[Show abstract][Hide abstract] ABSTRACT: 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.
[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; · 2.53 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; · 2.53 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; · 5.59 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; · 2.53 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. · 0.61 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.Methods. Data from two sequential prospective cohort studies were analyzed for subclinical and symptomatic DENV infections in school-children in Kamphaeng Phet, Thailand (1998-2002 and 2004-2007). Children experiencing at least one DENV infection were selected as the population for analysis (contributing 2169 person-years of followup).Results. 1696 children had at least one DENV infection detected during their enrollment; 268 experienced two 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 2.6 years between infections for DHF, 1.9 for DF, and 1.6 for subclinical infections).Conclusions. 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 in human subjects to suggest a window of cross-protection following DENV infection since Sabin's challenge studies in the 1940 s.
The Journal of Infectious Diseases 08/2013; · 5.85 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.
[Show abstract][Hide abstract] ABSTRACT: Revealing the patterns and determinants of the spread of dengue virus (DENV) at local scales is central to understanding the epidemiology and evolution of this major human pathogen. We performed a phylogenetic analysis of the envelope (E) genes of DENV-1, -2, -3, and -4 isolates (involving 97, 23, 5, and 74 newly collected sequences, respectively) sampled from school-based cohort and village-based cluster studies in Kamphaeng Phet, Thailand, between 2004 and 2007. With these data, we sought to describe the spatial and temporal patterns of DENV spread within a rural population where a future vaccine efficacy trial is planned. Our analysis revealed considerable genetic diversity within the study population, with multiple lineages within each serotype circulating for various lengths of time during the study period. These results suggest that DENV is frequently introduced into both semi-urban and rural areas in Kamphaeng Phet from other populations. In contrast, the persistence of viral lineages across sampling years was observed less frequently. Analysis of phylogenetic clustering indicated that DENV transmission was highly spatially and temporally focal, and that it occurred in homes rather than at school. Overall, the strength of temporal clustering suggests that seasonal bottlenecks in local DENV populations facilitate the invasion and establishment of viruses from outside of the study area, in turn reducing the extent of lineage persistence.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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).
[Show abstract][Hide abstract] ABSTRACT: It is well-known that the distribution of immunity in a population dictates the future incidence of infectious disease, but this process is generally understood at individual or macroscales. For example, herd immunity to multiple pathogens has been observed at national and city levels. However, the effects of population immunity have not previously been shown at scales smaller than the city (e.g., neighborhoods). In particular, no study has shown long-term effects of population immunity at scales consistent with the spatial scale of person-to-person transmission. Here, we use the location of dengue patients' homes in Bangkok with the serotype of the infecting pathogen to investigate the spatiotemporal distribution of disease risk at small spatial scales over a 5-y period. We find evidence for localized transmission at distances of under 1 km. We also observe patterns of spatiotemporal dependence consistent with the expected impacts of homotypic immunity, heterotypic immunity, and immune enhancement of disease at these distances. Our observations indicate that immunological memory of dengue serotypes occurs at the neighborhood level in this large urban setting. These methods have broad applications to studying the spatiotemporal structure of disease risk where pathogen serotype or genetic information is known.
Proceedings of the National Academy of Sciences 05/2012; 109(24):9535-8. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The understanding of dengue virus (DENV) transmission dynamics and the clinical spectrum of infection are critical to informing surveillance and control measures. Geographic cluster studies can elucidate these features in greater detail than cohort studies alone.
A 4-year longitudinal cohort and geographic cluster study was undertaken in rural Thailand. Cohort children underwent pre-/postseason serology and active school absence-based surveillance to detect inapparent and symptomatic dengue. Cluster investigations were triggered by cohort dengue and non-dengue febrile illnesses (positive and negative clusters, respectively).
The annual cohort incidence of symptomatic dengue ranged from 1.3% to 4.4%. DENV-4 predominated in the first 2 years, DENV-1 in the second 2 years. The inapparent-to-symptomatic infection ratio ranged from 1.1:1 to 2.9:1. Positive clusters had a 16.0% infection rate, negative clusters 1.1%. Of 119 infections in positive clusters, 59.7% were febrile, 20.2% were afebrile with other symptoms, and 20.2% were asymptomatic. Of 16 febrile children detected during cluster investigations who continued to attend school, 9 had detectable viremia.
Dengue transmission risk was high near viremic children in both high- and low-incidence years. Inapparent infections in the cohort overestimated the rate of asymptomatic infections. Ambulatory children with mild febrile viremic infections could represent an important component of dengue transmission.
The Journal of Infectious Diseases 05/2012; 206(3):389-98. · 5.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Seven commercial assays were evaluated to determine their suitability for the diagnosis of acute dengue infection: (i) the Panbio dengue virus Pan-E NS1 early enzyme-linked immunosorbent assay (ELISA), second generation (Alere, Australia); (ii) the Panbio dengue virus IgM capture ELISA (Alere, Australia); (iii) the Panbio dengue virus IgG capture ELISA (Alere, Australia); (iv) the Standard Diagnostics dengue virus NS1 antigen ELISA (Standard Diagnostics, South Korea); (v) the Standard Diagnostics dengue virus IgM ELISA (Standard Diagnostics, South Korea); (vi) the Standard Diagnostics dengue virus IgG ELISA (Standard Diagnostics, South Korea); and (vii) the Platelia NS1 antigen ELISA (Bio-Rad, France). Samples from 239 Thai patients confirmed to be dengue virus positive and 98 Sri Lankan patients negative for dengue virus infection were tested. The sensitivities and specificities of the NS1 antigen ELISAs ranged from 45 to 57% and 93 to 100% and those of the IgM antibody ELISAs ranged from 85 to 89% and 88 to 100%, respectively. Combining the NS1 antigen and IgM antibody results from the Standard Diagnostics ELISAs gave the best compromise between sensitivity and specificity (87 and 96%, respectively), as well as providing the best sensitivity for patients presenting at different times after fever onset. The Panbio IgG capture ELISA correctly classified 67% of secondary dengue infection cases. This study provides strong evidence of the value of combining dengue virus antigen- and antibody-based test results in the ELISA format for the diagnosis of acute dengue infection.
[Show abstract][Hide abstract] ABSTRACT: Dengue viruses (DENV; family Flaviviridae, genus Flavivirus) are transmitted by Aedes aegypti mosquitoes and can cause dengue fever (DF), a relatively benign disease, or more severe dengue haemorrhagic fever (DHF). Arthropod saliva contains proteins delivered into the bite wound that can modulate the host haemostatic and immune responses to facilitate the intake of a blood meal. The potential effects on DENV infection of previous exposure to Ae. aegypti salivary proteins have not been investigated. We collected Ae. aegypti saliva, concentrated the proteins and fractionated them by nondenaturing polyacrylamide gel electrophoresis (PAGE). By the use of immunoblots, we analysed reactivity with the mosquito salivary proteins (MSP) of sera from 96 Thai children diagnosed with secondary DENV infections leading either to DF or DHF, or with no DENV infection, and found that different proportions of each patient group had serum antibodies reactive to specific Ae. aegypti salivary proteins. Our results suggest that prior exposure to MSP might play a role in the outcome of DENV infection in humans.
[Show abstract][Hide abstract] ABSTRACT: The mosquito-borne Japanese encephalitis virus (JEV) causes encephalitis in man but not in pigs. Complete genomes of a human, mosquito and pig isolate from outbreaks in 1982 and 1985 in Thailand were sequenced with the aim of identifying determinants of virulence that may explain the differences in outcomes of JEV infection between pigs and man. Phylogenetic analysis revealed that five of these isolates belonged to genotype I, but the 1982 mosquito isolate belonged to genotype III. There was no evidence of recombination among the Thai isolates, but there were phylogenetic signals suggestive of recombination in a 1994 Korean isolate (K94P05). Two sites of the genome under positive selection were identified: codons 996 and 2296 (amino acids 175 of the non-structural protein NS1 and 24 of NS4B, respectively). A structurally significant substitution was seen at NS4B position 24 of the human isolate compared with the mosquito and pig isolates from the 1985 outbreak in Thailand. The potential importance of the two sites in the evolution and ecology of JEV merits further investigation.
Archives of Virology 01/2012; 157(1):75-83. · 2.03 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Dengue fever and dengue hemorrhagic fever are caused by dengue virus. Dengue infection remains a burning problem of many countries. To diagnose acute dengue in the early phase we improve the low cost, rapid SYBR green real time assay and compared the sensitivity and specificity with real time Taqman(®) assay and conventional nested PCR assay.
To develop low cost, rapid and reliable real time SYBR green diagnostic dengue assay and compare with Taqman real-time assay and conventional nested PCR (modified Lanciotti).
Eight cultured virus strains were diluted in tenth dilution down to undetectable level by the PCR to optimize the primer, temperature (annealing, and extension and to detect the limit of detection of the assay. Hundred and ninety three ELISA and PCR proved dengue clinical samples were tested with real time SYBR(®) Green assay, real time Taqman(®) assay to compare the sensitivity and specificity.
Sensitivity and specificity of real time SYBR® green dengue assay (84% and 66%, respectively) was almost comparable to those (81% and 74%) of Taqman real time PCR dengue assay. Real time SYBR(®) green RT-PCR was equally sensitive in primary and secondary infection while real time Taqman was less sensitive in the secondary infection. Sensitivity of real time Taqman on DENV3 (87%) was equal to SYBR green real time PCR dengue assay.
We developed low cost rapid diagnostic SYBR green dengue assay. Further study is needed to make duplex primer assay for the serotyping of dengue virus.
North American journal of medical sciences. 10/2011; 3(10):478-85.
[Show abstract][Hide abstract] ABSTRACT: 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.
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.
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.
[Show abstract][Hide abstract] ABSTRACT: 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).
The American journal of tropical medicine and hygiene 08/2011; 85(2):341-51. · 2.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
The American journal of tropical medicine and hygiene 02/2011; 84(2):218-23. · 2.53 Impact Factor