[Show abstract][Hide abstract] ABSTRACT: The effect of prior dengue virus (DENV) exposure on subsequent heterologous infection can be beneficial or detrimental depending on many factors including timing of infection. We sought to evaluate this effect by examining a large database of DENV infections captured by both active and passive surveillance encompassing a wide clinical spectrum of disease.
We evaluated datasets from 17 years of hospital-based passive surveillance and nine years of cohort studies, including clinical and subclinical DENV infections, to assess the outcomes of sequential heterologous infections. Chi square or Fisher's exact test was used to compare proportions of infection outcomes such as disease severity; ANOVA was used for continuous variables. Multivariate logistic regression was used to assess risk factors for infection outcomes.
Of 38,740 DENV infections, two or more infections were detected in 502 individuals; 14 had three infections. The mean ages at the time of the first and second detected infections were 7.6 ± 3.0 and 11.2 ± 3.0 years. The shortest time between sequential infections was 66 days. A longer time interval between sequential infections was associated with dengue hemorrhagic fever (DHF) in the second detected infection (OR 1.3, 95%CI 1.2-1.4). All possible sequential serotype pairs were observed among 201 subjects with DHF at the second detected infection, except DENV-4 followed by DENV-3. Among DENV infections detected in cohort subjects by active study surveillance and subsequent non-study hospital-based passive surveillance, hospitalization at the first detected infection increased the likelihood of hospitalization at the second detected infection.
Increasing time between sequential DENV infections was associated with greater severity of the second detected infection, supporting the role of heterotypic immunity in both protection and enhancement. Hospitalization was positively associated between the first and second detected infections, suggesting a possible predisposition in some individuals to more severe dengue disease.
BMC Public Health 12/2015; 15(1):1590. DOI:10.1186/s12889-015-1590-z · 2.26 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proper understanding of the long-term epidemiology of chikungunya has been hampered by poor surveillance. Outbreak years are unpredictable and cases often misdiagnosed. Here we analyzed age-specific data from two serological studies (from 1973 and 2012) in Cebu, Philippines, to reconstruct both the annual probability of infection and population-level immunity over a 60-year period (1952-2012). We also explored whether seroconversions during 2012-2013 were spatially clustered. Our models identified four discrete outbreaks separated by an average delay of 17 years. On average, 23% (95% CI: 16%-37%) of the susceptible population was infected per outbreak with >50% of the entire population remaining susceptible at any point. Participants who seroconverted during 2012-2013 were clustered at distances <230m suggesting focal transmission. Large-scale outbreaks of chikungunya did not result in sustained multi-year transmission. Nevertheless, we estimate >350,000 infections were missed by surveillance systems. Serological studies could supplement surveillance to provide important insights on pathogen circulation.
The Journal of Infectious Diseases 09/2015; DOI:10.1093/infdis/jiv470 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Chikungunya virus (CHIKV) is a globally re-emerging arbovirus for which previous studies have indicated the majority of infections result in symptomatic febrile illness. We sought to characterize the proportion of subclinical and symptomatic CHIKV infections in a prospective cohort study in a country with known CHIKV circulation.
A prospective longitudinal cohort of subjects ≥6 months old underwent community-based active surveillance for acute febrile illness in Cebu City, Philippines from 2012-13. Subjects with fever history were clinically evaluated at acute, 2, 5, and 8 day visits, and at a 3-week convalescent visit. Blood was collected at the acute and 3-week convalescent visits. Symptomatic CHIKV infections were identified by positive CHIKV PCR in acute blood samples and/or CHIKV IgM/IgG ELISA seroconversion in paired acute/convalescent samples. Enrollment and 12-month blood samples underwent plaque reduction neutralization test (PRNT) using CHIKV attenuated strain 181/clone25. Subclinical CHIKV infections were identified by ≥8-fold rise from a baseline enrollment PRNT titer <10 without symptomatic infection detected during the intervening surveillance period. Selected CHIKV PCR-positive samples underwent viral isolation and envelope protein-1 gene sequencing. Of 853 subjects who completed all study procedures at 12 months, 19 symptomatic infections (2.19 per 100 person-years) and 87 subclinical infections (10.03 per 100 person-years) occurred. The ratio of subclinical-to-symptomatic infections was 4.6:1 varying with age from 2:1 in 6 month-5 year olds to 12:1 in those >50 years old. Baseline CHIKV PRNT titer ≥10 was associated with 100% (95%CI: 46.1, 100.0) protection from symptomatic CHIKV infection. Phylogenetic analysis demonstrated Asian genotype closely related to strains from Asia and the Caribbean.
Subclinical infections accounted for a majority of total CHIKV infections. A positive baseline CHIKV PRNT titer was associated with protection from symptomatic CHIKV infection. These findings have implications for assessing disease burden, understanding virus transmission, and supporting vaccine development.
[Show abstract][Hide abstract] ABSTRACT: In Viet Nam, an inactivated, mouse brain-derived vaccine for Japanese encephalitis (JE) has been given exclusively to ≤5 years old children in 3 paediatric doses since 1997. However, JE incidence remained high, especially among children aged 5-9 years. We conducted a model JE immunization programme to assess the feasibility and impact of JE vaccine administered to 1-9 year(s) children in 3 standard-dose regimen: paediatric doses for children aged <3 years and adult doses for those aged ≥3 years. Of the targeted children, 96.2% were immunized with ≥2 doses of the vaccine. Compared to the national immunization programme, JE incidence rate declined sharply in districts with the model programme (11.32 to 0.87 per 100,000 in pre-versus post-vaccination period). The rate of reduction was most significant in the 5-9 years age-group. We recommend a policy change to include 5-9 years old children in the catch-up immunization campaign and administer a 4th dose to those aged 5-9 years, who had received 3 doses of the vaccine during the first 2-3 years of life.
Journal of Health Population and Nutrition 03/2015; 33(1):207-13. · 1.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
AFRIMS longitudinal dengue surveillance in Thailand depends on the nested RT-PCR and the dengue IgM/IgG ELISA.
To examine and improve the sensitivity of the nested RT-PCR using a panel of archived samples collected during dengue surveillance.
A retrospective analysis of 16,454 dengue IgM/IgG ELISA positive cases collected between 2000 and 2013 was done to investigate the sensitivity of the nested RT-PCR. From these cases, 318 acute serum specimens or extracted RNA, previously found to be negative by the nested RT-PCR, were tested using TaqMan real-time RT-PCR (TaqMan rRT-PCR). To improve the sensitivity of nested RT-PCR, we designed a new primer based on nucleotide sequences from contemporary strains found to be positive by the TaqMan rRT-PCR. Sensitivity of the new nested PCR was calculated using a panel of 87 samples collected during 2011-2013.
Results and conclusion:
The percentage of dengue IgM/IgG ELISA positive cases that were negative by the nested RT-PCR varied from 17% to 42% for all serotypes depending on the year. Using TaqMan rRT-PCR, dengue RNA was detected in 194 (61%) of the 318 acute sera or extracted RNA previously found to be negative by the nested RT-PCR. The newly designed DENV-1 specific primer increased the sensitivity of DENV-1 detection by the nested RT-PCR from 48% to 88%, and of all 4 serotypes from 73% to 87%. These findings demonstrate the impact of genetic diversity and signal erosion on the sensitivity of PCR-based methods.
[Show abstract][Hide abstract] ABSTRACT: The WHO 'Global Strategy for Dengue Prevention and Control, 2012-2020' addresses the growing need for the treatment of dengue, and targets a 25% reduction in morbidity and 50% in mortality (using 2010 estimates as baseline). Achieving these goals requires future dengue prevention strategies that will employ both potential vaccines and sustainable vector-control measures. Maternally transferred dengue antibody is an important factor in determining the optimal age for dengue vaccination.
To estimate the seroprevalence of dengue antibodies among mothers living in an area of high endemicity - Ban Pong, Ratchaburi Province - and to assess maternal dengue antibodies transferred to cord blood.
A cross-sectional study was conducted with 141 pregnant women who delivered at Ban Pong Hospital, Ratchaburi, Thailand. Maternal-cord paired sera were tested for dengue neutralizing (NT) antibody by PRNT50 assay. A ratio of ≥ 1:10 NT titer to dengue serotype was considered seropositive.
Most mothers (137/141, 97.2%) had NT antibodies to at least one dengue serotype in their sera. At birth, the proportion of cord sera with NT antibodies to DEN-1, DEN-2, DEN-3, and DEN-4, were high and similar to the sera of their mothers, at 93.6%, 97.2%, 97.9%, and 92.2%, respectively. The dengue geometric mean titers (GMT) in cord blood were significantly higher than the maternal antibodies (p<0.001): highest in DEN-2, followed by DEN-3, and then DEN-1. The GMT of DEN-4 was the lowest among all four serotypes.
Dengue infection is highly prevalent among pregnant women in this dengue-endemic area. Most of the cord blood had transferred dengue antibodies, which may have an impact on the disease burden in this population.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: Background
Currently, no dengue NS1 detection kit has regulatory approval for the diagnosis of acute dengue fever. Here we report the sensitivity and specificity of the InBios DEN Detect NS1 ELISA using a panel of well characterized human acute fever serum specimens.
The InBios DENV Detect NS1 ELISA was tested using a panel composed of 334 serum specimens collected from acute febrile patients seeking care in a Bangkok hospital in 2010 and 2011. Of these patients, 314 were found to have acute dengue by either RT-PCR and/or anti-dengue IgM/IgG ELISA. Alongside the InBios NS1 ELISA kit, we compared the performance characteristics of the BioRad Platelia NS1 antigen kit. The InBios NS1 ELISA Ag kit had a higher overall sensitivity (86% vs 72.8%) but equal specificity (100%) compared to the BioRad Platelia kit. The serological status of the patient significantly influenced the outcome. In primary infections, the InBios NS1 kit demonstrated a higher sensitivity (98.8%) than in secondary infections (83.5%). We found significant variation in the sensitivity of the InBios NS1 ELISA kit depending on the serotype of the dengue virus and also found decreasing sensitivity the longer after the onset of illness, showing 100% sensitivity early during illness, but dropping below 50% by Day 7.
The InBios NS1 ELISA kit demonstrated high accuracy when compared to the initial clinical diagnosis with greater than 85% agreement when patients were clinically diagnosed with dengue illness. Results presented here suggest the accurate detection of circulating dengue NS1 by the InBios DENV Detect NS1 ELISA can provide clinicians with a useful tool for diagnosis of early dengue infections.
[Show abstract][Hide abstract] ABSTRACT: Dengue is endemic to the rural province of Kamphaeng Phet, Northern Thailand. A decade of prospective cohort studies has provided important insights into the dengue viruses and their generated disease. However, as elsewhere, spatial dynamics of the pathogen remain poorly understood. In particular, the spatial scale of transmission and the scale of clustering are poorly characterized. This information is critical for effective deployment of spatially targeted interventions and for understanding the mechanisms that drive the dispersal of the virus.
[Show abstract][Hide abstract] ABSTRACT: Background
Accurate determination of neutralization antibody titers supports epidemiological studies of dengue virus transmission and vaccine trials. Neutralization titers measured using the plaque reduction neutralization test (PRNT) are believed to provide a key measure of immunity to dengue viruses, however, the assay's variability is poorly understood, making it difficult to interpret the significance of any assay reading. In addition there is limited standardization of the neutralization evaluation point or statistical model used to estimate titers across laboratories, with little understanding of the optimum approach.
We used repeated assays on the same two pools of serum using five different viruses (2,319 assays) to characterize the variability in the technique under identical experimental conditions. We also assessed the performance of multiple statistical models to interpolate continuous values of neutralization titer from discrete measurements from serial dilutions. We found that the variance in plaque reductions for individual dilutions was 0.016, equivalent to a 95% confidence interval of 0.45–0.95 for an observed plaque reduction of 0.7. We identified PRNT75 as the optimum evaluation point with a variance of 0.025 (log10 scale), indicating a titer reading of 1∶500 had 95% confidence intervals of 1∶240–1∶1000 (2.70±0.31 on a log10 scale). The choice of statistical model was not important for the calculation of relative titers, however, cloglog regression out-performed alternatives where absolute titers are of interest. Finally, we estimated that only 0.7% of assays would falsely detect a four-fold difference in titers between acute and convalescent sera where no true difference exists.
Estimating and reporting assay uncertainty will aid the interpretation of individual titers. Laboratories should perform a small number of repeat assays to generate their own variability estimates. These could be used to calculate confidence intervals for all reported titers and allow benchmarking of assay performance.
[Show abstract][Hide abstract] ABSTRACT: Safety and immunogenicity of two formulations of a live-attenuated tetravalent dengue virus (TDEN) vaccine produced using rederived master seeds from a precursor vaccine were tested against a placebo control in a phase II, randomized, double blind trial (NCT00370682). Two doses were administered 6 months apart to 120 healthy, predominantly flavivirus-primed adults (87.5% and 97.5% in the two vaccine groups and 92.5% in the placebo group). Symptoms and signs reported after vaccination were mild to moderate and transient. There were no vaccine-related serious adverse events or dengue cases reported. Asymptomatic, low-level viremia (dengue virus type 2 [DENV-2], DENV-3, or DENV-4) was detected in 5 of 80 vaccine recipients. One placebo recipient developed a subclinical natural DENV-1 infection. All flavivirus-unprimed subjects and at least 97.1% of flavivirus-primed subjects were seropositive to antibodies against all four DENV types 1 and 3 months post-TDEN dose 2. The TDEN vaccine was immunogenic with an acceptable safety profile in flavivirus-primed adults.
The American journal of tropical medicine and hygiene 05/2014; 91(1). DOI:10.4269/ajtmh.13-0452 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Indian Ocean chikungunya epidemic re-emerged in Thailand in August 2008. Forty-five adults with laboratory-confirmed chikungunya in Songkhla province, Thailand were clinically assessed and serially bled throughout the acute and convalescent phase of the disease. Patient symptoms, antibody responses, and viral kinetics were evaluated using observational assessments, polymerase chain reaction (PCR), and serological assays. All subjects experienced joint pain with 42 (93%) involving multiple joints; the interphalangeal most commonly affected in 91% of the subjects. The mean duration of joint pain was 5.8 days, 11 (25%) experiencing discomfort through the duration of the study. Rash was observed in 37 (82%) subjects a mean 3.5 days post onset of symptoms. Patents were positive by PCR for a mean of 5.9 days with sustained peak viral load through Day 5. The IgM antibodies appeared on Day 4 and peaked at Day 7 and IgG antibodies first appeared at Day 5 and rose steadily through Day 24.
The American journal of tropical medicine and hygiene 02/2014; 90(3). DOI:10.4269/ajtmh.12-0681 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue virus has traditionally caused substantial morbidity and mortality among children less than 15 years of age in Southeast Asia. Over the last 2 decades, a significant increase in the mean age of cases has been reported, and a once pediatric disease now causes substantial burden among the adult population. An age-stratified serological study (n = 1,736) was conducted in 2010 among schoolchildren in the Mueang Rayong district of Thailand, where a similar study had been conducted in 1980/1981. Serotype-specific forces of infection (λ(t)) and basic reproductive numbers (R0) of dengue were estimated for the periods 1969-1980 and 1993-2010. Despite a significant increase in the age at exposure and a decrease in λ(t) from 0.038/year to 0.019/year, R0 changed only from 3.3 to 3.2. Significant heterogeneity was observed across subdistricts and schools, with R0 ranging between 1.7 and 6.8. These findings are consistent with the idea that the observed age shift might be a consequence of the demographic transition in Thailand. Changes in critical vaccination fractions, estimated by using R0, have not accompanied the increase in age at exposure. These results have implications for dengue control interventions because multiple countries in Southeast Asia are undergoing similar demographic transitions. It is likely that dengue will never again be a disease exclusively of children.
American journal of epidemiology 11/2013; 179(3). DOI:10.1093/aje/kwt256 · 5.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A four-year longitudinal cohort and geographic cluster study in rural Thailand was conducted to characterize the clinical spectrum of dengue virus (DENV) infection. Symptomatic DENV infections in the cohort were detected by active school absence-based surveillance that triggered cluster investigations around ill cohort children. Data from 189 cohort children with symptomatic DENV infection and 126 contact children in the clusters with DENV infection were analyzed. Of infected contacts, only 19% were asymptomatic; 81% were symptomatic, but only 65.9% reported fever. Symptom-based case definitions were unreliable for diagnosis. Symptomatic infections in contacts were milder with lower DENV RNA levels than the cohort. Infections in contacts with fever history were more likely to have detectable DENV RNA than infections without fever history. Mild infections identified by cluster investigations account for a major proportion of all DENV infections. These findings are relevant for disease burden assessments, transmission modeling, and determination of vaccine impact.
The American journal of tropical medicine and hygiene 10/2013; 89(6). DOI:10.4269/ajtmh.13-0424 · 2.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Japanese encephalitis virus (JEV) is endemic in the Philippines but the incidence and burden of disease are not well established. We conducted a prospective hospital-based study at San Lazaro Hospital, a tertiary level hospital in Manila, from September 2005 to December 2006. Cases were determined using an in-house dengue and Japanese encephalitis (JE) enzyme-linked immunosorbent assay in order to detect the proportion of JE cases among the acute encephalitis syndrome (AES) cases admitted to our hospital. Fifteen patients were found to have AES, of whom 6 (40%) had confirmed JE. Of the JE cases, 4 were females and 2 were males with an age range of 3-14 years. Three of the 6 JE cases occurred during July. The most common signs and symptoms on admission among JE cases were: fever, headache, loss of appetite, neck rigidity and altered sensorium. JE likely comprises a significant proportion of hospitalized AES cases among children from Manila and nearby provinces. Further studies on the nation-wide prevalence and distribution of JE in the Philippines are needed to guide health authorities in disease control and prevention strategies.
The Southeast Asian journal of tropical medicine and public health 09/2013; 44(5):791-8. · 0.72 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Background:
Despite the strong association between secondary dengue virus (DENV) infections and dengue hemorrhagic fever (DHF), the majority of secondary infections are subclinical or mild. The determinants of clinical severity remain unclear, though studies indicate a titer-dependent and time-dependent role of cross-protective anti-DENV antibodies.
Data from 2 sequential prospective cohort studies were analyzed for subclinical and symptomatic DENV infections in schoolchildren in Kamphaeng Phet, Thailand (1998-2002 and 2004-2007). Children experiencing ≥ 1 DENV infection were selected as the population for analysis (contributing 2169 person-years of follow-up).
In total, 1696 children had ≥ 1 DENV infection detected during their enrollment; 268 experienced 2 or more infections. A shorter time interval between infections was associated with subclinical infection in children seronegative for DENV at enrollment, for whom a second-detected DENV infection is more likely to reflect a true second infection (average of 2.6 years between infections for DHF, 1.9 for DF, and 1.6 for subclinical infections).
These findings support a pathogenesis model where cross-reactive antibodies wane from higher-titer, protective levels to lower-titer, detrimental levels. This is one of the first studies of human subjects to suggest a window of cross-protection following DENV infection since Sabin's challenge studies in the 1940s.
The Journal of Infectious Diseases 08/2013; 209(3). DOI:10.1093/infdis/jit436 · 6.00 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Variation in the sequence of T cell epitopes between dengue virus (DENV) serotypes is believed to alter memory T cell responses during second heterologous infections. We identified a highly conserved, novel, HLA-B57-restricted epitope on the DENV NS1 protein. We predicted higher frequencies of B57-NS126-34 -specific CD8(+) T cells in PBMC from individuals undergoing secondary rather than primary DENV infection. However, high tetramer-positive T cell frequencies during acute infection were seen in only 1 of 9 subjects with secondary infection. B57-NS126-34 -specific and other DENV epitope-specific CD8(+) T cells, as well as total CD8(+) T cells, expressed an activated phenotype (CD69(+) and/or CD38(+) ) during acute infection. In contrast, expression of CD71 was largely limited to DENV epitope-specific CD8(+) T cells. In vitro stimulation of cell lines indicated that CD71 expression was differentially sensitive to stimulation by homologous and heterologous variant peptides. CD71 may represent a useful marker of antigen-specific T cell activation. This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: This paper describes an international collaboration to carry out studies that contributed to the understanding of pathogenesis, diagnosis, treatment, and prevention of several diseases of public health importance for Thailand and the United States. In Kamphaeng Phet Province, Thailand, febrile syndromes, including encephalitis, hepatitis, hemorrhagic fever, and influenza-like illnesses, occurred commonly and were clinically diagnosed, but the etiology was rarely confirmed. Since 1982, the Kamphaeng Phet Provincial Hospital, the Thai Ministry of Public Health, and the US Army Component of the Armed Forces Research Institute of Medical Sciences, along with vaccine manufacturers and universities, have collaborated on studies that evaluated and capitalized on improved diagnostic capabilities for infections caused by Japanese encephalitis, hepatitis A, dengue, and influenza viruses. The collaboration clarified clinical and epidemiological features of these infections and, in large clinical trials, demonstrated that vaccines against Japanese encephalitis and hepatitis A viruses were over 90% efficacious, supporting licensure of both vaccines. With the introduction of Japanese encephalitis vaccines in Thailand's Expanded Program of Immunization, reported encephalitis rates dropped substantially. Similarly, in the US, particularly in the military populations, rates of hepatitis A disease have dropped with the use of hepatitis A vaccine. Studies of the pathogenesis of dengue infections have increased understanding of the role of cellular immunity in responding to these infections, and epidemiological studies have prepared the province for studies of dengue vaccines. Approximately 80 publications resulted from this collaboration. Studies conducted in Kamphaeng Phet provided experience that contributed to clinical trials of hepatitis E and HIV vaccines, conducted elsewhere. To provide a base for continuing studies, The Kamphaeng Phet-AFRIMS Virology Research Unit (KAVRU) was established. This paper reviews the origins of the collaboration and the scientific observations made between 1982 and 2012.