in secondary infection, individuals with blood group AB were likely to have dengue hemorrhagic fever grade 3 than either grades 1 and 2 combined (corrected P value, <.0001; odds ratio, 0.097 [95% confidence interval, 0.03-0.33]) or dengue fever (corrected P value, <.0001; odds ratio, 0.119 [95% confidence interval, 0.04-0.37]). To our knowledge, this is the first report demonstrating an association between ABO blood group and the severity of dengue disease.

Previous studies have localized the region of the latency-associated transcript (LAT) of HSV-1 responsible for epinephrine-induced reactivation in the rabbit eye model to the first 1.5 kb of the primary transcript. This region extends from the 5prime prime or minute exon of the primary LAT transcript through the 5prime prime or minute half of the LAT 2.0-kb intron. To determine whether the 5prime prime or minute end of the LAT intron contributes to the induced reactivation phenotype, three recombinant viruses containing deletions within this portion of the LAT intron were constructed. The three recombinants, containing deletions spanning a combined region of 969 bp at the 5prime prime or minute end of the LAT intron, reactivated with the wild-type frequency of 17syn+. These results indicate that the elements governing induced reactivation reside within the first 699 bp of the primary LAT transcript encoding the 5prime prime or minute LAT exon.

Previous studies using cell culture systems to evaluate LAT expression demonstrated that the LAT promoter expresses at much higher levels in neuroblastoma cell lines than fibroblast lines. The high level of LAT expression in neuronal-derived cell lines correlates with the high level of LAT accumulation observed in sensory ganglia neurons during a latent infection. We have found that using LAT promoters to express reporter genes from recombinant viruses in vivo produces high levels of LAT promoter activity in the epithelium of the mouse foot. An analysis of LAT promoter activity during an acute infection in the mouse clearly demonstrates that in contrast to studies performed with selected cell lines, the LAT promoter expresses similar levels of reporter gene product in peripheral cells and in neurons. In addition, the amount of reporter gene product is higher when the LAT promoter is located within the R(L) as compared to the U(L) region, and when expression is adjusted for copy number of the reporter construct, expression is roughly the same. These results suggest the activity of the LAT promoter varies greatly according to cell type and that high levels of expression is not limited solely to neurons, especially in the in vivo setting., (Copyright 1999 Academic Press.)

It has become increasingly clear that the fate of herpes simplex virus (HSV) infections involves complex interactions between the virus and the specific cell types that comprise the tissues of the animal host. No reliable cell culture system for studying the establishment of latency and reactivation exists, and therefore these studies must be performed within animal models. One difficulty in elucidating the molecular regulation of these events is in determining the transcriptional activity of key viral genes during different stages of the infection in vivo. The heterogeneous cell types comprising infected tissues make PCR analysis of tissue homogenates difficult to interpret. The need to characterize expression of multiple transcriptional markers reliably and quantitatively at the level of individual cells is therefore key to determining the interplay between viral and cellular genes during latency and reactivation. Here we discuss the construction and evaluation of HSV reporter viruses that have been used in these analyses. HSV-1 recombinants have been engineered with representative viral promoters driving beta-galactosidase as a reporter. Methodology used to evaluate the levels of gene expression using (1) quantitative enzyme assays, (2) precipitatable substrate assays to localize the positive cells, and (3) immunohistochemistry and fluorescence assays to look at colocalization of markers during in vivo infection is presented. In addition to studying the molecular pathogenesis of HSV, the application of similar reporter viruses to evaluate promoters for targeting various differentiated tissues will be useful in developing these viruses as potential vectors for gene therapy., (Copyright 1998 Academic Press.)