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Chonlatip Pipattanaboon,1,3,8,* Tadahiro Sasaki,2,8,* Mitsuhiro Nishimura,2,8 Chayanee Setthapramote,1,8 Pannamthip Pitaksajjakul,1,4,8 Pornsawan Leaungwutiwong,1,3,8 Kriengsak Limkittikul,5,8 Orapim Puiprom,6 Mikiko Sasayama,6 Panjaporn Chaichana,6 Tamaki Okabayashi,6 Takeshi Kurosu,2,8 Ken-ichiro Ono,7,8 Pongrama Ramasoota,1,4,8 Kazuyoshi Ikuta2,8 1Center of Excellence for Antibody Research, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand; 2Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; 3Department of Microbiology and Immunology, 4Department of Social and Environmental Medicine, 5Department of Tropical Pediatrics, Faculty of Tropical Medicine, Mahidol University, 6Mahidol-Osaka Center for Infectious Diseases, Bangkok, Thailand; 7Medical and Biological Laboratories Corporation Ltd, Nagano, Japan; 8JST/JICA, Science and Technology Research Partnership for Sustainable Development, Tokyo, Japan *These authors made an equal contribution to this study Background: Hybridomas that produce human monoclonal antibodies (HuMAbs) against Dengue virus (DV) had been prepared previously using peripheral blood lymphocytes from patients with DV during the acute and convalescent phases of a secondary infection. Anti-DV envelope glycoprotein (E) 99 clones, anti-DV premembrane protein (prM) 8 clones, and anti-DV nonstructural protein 1 (NS1) 4 clones were derived from four acute-phase patients, and anti-DV E 2 clones, anti-DV prM 2 clones, and anti-DV NS1 8 clones were derived from five convalescent-phase patients. Methods and results: In the present study, we examined whether these clones cross-reacted with Japanese encephalitis virus (JEV), which belongs to the same virus family. Forty-six of the above-described 99 (46/99) anti-E, 0/8 anti-prM, and 2/4 anti-NS1 HuMAbs from acute-phase, and 0/2 anti-E, 0/2 anti-prM, and 5/8 anti-NS1 HuMAbs from convalescent-phase showed neutralizing activity against JEV. Thus, most of the anti-E and anti-NS1 (but not the anti-prM) antibodies cross-reacted with JEV and neutralized this virus. Interestingly, 3/46 anti-E HuMAbs derived from acute-phase patients and 3/5 anti-NS1 HuMAbs from convalescent-phase patients showed particularly high neutralizing activity against JEV. Consequently, the HuMAbs showing neutralization against JEV mostly consisted of two populations: one was HuMAbs recognizing DV E and showing neutralization activity against all four DV serotypes (complex-type) and the other was HuMAbs recognizing DV NS1 and showing subcomplex-type cross-reaction with DV. Conclusion: Anti-DV E from acute phase (46/99) and anti-DV NS1 (7/12) indicate neutralizing activity against JEV. In particular, three of 46 anti-DV E clones from acute phase and three of five anti-NS1 clones from convalescent phase showed strong neutralizing activity against JEV. Keywords: Dengue virus, Japanese encephalitis virus, viral neutralization, human monoclonal antibody, envelope, nonstructural protein 1

The pharmacokinetics of albendazole/albendazole sulphoxide and praziquantel were investigated in Thai children with Giardia infection. Twenty school-age children were randomly allocated to receive either a single oral dose of albendazole (400 mg/child) or the same dose of albendazole given concurrently with a single oral dose of praziquantel (20 mg/kg). The concentrations of albendazole/albendazole sulphoxide and praziquantel in plasma samples, collected at intervals in the first 24 h post-treatment, were then quantified using HPLC with ultra-violet detection. No significant pharmacokinetic interaction between the albendazole and praziquantel was demonstrated. For albendazole sulphoxide, the active metabolite of albendazole, there was marked inter-individual variation in the maximum plasma concentration and the 'area under the curve'. The pharmacokinetics of albendazole sulphoxide were similar whether albendazole was given alone or in combination with praziquantel. [ABSTRACT FROM AUTHOR]

Background: A candidate tetravalent dengue vaccine is being assessed in three clinical trials involving more than 35,000 children between the ages of 2 and 16 years in Asian-Pacific and Latin American countries. We report the results of long-term follow-up interim analyses and integrated efficacy analyses.Methods: We are assessing the incidence of hospitalization for virologically confirmed dengue as a surrogate safety end point during follow-up in years 3 to 6 of two phase 3 trials, CYD14 and CYD15, and a phase 2b trial, CYD23/57. We estimated vaccine efficacy using pooled data from the first 25 months of CYD14 and CYD15.Results: Follow-up data were available for 10,165 of 10,275 participants (99%) in CYD14 and 19,898 of 20,869 participants (95%) in CYD15. Data were available for 3203 of the 4002 participants (80%) in the CYD23 trial included in CYD57. During year 3 in the CYD14, CYD15, and CYD57 trials combined, hospitalization for virologically confirmed dengue occurred in 65 of 22,177 participants in the vaccine group and 39 of 11,089 participants in the control group. Pooled relative risks of hospitalization for dengue were 0.84 (95% confidence interval [CI], 0.56 to 1.24) among all participants, 1.58 (95% CI, 0.83 to 3.02) among those under the age of 9 years, and 0.50 (95% CI, 0.29 to 0.86) among those 9 years of age or older. During year 3, hospitalization for severe dengue, as defined by the independent data monitoring committee criteria, occurred in 18 of 22,177 participants in the vaccine group and 6 of 11,089 participants in the control group. Pooled rates of efficacy for symptomatic dengue during the first 25 months were 60.3% (95% CI, 55.7 to 64.5) for all participants, 65.6% (95% CI, 60.7 to 69.9) for those 9 years of age or older, and 44.6% (95% CI, 31.6 to 55.0) for those younger than 9 years of age.Conclusions: Although the unexplained higher incidence of hospitalization for dengue in year 3 among children younger than 9 years of age needs to be carefully monitored during long-term follow-up, the risk among children 2 to 16 years of age was lower in the vaccine group than in the control group. (Funded by Sanofi Pasteur; ClinicalTrials.gov numbers, NCT00842530, NCT01983553, NCT01373281, and NCT01374516.). [ABSTRACT FROM AUTHOR]

Background: A serum-free, highly purified Vero rabies vaccine-next generation (PVRV-NG2) is under development. We conducted a phase III trial to describe the safety and immunogenicity profile of PVRV-NG2 compared with those of licensed purified Vero rabies vaccine (PVRV) in a simulated rabies postexposure prophylaxis (PEP) Zagreb regimen in Thailand., Methods: Healthy adults aged ≥18 years (n = 201) were randomized in a 2:1 ratio to receive PVRV-NG2 or PVRV in a rabies PEP Zagreb (days 0, 7, 21 [2-1-1]) regimen, with concomitant human rabies immunoglobulin (HRIG) at day 0. Immunogenicity end points included the proportion of participants with rabies virus-neutralizing antibody (RVNA) titers ≥0.5 IU/mL at days 0, 14, and 35. Safety outcomes were also assessed., Results: A total of 199 participants completed the study (PVRV-NG2 n = 133, PVRV n = 66). In the PVRV-NG2 group and PVRV group, respectively, 91.0% (95% CI, 84.1%-95.6%) and 94.6% (95% CI, 85.1%-98.9%) had RVNA titers ≥0.5 IU/mL at day 14, increasing to 100% (95% CI, 96.8%-100%) and 100% (95% CI, 93.5%-100%) by day 35. The vaccines had similar safety profiles, and there were no safety concerns., Conclusions: PVRV-NG2 showed acceptable safety and immunogenicity profiles when co-administered with HRIG in a simulated PEP Zagreb regimen in healthy adults in Thailand., Competing Interests: Potential conflicts of interest. D.C., C.P., F.G.M., C.F., and A.C.P.P. are employees of Sanofi and may hold shares and/or stock options in the company. Y.W. was an employee of Sanofi at the time of the study and may have held stock and/or share options in the company. All other authors report no potential conflicts., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Animal rabies is a potentially fatal infectious disease in mammals, especially dogs. Currently, the number of rabies cases in pet dogs is increasing in several regions of Thailand. However, no passive postexposure prophylaxis (PEP) has been developed to combat rabies infection in animals. As monoclonal antibodies (MAbs) are promising biological therapies for postinfection, we developed a canine-neutralizing MAb against rabies virus (RABV) via the single-chain variable fragment (scFv) platform. Immunized phage-displaying scFv libraries were constructed from PBMCs via the pComb3XSS system. Diverse canine VHVLκ and VHVLλ libraries containing 2.4 × 10 8 and 1.3 × 10 6 clones, respectively, were constructed. Five unique clones that show binding affinity with the RABV glycoprotein were then selected, of which K9RABVscFv1 and K9RABVscFv16 showed rapid fluorescent foci inhibition test (RFFIT) neutralizing titers above the human protective level of 0.5 IU/ml. Finally, in silico docking predictions revealed that the residues on the CDRs of these neutralizing clones interact mainly with similar antigenic sites II and III on the RABV glycoprotein. These candidates may be used to develop complete anti-RABV MAbs as a novel PEP protocol in pet dogs and other animals., (© 2024. The Author(s).)

Background: Dengue disease is caused by dengue virus, which is transmitted by Aedes mosquitoes in tropical and subtropical regions worldwide. Although most infected individuals have benign febrile illness or no apparent symptoms, a small percentage develop severe dengue, a potentially fatal condition that occurs after a febrile stage. Many studies have identified factors predicting dengue severity among different populations and time courses. To help find practical approaches applicable in remote settings, we focused on the investigation of early factors associated with severe dengue in Thai-Myanmar cross-border region., Methods: This retrospective case-control study was performed to determine factors contributing to severe dengue in the pediatric population. We reviewed the hospital records of patients with dengue infection aged 0-19 years who were admitted to Maesot General Hospital, situated near the Thai-Myanmar cross-border region, between 2017 and 2022. Medical data during the first 5 days of illness and outcomes were collected and analyzed., Results: This study included 144 patients with a serologically confirmed diagnosis of dengue infection, with 43 severe and 101 non-severe cases. Among biological factors, being an infant and belonging to an ethnic group in Myanmar showed a significant association with severe dengue in the univariable analysis. Multivariable logistic regression revealed that the presence of mucosal bleeding (adjusted OR 5.39, 95% CI 1.06-27.52, P = 0.043), a change in hematocrit ≥ 10% (adjusted OR 3.68, 95% CI 1.15-11.74, P = 0.028), and serum albumin < 35 g/L (adjusted OR 8.10, 95% CI 2.55-25.72, P < 0.001) during the first 5 days of illness were significantly associated with developing severe dengue., Conclusions: This study supports the use of certain WHO warning signs and hematocrit change during febrile phase to predict pediatric severe dengue in low-resource settings. Potential factors such as very young age and ethnic groups warrant further exploration to identify risks contributing to severe dengue infection., (© 2024. The Author(s).)

The frequency of Zika virus (ZIKV)-specific IgA and IgM and the cytokine expression profile of ZIKV-infected patients in hyperendemic areas remain unclear. This study investigated the rates of ZIKV non-structural protein 1 (NS1)-specific IgA and IgM and evaluated serum cytokine levels of ZIKV and Dengue virus (DENV) cases in Thailand to identify potential diagnostic biomarkers, elucidate the immunity against ZIKV and DENV, and investigate the association between cytokine levels and ZIKV symptoms. Low rates of positivity for ZIKV NS1-specific IgA and IgM were detected in our study. ZIKV NS1 IgA/M (11%, 11/101) in combination was more frequently detected than ZIKV NS1 IgM (2%, 2/101) or ZIKV NS1 IgA (4%, 4/96) alone, especially in acute ZIKV cases with previous DENV exposure (14%, 10/72). Cytokine analysis showed that both ZIKV and DENV infections induced polyfunctional immunity, and the latter triggered more prolonged responses. The existence of significant differences in IL-4 and IL-10 levels between acute ZIKV and acute DENV cases suggested that IL-4 ( p = 0.0176) and IL-10 ( p = 0.0003) may represent biomarkers for acute ZIKV and acute DENV infections, respectively. Analysis of the association between increased cytokine levels and ZIKV symptoms indicated that CXCL10 ( p = 0.0029) was associated with exanthema, while IL-5 ( p = 0.0496) was linked to headache. The detection of ZIKV NS1 IgA and IgM in combination may enhance the diagnosis of early ZIKV infection, particularly when levels of IgM or IgA alone are low or undetectable. IL-4 and IL-10 may serve as targets for the development of diagnostic tools to detect ZIKV and DENV infections early, respectively, in flavivirus-endemic regions.

Circulating hsa-miRNA-126 (CmiR-126) has been reported to involve in the pathogenesis of many infectious diseases including dengue virus infection. However, no prior study has been conducted to describe more details in dengue-infected pediatric patients. This study aimed to describe CmiR-126-3p in dengue-infected pediatric patients during the febrile and convalescent phases. Additionally, the correlations between CmiR-126-3p and other relevant clinical laboratory factors were investigated. Sixty paired-serum specimens collected during febrile and convalescent phases were retrieved from patients with dengue fever (DF) (n = 30) and dengue hemorrhagic fever (DHF) (n = 30). Thirty paired-serum specimens collected from non-dengue acute febrile illness patients (AFI) were included as the control group. CmiR-126-3p was determined using reverse transcription quantitative real-time polymerase-chain reaction (RT-qPCR). Relative miRNA expression was calculated as 2 -ΔCt using CmiR-16-5p for data normalization. CmiR-126-3p expression during febrile and convalescent phases in dengue-infected patients was significantly lower than AFI ( p < 0.05). However, miRNA levels were not different ( p > 0.05) compared between DF and DHF and between primary and secondary infection. CmiR-126-3p levels in DF in the convalescent were significantly higher than in the febrile phase ( p = 0.025). No association between CmiR-126-3p and hematocrit, WBC level, platelet count, WBC differential count or dengue viral load was observed ( p > 0.05). The data suggest that hsa-miR-126-3p involved in pathogenesis of dengue infection and may be a promising early and late biomarker for DENV infection. However, hsa-miR-126-3p alone cannot be used as a predictor for dengue severity.

Japanese encephalitis is the main cause of viral encephalitis in Asia. In a previous single-arm vaccine trial, an inactivated chromatographically purified Japanese encephalitis Vero cell vaccine (CVI-JE; JEVAC TM ) was safe and immunogenic in 152 Thai children aged 1-3 years receiving a 2-dose primary immunization and booster dose 1 year later. We conducted a 5-year follow-up assessment of the persistence of the immune response the 144 children remaining in this cohort after first booster dose. Immunity was assessed by 50% plaque reduction neutralization test annually for up to 5 years post-booster. Seroprotection rates (95%CI) decreased from 100% (97.1-100) at 1 year post-booster to 93% (85.0-98.3) at 5 years post-booster. No serious vaccine-related adverse events or Japanese encephalitis infections were reported. A 2-dose primary immunization and booster 1 year later with CVI-JE provided long-lasting immunity in the majority of children.

Arboviruses, particularly dengue virus (DENV), Zika virus (ZIKV), and Chikungunya virus (CHIKV), pose a growing threat to global public health. For disease burden estimation and disease control, seroprevalence studies are paramount. This study was performed to determine the prevalence of DENV, ZIKV, and CHIKV on healthy individuals aged from 1-55 years old in Bangphae district, Ratchaburi province, Thailand. Enzyme-linked immunosorbent assays (ELISAs) and rapid diagnostic tests (RDTs) were performed on archived samples from a dengue serological survey conducted from 2012-2015. All 2012 samples had been previously tested using an anti-DENV immunoglobulin (Ig)G ELISA, and 400 randomly selected samples stratified by age, sex, and residential area were assessed by an in-house anti-ZIKV IgG ELISA and a commercial anti-CHIKV IgG ELISA to determine virus-specific antibody levels. An RDT (Chembio DPP ® ZCD IgM/IgG System) was also used to investigate the presence of antibodies against DENV, ZIKV, or CHIKV. The ELISA results indicate that the seroprevalences of DENV, ZIKV, and CHIKV were 84.3%, 58.0%, and 22.5%, respectively. The youngest age group had the lowest seroprevalence for all three arboviruses, and the seroprevalences for these viruses were progressively higher with increasing participant age. The DPP ® IgG sensitivities, as compared with ELISAs, for DENV, ZIKV, and CHIKV were relatively low, only 43.92%, 25.86%, and 37.78%, respectively. The ELISA results indicate that 16% of the study population was seropositive for all three viruses. DENV had the highest seroprevalence. ZIKV and CHIKV were also circulating in Bangphae district, Ratchaburi province, Thailand. The DPP ® ZCD rapid test is not sensitive enough for use in seroprevalence studies.

Zika virus (ZIKV) is a mosquito-borne flavivirus that has recently emerged as a global health threat. The rise in ZIKV infections has driven an increased incidence of neonates born with microcephaly or other neurological malformations. Therefore, screening for ZIKV infection can considerably impact pregnant women, especially during the first trimester. The majority of ZIKV infections are mild or asymptomatic, and clinical diagnosis is inaccurate. Moreover, given the high level of cross-reactivity among flaviviruses, serological approaches to distinguish ZIKV from dengue virus (DENV) infections are complicated. We used the combination of DENV and ZIKV nonstructural protein 1 (NS1) IgG enzyme-linked immunosorbent assay (ELISA) and ZIKV NS1 blockade-of-binding (BOB) ELISA to test the convalescent sera of non-flavivirus, primary DENV, secondary DENV, and ZIKV infections. Our findings indicate that primary testing using a ZIKV NS1 IgG ELISA, the test of choice for large-scale ZIKV serosurvey studies, provided relatively high sensitivity. Moreover, the confirmation of positive ELISA results using the ZIKV NS1 BOB ELISA increased average specificity to 94.59% across serum samples. The combined use of two simple ELISAs for ZIKV serosurveys and the monitoring of ZIKV infection during pregnancy can elucidate the epidemiology, pathogenesis, and complications of ZIKV in DENV-endemic areas.

Most cases of dengue virus infection are mild, but severe cases can be fatal. Therefore, identification of factors associated with dengue severity is essential to improve patient outcomes and reduce mortality. The objective of this study was to assess associations between nutritional status and dengue severity among Thai children and adolescents. This retrospective cross-sectional study was based on the medical records of 355 patients with dengue treated at the Hospital for Tropical Disease (Bangkok, Thailand) from 2017 to 2019. Subjects were Thai children aged less than 18 years with dengue virus infection confirmed by positive NS1 antigen or IgM. The 1997 and 2009 World Health Organization (WHO) dengue classifications were used to define disease severity and body mass index for age while the WHO growth chart was used to classify nutritional status. The proportions of patients with dengue fever who were underweight, normal weight, and overweight were 8.8%, 61.5%, and 29.7%, respectively. The proportions of patients with dengue haemorrhagic fever (DHF) who were underweight, normal weight, and overweight were 10.2%, 66.1%, and 23.7%, respectively. The proportions of patients with non-severe dengue who were underweight, normal weight, and overweight were 8.6%, 60.9%, and 30.5%, respectively; the same proportions of patients with severe dengue were 10.5%, 67.1%, and 22.4%, respectively. Higher proportions of patients with severe plasma leakage (DHF grade III and IV) were overweight compared with those with mild plasma leakage (DHF grade I and II) (45.5% vs. 18.8%). No difference in nutritional status was observed in patients with different dengue severity., Competing Interests: The authors have declared that no competing interests exist.

Purpose: The purpose of this study was to identify the risk factors for recurrent abdominal pain (RAP) in children who presented with nonorganic acute abdominal pain., Methods: A retrospective, single study was conducted on 2-15-year-old children diagnosed with nonorganic acute abdominal pain at the pediatric outpatient department of Vajira Hospital, Nawamindradhiraj University, between January 2015 and December 2019. The potential risk factors were analyzed using univariate and multivariate analyses., Results: Of the 367 patients with nonorganic acute abdominal pain, 94 (25.6%) experienced RAP within three months. In this group with RAP, 76 patients (80.8%) were diagnosed with functional gastrointestinal disorders, including functional dyspepsia, irritable bowel syndrome, functional abdominal pain-not otherwise specified, and functional constipation. History of gastrointestinal infection ( p =0.011), mental health problems ( p =0.022), abdominal pain lasting ≥7 days ( p <0.001), and change in stool frequency ( p =0.001) were the independent risk factors associated with RAP in children with nonorganic acute abdominal pain; their odds ratios and 95% confidence intervals were 3.364 (1.314-8.162), 3.052 (1.172-7.949), 3.706 (1.847-7.435), and 2.649 (1.477-4.750), respectively., Conclusion: RAP is a common problem among children who first present with nonorganic acute abdominal pain. The identification of risk factors may provide proper management, especially follow-up plans for this group in the future., Competing Interests: Conflict of Interest: The authors have no financial conflicts of interest., (Copyright © 2022 by The Korean Society of Pediatric Gastroenterology, Hepatology and Nutrition.)

In 2016, Zika virus (ZIKV) infection was declared a public health emergency of international concern because of the neurological consequences in babies born to infected people. Because of the mild and nonspecific symptoms, serological tests are essential in epidemiological studies. However, cross-reactive antibodies between other Flaviviridae members may complicate the interpretation of results of these tests. This study investigated the seroprevalence of ZIKV infection in Samut Songkhram in central Thailand which was affected by the Zika outbreak of 2016. Three hundred and fifty volunteers aged 5-50 years in Amphawa District, Samut Songkhram, were enrolled between April 2017 and April 2018. ZIKV nonstructural protein 1 (NS1) immunoglobulin G enzyme-linked immunosorbent assay (ELISA) was used to screen serum samples collected on the first day of enrollment and after 6 and 12 months. The seroprevalence and seroconversion of ZIKV were assessed. Cases of ZIKV seroconversion were verified as evidence of ZIKV infection by NS1 blockade-of-binding ELISA and plaque reduction neutralization test (PRNT50). ZIKV seroprevalence in Amphawa was 15.1-17.8% with no significant change over the year. The total seroconversion rate throughout the year was 7/100 person-years. The ratio of asymptomatic to symptomatic infections was 4.5:1. The cases in our study confirmed the occurrence of occult ZIKV infections in the community. These undetected infections might promote the spread of ZIKV in vulnerable groups of the community.

Background: To determine the seroprevalence and transmission dynamics of dengue virus (DENV), age-stratified longitudinal serological surveys were conducted in Bangphae district, Ratchaburi province, Thailand, for 3 years between April 2012 and April 2015., Methodology: The surveys enrolled 2012 healthy children and adults between 1 and 55 years-of-age, and a longitudinal serosurvey of six repeated bleeds of the same cohort of individuals was conducted every 8 months for the first 2 years (M0, M8, M16) and every half a year (M24, M30, M36) for the rest of the study period. All samples were tested using in-house indirect sandwich dengue IgG ELISA to determine DENV antibody titer, and 640 paired samples which showed rising of DENV IgG titers in paired serum were further tested using in-house neutralization assay, Plaque Reduction Neutralization Test (PRNT50)., Principal Findings: When compared against the gold standard based on the results of PRNT50, sensitivity and specificity of indirect ELISA were found to be both about 85%. The overall DENV IgG positivity determined by ELISA was 74.3% in 2012 and increased to 79.4% by the final sample collection in 2015. In our study sample, more than 98% of subjects older than 25 years were found to be seropositive. Among 518 IgG negative subjects at enrollment, the seroconversion rates were measured in paired bleeds; the rates (between successive visits, approximately 6 months) ranged between 4.8% (between M16 and M24) and 14.7% (between M0 and M8). The dominant serotype of primary DENV infection cases based on seroconversion was identified from the PRNT results and it was DENV-2., Conclusions: Our study documented high levels of seroprevalence and rate of transmission. Given the importance of the serostatus and disease burden in consideration for dengue vaccine introduction, our data could be used in decision-making on implementation of various dengue control and preventive measures., Competing Interests: The authors have declared that no competing interests exist.

The recombinant, live, attenuated, tetravalent dengue vaccine CYD-TDV has shown efficacy against all four dengue serotypes. In this exploratory study (CYD59, NCT02827162), we evaluated potential associations of host human leukocyte antigen (HLA) alleles with dengue antibody responses, CYD-TDV vaccine efficacy, and virologically-confirmed dengue (VCD) cases. Children 4-11 years old, who previously completed a phase 2b efficacy study of CYD-TDV in a single center in Thailand, were included in the study. Genotyping of HLA class I and II loci was performed by next-generation sequencing from DNA obtained from 335 saliva samples. Dengue neutralizing antibody titers (NAb) were assessed as a correlate of risk and protection. Regression analyses were used to assess associations between HLA alleles and NAb responses, vaccine efficacy, and dengue outcomes. Month 13 NAb log geometric mean titers (GMTs) were associated with decreased risk of VCD. In the vaccine group, HLA-DRB1*11 was significantly associated with higher NAb log GMT levels (beta: 0.76; p = 0.002, q = 0.13). Additionally, in the absence of vaccination, HLA associations were observed between the presence of DPB1*03:01 and increased NAb log GMT levels (beta: 1.24; p = 0.005, q = 0.17), and between DPB1*05:01 and reduced NAb log GMT levels (beta: -1.1; p = 0.001, q = 0.07). This study suggests associations of HLA alleles with NAb titers in the context of dengue outcomes. This study was registered with clinicaltrials.gov: NCT02827162., Competing Interests: Declaration of Competing Interest D.C., C.F., A.B., F.B., S.G. are employees of Sanofi Pasteur and may hold shares and/or stock options in the company. R.T., K.L., W.H., S.S., W.T., R.G.J., A.G., A.S., P.E., N.L.M., C.B. have no conflict of interest to declare. P.B.G., Z.M., M.J., and S.S.L. received a contract from Sanofi Pasteur to conduct the statistical analysis work., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Zika virus (ZIKV) is the mosquito-transmitted virus that the WHO declared a Public Health Emergency of International Concern in 2016 due to the consequence of microcephaly from infected pregnancies. The incidence of Zika infection has been unclear in many countries because most infected people have nonspecific febrile illnesses. This study's aim is to investigate the incidence of symptomatic Zika virus infections from the archived samples of a dengue cohort study of children in central Thailand from 2006 to 2009. We performed Zika NS1 immunoglobulin (Ig)G enzyme-linked immunosorbent assay (ELISA) screening to identify symptomatic Zika infections in paired acute/convalescent serum samples. Symptomatic Zika infections were confirmed by reverse transcription polymerase chain reactions (RT-PCR) of acute serum samples. The comparison of the Zika NS1 IgG ELISA results between acute and convalescent samples showed 290/955 (30.4%) seropositive cases. Zika RT-PCR results were positive in 28 febrile cases (15 females, 13 males). Zika RT-PCR showed that symptomatic Zika infection occurred in children aged 4-11 years in Ratchaburi province, Thailand (2007-2009, first case in April 2007), and the symptomatic Zika:dengue infection ratio was 28 Zika:394 dengue (1:14). Phylogenetic analysis showed that all Zika viruses were of Asian lineage. Zika NS1 IgG ELISA identified Zika-infected patients and showed a low Zika:dengue ratio.

Background: Dengue is a major public health problem in Thailand, but data are often focused on certain dengue-endemic areas. Methods: To better understand dengue epidemiology and clinical characteristics in Thailand, a fever surveillance study was conducted among patients aged 1-55 years, who presented with non-localized febrile illness at Bang Phae Community Hospital in Ratchaburi province, Thailand from October 2011 to September 2016., Results: Among 951 febrile episodes, 130 were dengue-confirmed. Individuals aged 10-14 years were mostly affected, followed by those 15-19 years-of-age, with about 15% of dengue-confirmed cases from adults 25 years and older. There were annual peaks of dengue occurrence between June-November. Most prevalent serotype in circulation was DENV-2 in 2012, DENV-3 in 2014, and DENV-4 & -3 in 2015. Among dengue cases, 65% were accurately detected using the dengue NS1 RDT. Detection rate was similar between secondary and primary dengue cases where 66% of secondary vs. 60% of primary dengue cases had positive results on the NS1 RDT. Among dengue cases, 66% were clinically diagnosed with suspected dengue or DHF, prior to lab confirmation. Dengue was positively associated with rash, headache, hematemesis and alterations to consciousness, when compared to non-dengue. Dengue patients were 10.6 times more likely to be hospitalized, compared to non-dengue cases. Among dengue cases, 95 were secondary and 35 were primary infections. There were 8 suspected DHF cases and all were identified to be secondary dengue. Secondary dengue cases were 3.5 times more likely to be hospitalized compared to primary dengue cases. Although the majority of our dengue-positive patients were secondary dengue cases, with few patients showing manifestations of DHF, our dengue cases were mostly mild disease. Even among children < 10 years-of-age, 61% had secondary infection and the rate of secondary infection increased with age., Conclusion: While the majority of dengue-confirmed cases were children, almost three-quarters of dengue-confirmed cases in this study were secondary dengue. Our study results consistent with previous data from the country confirm the hyperendemic transmission of DENV in Thailand, even in the non-epidemic years. With various interventions becoming available for dengue prevention and control, including dengue vaccines, decision-making on future implementation strategies should be based on such burden of disease data., Competing Interests: The authors have declared that no competing interests exist.

Objectives: This study aimed to evaluate a microfluidic paper-based analytical device (DEN-NS1-PAD) based on a rapid NS1 antigen test for diagnosing dengue at the point of care., Methods: 219 serum samples from suspected dengue cases were tested with the developed DEN-NS1-PAD and commercial RDT by SD BIOLINE. The results were compared with the nested-PCR results., Results: The limit of detection of DEN-NS1-PAD was 0.78 ng mL -1 . It showed 88.89% sensitivity, 86.67% specificity, and a substantial agreement correlation (κ = 0.7522) compared with nested-PCR. In contrast, SD BIOLINE for NS1 (SD-NS1) detection showed 87.88% sensitivity, 90.00% specificity, and had a substantial agreement correlation with nested-PCR (κ = 0.7788)., Conclusions: DEN-NS1-PAD is a valuable tool for diagnosing DENV infections, especially for diagnosed patients with early acute phase samples with high viral load. DEN-NS1-PAD has better sensitivity than SD-NS1 but less specificity., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The diagnosis of dengue infection is still a critical factor determining success in the clinical management and treatment of patients. Here, the development of microfluidic paper-based analytical devices (μPADs) utilizing a sandwich immunoassay on wax patterned paper functionalized with anti-dengue NS1 monoclonal antibodies for point-of-care detection of dengue NS1 (DEN-NS1-PAD) is reported. Various assay conditions, including the length of the channel and diluent, were optimized, and the response detected by the naked eye and digitized images within 20-30 min. The DEN-NS1-PAD was successfully tested in the field for detecting dengue NS1 in buffer, cell culture media, and human serum. The limit of detection (LoD) of the DEN-NS1-PAD obtained with the naked eye, scanner, and a smartphone camera was 200, 46.7, and 74.8 ng mL -1 , respectively. The repeatability, reproducibility, and stability of the DEN-NS1-PAD were also evaluated. High true specificity and sensitivity in the serum of pediatric patients were observed. These evaluation results confirm that the DEN-NS1-PAD can potentially be used in point-of-care dengue diagnostics, which can significantly impact on the spreading of mosquito-borne diseases, which are likely to become more prevalent with the effects of global warming. Graphical Abstract.

Background: Dengue is an important mosquito-borne disease. There is currently only one licensed vaccine for dengue prevention. The vaccine provides higher efficacy in pre-vaccination dengue-seropositive persons but a higher risk of subsequent more severe dengue in dengue-seronegative persons. It is recommended that the dengue vaccine may be given in dengue-seropositive individuals or as mass vaccination without individual pre-vaccination screening in areas where the dengue seroprevalence is > 80% in children aged 9 years. We evaluated a dengue specific immunoglobulin G monoclonal antibody-based capture enzyme-linked immunosorbent assay (MAb-ELISA) in the diagnosis of previous dengue infection using serum samples from the cohort study in Ratchaburi Province, Thailand., Methods: The MAb-ELISA was compared to 70% plaque reduction neutralization test (PRNT70) in 453 serum samples from children aged 3-11 years in Ratchaburi Province, Thailand., Results: The sensitivity and specificity of MAb-ELISA at the positive to negative (P/N) ratio cut-off level of > 3 were both 0.91 in the diagnosis of previous dengue infection, compared to PRNT70. The false positivity was mainly in Japanese encephalitis (JE) seropositive subjects., Conclusions: This research provides evidence that MAb-ELISA is useful for dengue seroprevalence study and dengue pre-vaccination screening. JE seropositivity was the major cause of false positive result in the study population.

The human leukocyte antigen (HLA) genes are highly variable and are known to play an important role in disease outcomes, including infectious diseases. Prior knowledge of HLA polymorphisms in a population usually forms the basis for an effective case-control study design. As a prelude to future disease association analyses, we report HLA class I and II diversity in 334 unrelated donors from a Dengue vaccine efficacy trial conducted in Thailand. Long-range PCR amplification of six HLA loci was performed on DNA extracted from saliva samples. HLA-A, -B, -C, -DPB1, -DQB1 and -DRB1 were genotyped using a next-generation sequencing method presented at the 17th International HLA and Immunogenetics Workshop. In total, we identified 201 HLA alleles, including 35 HLA-A, 57 HLA-B, 28 HLA-C, 24 HLA-DPB1, 21 HLA-DQB1 and 36 HLA-DRB1 alleles. Very common HLA alleles with frequencies greater than 10 percent were A∗11:01:01, A∗33:03:01, A∗24:02:01, B∗46:01:01, C∗07:02:01, C∗01:02:01, C∗08:01:01, DPB1∗05:01:01, DPB1∗13:01:01, DPB1∗04:01:01, DPB1∗02:01:02, DQB1∗03:01:01, DQB1∗05:02:01, DQB1∗03:03:02, DRB1∗12:02:01, DRB1∗09:01:02, and DRB1∗15:02:01. A novel HLA allele, B∗15:450, had a non-synonymous substitution and occurred in more than one donor. Population-based full-length NGS HLA typing is more conclusive and provides a sound foundation for exploring disease association in a given population., (Copyright © 2018. Published by Elsevier Inc.)

Inactivated mouse-brain-derived Japanese encephalitis vaccine has a worrisome safety profile and the live attenuated vaccine is unsuitable in immunodeficiency. This study aimed to evaluate the immunogenicity and safety of an inactivated chromatographically purified Vero-cell-derived JE vaccine (CVI-JE, Beijing P-3 strain) in children. 152 healthy Thai children, with an average (SD) age of 14.4 (3.8) months, received 3 doses of CVI-JE on days 0, 7-28, and one year. Homologous JE neutralizing antibody titers (NT) were measured. All subjects had seroprotection [geometric mean titer (GMT) 150] 28 days' post 2nd vaccination. The seroprotection rates at 1 year after primary series and and 1 month after the booster were 89.3% (GMT 49) and 100% (GMT 621), respectively. Local and systemic reactions-fever (17.6%), vomiting (8%), and poor appetite (5.3%)-were noted within 28 days' post-vaccination. All these symptoms were self-limited., Conclusions: CVI-JE is safe, immunogenic, and provided high NT.

Background: The pathogenic mechanisms underlying the increased vascular permeability in dengue hemorrhagic fever (DHF) are not well understood. Enhanced cellular immune activation, especially activation of serotype-cross reactive T cells, has been implicated in plasma leakage in DHF. Changes in several biological markers and mediators including cytokines, chemokines, angiogenic factors and their receptors have been shown to correlate with disease severity. A decline in plasma levels of a soluble form of vascular endothelial growth factor receptor 2 (VEGFR2), a receptor of vascular endothelial growth factor (VEGF), has been associated with plasma leakage in dengue patients., Objective: We aimed to investigate the effect of dengue virus (DV)-specific CD8⁺ T cells on the expression of VEGFR2 on endothelial cells., Methods: An in vitro model was developed in which dengue virus-specific CD8⁺ T cells generated from peripheral blood mononuclear cells (PBMCs) of DHF patients were co-cultured with antigen-presenting cells, human umbilical vein endothelial cells (HUVECs) and activated with DV non-structural protein 3 (NS3) peptides. The expression of VEGFR2 by endothelial cells was measured., Results: DV-specific CD8⁺ T cells were serotype cross-reactive. Activation of DV-specific CD8⁺ T cells resulted in down-regulation of soluble VEGFR2 production and an up-regulation of cell-associated VEGFR2., Conclusions: Our findings indicate that activation of DV-specific T cell is associated with modulation of VEGFR2 expression that may contribute to increased VEGF responsiveness and vascular permeability.

The recent development of a nanopore-type portable DNA sequencer has changed the way we think about DNA sequencing. We can perform sequencing directly in the field, where we collect the samples. Here, we report the development of a novel method to detect and genotype tropical disease pathogens, using dengue fever as a model. By combining the sequencer with isothermal amplification that only requires a water bath, we were able to amplify and sequence target viral genomes with ease. Starting from a serum sample, the entire procedure could be finished in a single day. The analysis of blood samples collected from 141 Indonesian patients demonstrated that this method enables the clinical identification and serotyping of the dengue virus with high sensitivity and specificity. The overall successful detection rate was 79%, and a total of 58 SNVs were detected. Similar analyses were conducted on 80 Vietnamese and 12 Thai samples with similar performance. Based on the obtained sequence information, we demonstrated that this approach is able to produce indispensable information for etiologically analyzing annual or regional diversifications of the pathogens.

Rabies is a viral zoonosis affecting around 16,000-39,000 people annually. Pre-exposure vaccination should be offered to individuals at high risk of rabies exposure and is suggested for children living in rabies endemic areas.The incidence rate of dog bite in the test group was 14.1/1,000 person-years. Intradermal (ID) route of administration is considered an alternative to the standard intramuscular (IM) administration, and reduces vaccination cost. A 3-year clinical study of a rabies vaccine administered IM or ID to 12–18-month-old Thai children, simultaneously with a Japanese encephalitis (JE) vaccine (M49P2 study), revealed that all regimens used elicited an adequate immune response. In order to determine the long-term (4-8 years) rabies neutralizing antibody titers induced by the pre-exposure regimens, blood was collected annually from 68 from the M49P2 study and analyzed using a rapid fluorescence focus inhibition test. Full- IM(three doses of 1 ml/dose), half-IM(three doses of 0.5 ml/dose), and 3-ID(three doses of 0.1 ml/dose) regimens induced antibody titers above the seroprotective level throughout the study period. However, the 2-ID(two doses of 0.1 ml/dose) group had sub-seroprotective titer of 6.7%, 13.4%, 25.0%, and 36.4% in year 5, 6, 7, and 8, respectively. A secondary immune response was induced by rabies booster vaccination. This study demonstrates a reduced-dose rabies regimen may lower the cost of long-term protection against rabies for vulnerable populations, thus improving the cost-effectiveness of pre-exposure rabies vaccination in children.

Background: An acellular Pertussis (aP) vaccine containing recombinant genetically detoxified Pertussis Toxin (PTgen), Filamentous Hemagglutinin (FHA) and Pertactin (PRN) has been developed by BioNet-Asia (BioNet). We present here the results of the first clinical study of this recombinant aP vaccine formulated alone or in combination with tetanus and diphtheria toxoids (TdaP)., Methods: A phase I/II, observer-blind, randomized controlled trial was conducted at Mahidol University in Bangkok, Thailand in healthy adult volunteers aged 18-35 y. The eligible volunteers were randomized to receive one dose of either BioNet's aP or Tetanus toxoid-reduced Diphtheria toxoid-acellular Pertussis (TdaP) vaccine, or the Tdap Adacel® vaccine in a 1:1:1 ratio. Safety follow-up was performed for one month. Immunogenicity was assessed at baseline, at 7 and 28 d after vaccination. Anti-PT, anti-FHA, anti-PRN, anti-tetanus and anti-diphtheria IgG antibodies were assessed by ELISA. Anti-PT neutralizing antibodies were assessed also by CHO cell assay., Results: A total of 60 subjects (20 per each vaccine group) were enrolled and included in the safety analysis. Safety laboratory parameters, incidence of local and systemic post-immunization reactions during 7 d after vaccination and incidence of adverse events during one month after vaccination were similar in the 3 vaccine groups. One month after vaccination, seroresponse rates of anti-PT, anti-FHA and anti-PRN IgG antibodies exceeded 78% in all vaccine groups. The anti-PT IgG, anti-FHA IgG, and anti-PT neutralizing antibody geometric mean titers (GMTs) were significantly higher following immunization with BioNet's aP and BioNet's TdaP than Adacel® (P< 0.05). The anti-PRN IgG, anti-tetanus and anti-diphtheria GMTs at one month after immunization were comparable in all vaccine groups. All subjects had seroprotective titers of anti-tetanus and anti-diphtheria antibodies at baseline., Conclusion: In this first clinical study, PTgen-based BioNet's aP and TdaP vaccines showed a similar tolerability and safety profile to Adacel® and elicited significantly higher immune responses to PT and FHA.

Background: We previously reported that a clinical isolate of dengue virus (DENV) is capable of causing acute-phase systemic infection in mice harboring knockouts of the genes encoding type-I and -II interferon IFN receptors (IFN-α/β/γR KO mice); in contrast, other virulent DENV isolates exhibited slow disease progression in this mice, yielding lethal infection around 20 days post-infection (p.i.). In the present study, we sought to clarify the dynamics of slow disease progression by examining disease progression of a type-2 DENV clinical isolate (DV2P04/08) in mice., Methods: The tissue distributions of DV2P04/08 in several organs of infeted mice were examined at different time points. Whole genome viral sequences from organs were determined., Results: At day 6 p.i., high levels of viral RNA (vRNA) were detected in non-neuronal organs (including peritoneal exudate cells (PECs), spleen, kidney, liver, lung, and bone marrow) but not in brain. By day 14 p.i, vRNA levels subsequently decreased in most organs, with the exception of thymus and brain. Sequence analysis of the whole genome of the original P04/08 and those of viruses recovered from mouse brain and thymus demonstrated the presence of both synonymous and non-synonymous mutations. Individual mice showed different virus populations in the brain. The vRNA sequence derived from brain of one mouse was nearly identical to the original DV2P04/08 inoculum, suggesting that there was no need for adaptation of DV2P04/08 for growth in the brain. However, quasispecies (that is, mixed populations, detected as apparent nucleotide mixtures during sequencing) were observed in the thymus of another mouse, and interestingly only mutant population invaded the brain at a late stage of infection., Conclusions: These results suggested that the mouse nearly succeeded in eliminating virus from non-neuronal organs but failed to do so from brain. Although the cause of death by DV2P04/08 infection is likely to be the result of virus invasion to brain, its processes to the death are different in individual mice. This study will provide a new insight into disease progression of DENV in mice.

Background: Dengue diagnosis confirmation and surveillance are widely based on serological assays to detect anti-dengue IgM or IgG antibodies since such tests are affordable/user-friendly. The World Health Organization identified serological based diagnosis as a potential tool to define probable dengue cases in the context of vaccine trials, while acknowledging that this may have to be interpreted with caution., Methods: In a phase IIb randomized, placebo-controlled trial assessing the efficacy of a tetravalent dengue vaccine (CYD-TDV) in Thai schoolchildren, case definition was based on virological confirmation by either serotype-specific RT-PCRs or by NS1-antigen ELISA (Clinicaltrials.gov NCT00842530). Here, we characterized suspected dengue cases using IgM and IgG ELISA to assess their utility in evaluating probable dengue cases in the context of vaccine efficacy trials, comparing virologically-confirmed and serologically diagnosed dengue in the vaccine and placebo groups. Serologically probable cases were defined as: (1) IgM positive acute- or convalescent-phase samples, or (2) IgG positive acute-phase sample and ≥4-fold IgG increase between acute and convalescent-phase samples., Results: Serological diagnosis had good sensitivity (97.1%), but low specificity (85.1%) compared to virological confirmation. A high level of false positivity through serology diagnosis particularly in the 2 months post-vaccination was observed, and is most likely related to detection of the immune response to the dengue vaccine. This lack of specificity and bias with vaccination demonstrates the limitation of using IgM and IgG antibody responses to explore vaccine efficacy., Conclusion: Reliance on serological assessments would lead to a significant number of false positives during routine clinical practice and surveillance following the introduction of the dengue vaccine., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Severe dengue is caused by host responses to viral infection, but the pathogenesis remains unknown. This is, in part, due to the lack of suitable animal models. Here, we report a non-mouse-adapted low-passage DENV-3 clinical isolate, DV3P12/08, derived from recently infected patients. DV3P12/08 caused a lethal systemic infection in type I and II IFN receptor KO mice (IFN-α/β/γR KO mice), which have the C57/BL6 background. Infection with DV3P12/08 induced a cytokine storm, resulting in severe vascular leakage (mainly in the liver, kidney and intestine) and organ damage, leading to extensive hemorrhage and rapid death. DV3P12/08 infection triggered the release of large amounts of TNF-α, IL-6, and MCP-1. Treatment with a neutralizing anti-TNF-α antibody (Ab) extended survival and reduced liver damage without affecting virus production. Anti-IL-6 neutralizing Ab partly prolonged mouse survival. The anti-TNF-α Ab suppressed IL-6, MCP-1, and IFN-γ levels, suggesting that the severe response to infection was triggered by TNF-α. High levels of TNF-α mRNA were expressed in the liver and kidneys, but not in the small intestine, of infected mice. Conversely, high levels of IL-6 mRNA were expressed in the intestine. Importantly, treatment with Angiopoietin-1, which is known to stabilize blood vessels, prolonged the survival of DV3P12/08-infected mice. Taken together, the results suggest that an increased level of TNF-α together with concomitant upregulation of Tie2/Angiopoietin signaling have critical roles in severe dengue infection.

Background: The rise in dengue fever cases and the absence of dengue vaccines will likely cause governments to consider various types of effective means for controlling the disease. Given strong public interests in potential dengue vaccines, it is essential to understand the private economic benefits of dengue vaccines for accelerated introduction of vaccines into the public sector program and private markets of high-risk countries., Methodology/principal Findings: A contingent valuation study for a hypothetical dengue vaccine was administered to 400 households in a multi-country setting: Vietnam, Thailand, and Colombia. All respondents received a description of the hypothetical dengue vaccine scenarios of 70% or 95% effectiveness for 10 or 30 years with a three dose series. Five price points were determined after pilot tests in order to reflect different local situations such as household income levels and general perceptions towards dengue fever. We adopted either Poisson or negative binomial regression models to calculate average willingness-to-pay (WTP), as well as median WTP. We found that there is a significant demand for dengue vaccines. The parametric median WTP is $26.4 ($8.8 per dose) in Vietnam, $70.3 ($23.4 per dose) in Thailand, and $23 ($7.7 per dose) in Colombia. Our study also suggests that respondents place more value on vaccinating young children than school age children and adults., Conclusions/significance: Knowing that dengue vaccines are not yet available, our study provides critical information to both public and private sectors. The study results can be used to ensure broad coverage with an affordable price and incorporated into cost benefit analyses, which can inform prioritization of alternative health interventions at the national level.

The "standard" methods of isolating dengue virus (DENV) utilize the mosquito cell line C6/36, monkey kidney LLC-MK2 cells, Vero cells, or baby hamster kidney (BHK-21) cells. However, these cells lines lack a particular DENV receptor, known as dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which is expressed on immature dendritic cells and monocytes/macrophages. This may result in less efficient virus isolation and propagation. The present study used a lentivirus vector to establish Vero and BHK-21 cell lines (Vero-DC and BHK-DC) that express human DC-SIGN stably. Five DENV strains, each passaged several times in C6/36 cells, replicated more efficiently in Vero-DC and BHK-DC than in the parental Vero or BHK-21 cells. Vero/Vero-DC and BHK-21/BHK-DC were used to isolate virus from buffy coats and plasma samples derived from 13 patients infected with DENV. Most of the viruses showed increased production in cell lines expressing DC-SIGN. However, the isolation rate was lower (15.4-46.2%) than that from C6/36 cells (84.6%). Interestingly, when the viruses were isolated in C6/36 cells prior to infecting Vero/Vero-DC and BHK-21/BHK-DC, the rate of virus production increased markedly, reaching levels higher than those initially achieved in C6/36 cells. These data suggest that Vero-DC and BHK-DC could be useful tools for virus propagation, and that human specimens may contain a factor that interferes with virus growth in mammalian cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Unlabelled: A literature survey and analysis was conducted to describe the epidemiology of dengue disease in Thailand reported between 2000 and 2011. The literature search identified 610 relevant sources, 40 of which fulfilled the inclusion criteria defined in the review protocol. Peaks in the number of cases occurred during the review period in 2001, 2002, 2008 and 2010. A shift in age group predominance towards older ages continued through the review period. Disease incidence and deaths remained highest in children aged ≤ 15 years and case fatality rates were highest in young children. Heterogeneous geographical patterns were observed with higher incidence rates reported in the Southern region and serotype distribution varied in time and place. Gaps identified in epidemiological knowledge regarding dengue disease in Thailand provide several avenues for future research, in particular studies of seroprevalence., Protocol Registration: PROSPERO CRD42012002170.

Background: The plaque reduction neutralization test (PRNT) is currently the best and most widely accepted approach to measuring virus-neutralizing and protective antibodies to dengue virus, and in assessing the immunogenicity of a dengue vaccine. However, the correlation between presence of dengue-neutralizing antibody and protection from infection is not absolute., Findings: In a cohort study in Ratchaburi Province, Thailand, 48 subjects with serologically confirmed symptomatic dengue infection were tested for pre-existing dengue neutralizing antibody using PRNT. Nine subjects had quite high pre-existing PRNT50 titers (titer >90) to subsequent infecting dengue serotypes, but still had symptomatic infections., Conclusion: This report provides evidence that PRNT may not be a good test for predicting protection against subsequent dengue infection.

Background: Commonly used trivalent vaccines contain one influenza B virus lineage and may be ineffective against viruses of the other B lineage. We evaluated the efficacy of a candidate inactivated quadrivalent influenza vaccine (QIV) containing both B lineages., Methods: In this multinational, phase 3, observer-blinded study, we randomly assigned children 3 to 8 years of age, in a 1:1 ratio, to receive the QIV or a hepatitis A vaccine (control). The primary end point was influenza A or B confirmed by real-time polymerase chain reaction (rt-PCR). Secondary end points were rt-PCR-confirmed, moderate-to-severe influenza and rt-PCR-positive, culture-confirmed influenza. The vaccine efficacy and the effect of vaccination on daily activities and utilization of health care resources were assessed in the total vaccinated cohort (2584 children in each group) and the per-protocol cohort (2379 children in the QIV group and 2398 in the control group)., Results: In the total vaccinated cohort, 62 children in the QIV group (2.40%) and 148 in the control group (5.73%) had rt-PCR-confirmed influenza, representing a QIV efficacy of 59.3% (95% confidence interval [CI], 45.2 to 69.7), with efficacy against culture-confirmed influenza of 59.1% (97.5% CI, 41.2 to 71.5). For moderate-to-severe rt-PCR-confirmed influenza, the attack rate was 0.62% (16 cases) in the QIV group and 2.36% (61 cases) in the control group, representing a QIV efficacy of 74.2% (97.5% CI, 51.5 to 86.2). In the per-protocol cohort, the QIV efficacy was 55.4% (95% CI, 39.1 to 67.3), and the efficacy against culture-confirmed influenza 55.9% (97.5% CI, 35.4 to 69.9); the efficacy among children with moderate-to-severe influenza was 73.1% (97.5% CI, 47.1 to 86.3). The QIV was associated with reduced risks of a body temperature above 39°C and lower respiratory tract illness, as compared with the control vaccine, in the per-protocol cohort (relative risk, 0.29 [95% CI, 0.16 to 0.56] and 0.20 [95% CI, 0.04 to 0.92], respectively). The QIV was immunogenic against all four strains. Serious adverse events occurred in 36 children in the QIV group (1.4%) and in 24 children in the control group (0.9%)., Conclusions: The QIV was efficacious in preventing influenza in children. (Funded by GlaxoSmithKline Biologicals; ClinicalTrials.gov number, NCT01218308.).

The non-structural protein NS2B/NS3 serine-protease complex of the dengue virus (DENV) is required for the maturation of the viral polyprotein. Dissociation of the NS2B cofactor from NS3 diminishes the enzymatic activity of the complex. In this study, we identified a small molecule inhibitor that interferes with the interaction between NS2B and NS3 using structure-based screening and a cell-based viral replication assay. A library containing 661,417 small compounds derived from the Molecular Operating Environment lead-like database was docked to the NS2B/NS3 structural model. Thirty-nine compounds with high scores were tested in a secondary screening using a cell-based viral replication assay. SK-12 was found to inhibit replication of all DENV serotypes (EC50=0.74-4.92 μM). In silico studies predicted that SK-12 pre-occupies the NS2B-binding site of NS3. Steady-state kinetics using a fluorogenic short peptide substrate demonstrated that SK-12 is a noncompetitive inhibitor against the NS2B/NS3 protease. Inhibition to Japanese encephalitis virus by SK-12 was relatively weak (EC50=29.81 μM), and this lower sensitivity was due to difference in amino acid at position 27 of NS3. SK-12 is the promising small-molecule inhibitor that targets the interaction between NS2B and NS3., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In this study, we compared the immunogenicities of two lots of meningococcal ACWY-tetanus toxoid conjugate vaccine (MenACWY-TT) that differed in serogroup A polysaccharide (PS) O-acetylation levels and evaluated their immunogenicities and safety in comparison to a licensed ACWY polysaccharide vaccine (Men-PS). In this phase III, partially blinded, controlled study, 1,170 healthy subjects aged 18 to 25 years were randomized (1:1:1) to receive one dose of MenACWY-TT lot A (ACWY-A) (68% O-acetylation), MenACWY-TT lot B (ACWY-B) (92% O-acetylation), or Men-PS (82% O-acetylation). Immunogenicity was evaluated in terms of serum bactericidal activity using rabbit complement (i.e., rabbit serum bactericidal activity [rSBA]). Solicited symptoms, unsolicited adverse events (AEs), and serious AEs (SAEs) were recorded. The immunogenicities, in terms of rSBA geometric mean titers, were comparable for both lots of MenACWY-TT. The vaccine response rates across the serogroups were 79.1 to 97.0% in the two ACWY groups and 73.7 to 94.1% in the Men-PS group. All subjects achieved rSBA titers of ≥1:8 for all serogroups. All subjects in the two ACWY groups and 99.5 to 100% in the Men-PS group achieved rSBA titers of ≥1:128. Pain was the most common solicited local symptom and was reported more frequently in the ACWY group (53.9 to 54.7%) than in the Men-PS group (36.8%). The most common solicited general symptoms were fatigue and headache, which were reported by 28.6 to 30.3% and 26.9 to 31.0% of subjects, respectively. Two subjects reported SAEs; one SAE was considered to be related to vaccination (blighted ovum; ACWY-B group). The level of serogroup A PS O-acetylation did not affect vaccine immunogenicity. MenACWY-TT (lot A) was not inferior to Men-PS in terms of vaccine response and was well tolerated.

Public health concern about dengue diseases, caused by mosquito-borne infections with four serotypes of dengue virus (DENV-1-DENV-4), is escalating in tropical and subtropical countries. Most of the severe dengue cases occur in patients experiencing a secondary infection with a serotype that is different from the first infection. This is believed to be due to antibody-dependent enhancement (ADE), by which one DENV serotype uses pre-existing anti-DENV antibodies elicited in the primary infection to facilitate entry of a different DENV serotype into the Fc receptor-positive macrophages. Recently, we prepared a number of hybridomas producing human monoclonal antibodies (HuMAbs) by using peripheral blood lymphocytes from Thai patients at acute phase of secondary infection with DENV-2. Here, we characterized 17 HuMAbs prepared from two patients with dengue fever (DF) and one patient with dengue hemorrhagic fever (DHF) that were selected as antibodies recognizing viral envelope protein and showing higher neutralization activity to all serotypes. In vivo evaluation using suckling mice revealed near perfect activity to prevent mouse lethality following intracerebral DENV-2 inoculation. In a THP-1 cell assay, these HuMAbs showed ADE activities against DENV-2 at similar levels between HuMAbs derived from DF and DHF patients. However, the F(ab')2 fragment of the HuMAb showed a similar virus neutralization activity as original, with no ADE activity. Thus, these HuMAbs could be one of the therapeutic candidates against DENV infection., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We conducted this study to identify the clinical features and risk factors for atypical acute post-streptococcal glomerulonephritis (APSGN). Thirty-five cases of atypical APSGN treated at Srinagarind Hospital during 2002-2009 were compared with 27 typical cases. The clinical symptoms, anti-streptococcal antibody titers, and laboratory data at the first hospital visit were compared between the two groups. A marked elevation in anti-streptolysin O (ASO) titer was seen more commonly in the atypical APSGN group than in the typical APSGN group (p=0.025). Significantly more patients in the atypical APSGN group had a high urine specific gravity, hematuria and pyuria than patients in the typical APSGN group (p<0.01, p<0.031, and p<0.046, respectively). A high ASO titer, high urine specific gravity, severe hematuria and pyuria early in the illness were suggestive of a higher risk for an atypical presentation.