Proceedings of the 6th Asia Dengue Summit, June 2023.

The 6th Asia Dengue Summit (ADS) themed "Road Map to Zero Dengue Death" was held in Thailand from 15th-16th June 2023. The summit was hosted by Tropical Medicine Cluster, Chulalongkorn University, Bangkok, Thailand in conjunction with Queen Saovabha Memorial Institute, The Thai Red Cross Society; Faculty of Tropical Medicine, Mahidol University; and the Ministry of Public Health. The 6th ADS was convened by Asia Dengue Voice and Action (ADVA); Global Dengue and Aedes Transmitted Diseases Consortium (GDAC); Southeast Asian Ministers of Education Tropical Medicine and Public Health Network (SEAMEO TROPMED); Fondation Mérieux (FMx) and the International Society for Neglected Tropical Diseases (ISNTD). Dengue experts from academia and research, and representatives from the Ministries of Health, Regional and Global World Health Organization (WHO) and International Vaccine Institute (IVI) participated in the three-day summit. With more than 51 speakers and 451 delegates from over 24 countries, 10 symposiums, and 2 full days, the 6th ADS highlighted the growing threat of dengue and its antigenic evolution, flagged the urgent need to overcome vaccine hesitancy and misinformation crisis, and focused on dengue control policies, newer diagnostics, therapeutics and vaccines, travel-associated dengue, and strategies to improve community involvement.

Intra-serotypic antigenic diversity of dengue virus serotype 3 in Thailand during 2004-2015.

In addition to the well-known differences among the four dengue serotypes, intra-serotypic antigenic diversity has been proposed to play a role in viral evolution and epidemic fluctuation. A replacement of genotype II by genotype III of dengue virus serotype 3 (DENV3) occurred in Thailand during 2007-2014, raising questions about the role of intra-serotypic antigenic differences in this genotype shift. We characterized the antigenic difference of DENV3 of genotypes II and III in Thailand, utilizing a neutralizing antibody assay with DENV3 vaccine sera and monotypic DENV3 sera. Although there was significant antigenic diversity among the DENV3, it did not clearly associate with the genotype. Our data therefore do not support the role of intra-serotypic antigenic difference in the genotype replacement. Amino acid alignment showed that eight positions are potentially associated with diversity between distinct antigenic subgroups. Most of these amino acids were found in envelope domain II. Some positions (aa81, aa124, and aa172) were located on the surface of virus particles, probably involving the neutralization sensitivity. Notably, the strains of both genotypes II and III showed clear antigenic differences from the vaccine genotype I strain. Whether this differencewill affect vaccine efficacy requires further studies.

Heterologous prime-boost immunization induces protection against dengue virus infection in cynomolgus macaques.

IMPORTANCE: Currently licensed dengue vaccines do not induce long-term protection in children without previous exposure to dengue viruses in nature. These vaccines are based on selected attenuated strains of the four dengue serotypes and employed in combination for two or three consecutive doses. In our search for a better dengue vaccine candidate, live attenuated strains were followed by non-infectious virus-like particles or the plasmids that generate these particles upon injection into the body. This heterologous prime-boost immunization induced elevated levels of virus-specific antibodies and helped to prevent dengue virus infection in a high proportion of vaccinated macaques. In macaques that remained susceptible to dengue virus, distinct mechanisms were found to account for the immunization failures, providing a better understanding of vaccine actions. Additional studies in humans in the future may help to establish whether this combination approach represents a more effective means of preventing dengue by vaccination.

Molecular characterization and geographical distribution of Zika virus worldwide from 1947 to 2022.

OBJECTIVES: We conducted molecular characterization, demonstrated the geographical distribution of Zika virus (ZIKV) circulating worldwide from 1947 to 2022 and explored the potential genetic recombination site in the Thailand ZIKV genomes. METHODS: We constructed phylogenetic trees based on ZIKV coding sequences (CDS) and determined the geographical distribution of the representative viruses by genetic relationship and timeline. We determined genetic recombination among ZIKV and between ZIKV and other flaviviruses using similarity plot and bootscan analyzes, together with the phylogeny encompassing the CDS and eight subgenomic regions. RESULTS: The phylogenetic trees comprising 717 CDS showed two distinct African and Asian lineages. ZIKV in the African lineage formed two sublineages, and ZIKV in the Asian lineage diversified into the Asian and American sublineages. The 1966 Malaysian isolate was designated the prototype of the Asian sublineage and formed a node of only one member, while the newer viruses formed a distinct node. We detected no genetic recombination in the Thailand ZIKV. CONCLUSION: Five Thailand isolates discovered in 2006 were the second oldest ZIKV after the Malaysian prototype. Our result suggested two independent routes of ZIKV spread from Southeast Asia to Micronesia in 2007 and French Polynesia in 2013 before further spreading to South American countries.

Blockade-of-Binding Activities toward Envelope-Associated, Type-Specific Epitopes as a Correlative Marker for Dengue Virus-Neutralizing Antibody.

Humans infected with dengue virus (DENV) acquire long-term protection against the infecting serotype, whereas cross-protection against other serotypes is short-lived. Long-term protection induced by low levels of type-specific neutralizing antibodies can be assessed using the virus-neutralizing antibody test. However, this test is laborious and time-consuming. In this study, a blockade-of-binding enzyme-linked immunoassay was developed to assess antibody activity by using a set of neutralizing anti-E monoclonal antibodies and blood samples from dengue virus-infected or -immunized macaques. Diluted blood samples were incubated with plate-bound dengue virus particles before the addition of an enzyme-conjugated antibody specific to the epitope of interest. Based on blocking reference curves constructed using autologous purified antibodies, sample blocking activity was determined as the relative concentration of unconjugated antibody that resulted in the same percent signal reduction. In separate DENV-1-, -2-, -3-, and -4-related sets of samples, moderate to strong correlations of the blocking activity with neutralizing antibody titers were found with the four type-specific antibodies 1F4, 3H5, 8A1, and 5H2, respectively. Significant correlations were observed for single samples taken 1 month after infection as well as samples drawn before and at various time points after infection/immunization. Similar testing using a cross-reactive EDE-1 antibody revealed a moderate correlation between the blocking activity and the neutralizing antibody titer only for the DENV-2-related set. The potential usefulness of the blockade-of-binding activity as a correlative marker of neutralizing antibodies against dengue viruses needs to be validated in humans. IMPORTANCE This study describes a blockade-of-binding assay for the determination of antibodies that recognize a selected set of serotype-specific or group-reactive epitopes in the envelope of dengue virus. By employing blood samples collected from dengue virus-infected or -immunized macaques, moderate to strong correlations of the epitope-blocking activities with the virus-neutralizing antibody titers were observed with serotype-specific blocking activities for each of the four dengue serotypes. This simple, rapid, and less laborious method should be useful for the evaluation of antibody responses to dengue virus infection and may serve as, or be a component of, an in vitro correlate of protection against dengue in the future.

Proceedings of the 5th Asia Dengue Summit.

The 5th Asia Dengue Summit, themed "Roll Back Dengue", was held in Singapore from 13 to 15 June 2022. The summit was co-convened by Asia Dengue Voice and Action (ADVA), Global Dengue and Aedes transmitted Diseases Consortium (GDAC), Southeast Asian Ministers of Education Tropical Medicine and Public Health Network (SEAMEO TROPMED), and the Fondation Mérieux (FMx). Dengue experts from academia and research and representatives from the Ministries of Health, Regional and Global World Health Organization (WHO), and International Vaccine Institute (IVI) participated in the three-day summit. With more than 270 speakers and delegates from over 14 countries, 12 symposiums, and 3 full days, the 5th ADS highlighted the growing threat of dengue, shared innovations and strategies for successful dengue control, and emphasized the need for multi-sectoral collaboration to control dengue.

Seroepidemiological study of Japanese encephalitis virus in Chiang Mai: Immunity and susceptibility 28 years after introduction of a vaccination programme.

BACKGROUND: Thailand has introduced a nationwide vaccination against Japanese encephalitis virus (JEV) into National Immunization Programme since the 1990's. To improve the understanding of immunity and susceptibility of the population after 28 years of a vaccination programme, we conducted a JEV seroepidemiological study in a JEV-endemic area of Thailand. METHODS: An age-stratified, population-based, seroepidemiological study was conducted in Chiang Mai, Thailand-a northern Thai province where is an endemic area of Japanese encephalitis. Nine districts were chosen based on administrative definition: rural (n = 3); urban (n = 3); and peri-urban (n = 3). Within each district, eligible participants were randomly selected from 3 age groups: adolescents (10-20 years); adults (21-50 years); and older adults/elderly (≥51 years) by computer randomization. Plaque reduction neutralization tests (PRNT50 and PRNT90) were performed to measure neutralizing antibodies to JEV. To account for the cross-reactivity of JEV and other flaviviruses, JEV seroprotection was defined according to age, previous history of JEV vaccination, and PRNT50/PRNT90 levels of study participants. RESULTS: Overall, 279 adolescents, 297 adults, and 297 older adults/elderly were enrolled from nine districts. Age-stratified, protocol-defined, cluster-adjusted JEV seroprotection rates were 61% (95% CI: 48-73%), 43% (95% CI: 31-57%), and 52% (95% CI: 37-67%) for adolescents, adults, and older adults/elderly, respectively. Living in peri-urban districts, having a history of prior dengue virus infection, and previously receiving mouse brain-derived JEV vaccine were significantly associated with seroprotection to JEV in adolescents. Older age and male sex were associated with seroprotection for adults; and only male sex was the associated factor for older adults/elderly (P <0.05). CONCLUSIONS: Approximately half of population living in a JEV-endemic area demonstrated seroprotection to JEV. Ongoing nationwide surveillance on JEV seropepidemiology is an important strategy to understand the evolving population-level immunity to JEV, and to help formulating the appropriate recommendations on JE immunization.

The helper oligonucleotides enable detection of folded single-stranded DNA by lateral flow immunoassay after HCR signal amplification.

A combination of Hybridization Chain Reaction (HCR) and Lateral Flow Immunoassay (LFIA) is an attractive strategy for a simple signal amplification DNA/RNA detection. The present study aimed to report a strategy used to solve a problem encountered when the target DNA contained folded secondary structure during HCR, enabling HCR hairpin probes to easily access the target site. The 24-nt conserved sequence within 3'-UTR, present only in dengue virus genome but not in other species, is an ideal target to use as a probe binding site for pan-dengue virus detection. Thus, the 105-nt target containing the 24-nt target sequence was chosen as a target with secondary structures. The 24-nucleotide (nt) synthetic target DNA successfully induced HCR reaction within 5 min at room temperature. However, the HCR detection of the 105-nt synthetic target DNA with secondary structures was problematic. The probe hybridization was prevented by the secondary structures of the target, resulting in a failure to generate HCR product. To solve this problem, two helper oligonucleotides (helper1 and helper2) were designed to linearize the folded structure of the 105-nt target through strand-displacement mechanism, allowing the HCR hairpin probes to easily access the target site. The HCR product with the labeled helper oligonucleotides and the labeled probes were successfully detected by LFIA. With this strategy, the combination of the helper-enhanced HCR and LFIA exhibited a limit of detection (LOD) in a nanomolar range of the 105-nt DENV synthetic target DNA. Our study demonstrated that signal amplification by the combination of HCR and LFIA could successfully detect the target DNA with secondary structure, but not target RNA with secondary structure. In summary, this work provided a proof of concept of two main issues including probe hybridization enhancement by helper oligonucleotide for the target with complicated secondary structure and the advantage of a combination of labeled helper and HCR probes design for LFIA to overcome the false positive result from HCR probe leakage. Our findings on the use of helper oligonucleotides may be beneficial for the development of other isothermal amplification, since the secondary structure of the target is one of the major obstacles among hybridization-based methods.

A Small-Plaque Isolate of the Zika Virus with Envelope Domain III Mutations Affect Viral Entry and Replication in Mammalian but Not Mosquito Cells.

An Asian Zika virus (ZIKV) isolated from a Thai patient that was serially passaged in Primary Dog Kidney (PDK) cells for attenuation displayed both big and small plaque-forming viruses by the 7th passage. Two small-plaque isolates were selected and purified for characterization as attenuated ZIKV candidates. In vitro growth kinetics showed significantly reduced titers for small-plaque isolates in Vero cells early post-infection compared to the parental ZIKV and a big-plaque isolate, but no significant difference was observed in C6/36 cells. Viral entry experiments elucidate that titer reduction likely occurred due to the diminished entry capabilities of a small-plaque isolate. Additionally, a small-plaque isolate displayed lowered neurovirulence in newborn mice compared to 100% lethality from infection with the parental ZIKV. Genomic analysis revealed the same three unique non-synonymous mutations for both small-plaque isolates: two on the envelope (E) protein at residues 310, alanine to glutamic acid (A310E), and 393, glutamic acid to lysine (E393K), and one on residue 355 of NS3, histidine to tyrosine (H355Y). Three-dimensional (3D) mapping suggests that the E protein mutations located on the receptor-binding and fusion domain III likely affect cell entry, tropism, and virulence. These ZIKV isolates and genotypic markers will be beneficial for vaccine development.

Antibody persistence upto 5 years after primary immunization and booster with an inactivated chromatographically purified Vero cell-derived Japanese encephalitis vaccine in Thai children.

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; JEVACTM) 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.

Feasibility of chitosan-based nanoparticles approach for intranasal immunisation of live attenuated Japanese encephalitis vaccine.

Intranasal (IN) administration, a non-invasive route, is explored to overcome the limitations of conventional subcutaneous (SC) injection for Japanese encephalitis (JE) immunisation. Mucoadhesive nanoparticles (NPs) are recognised for the benefits they offer via IN delivery, such as extended retention time of the vaccine on the mucosa. The purpose of this study was to evaluate immunisation effect of live attenuated Japanese encephalitis-chimeric virus vaccine (JE-CV)-loaded mucoadhesive NPs based on chitosan (CS) or chitosan maleimide (CM), a novel mucoadhesive polymer, via the IN route to improve the mucosal immunisation against JE. The results revealed that IN immunisation stimulated seroprotection following PRNT50 evaluation. Moreover, compared with SC immunisation, IN immunisation in mice provided a higher sIgA level, leading to improved mucosal immune response. In addition, chitosan-based NPs showed an adjuvant effect on the IN vaccine due to their mucoadhesive and antigen-uptaken properties. CM NPs successfully induced sIgA. In contrast, SC JE-CV immunisation induced negligible mucosal immunity. These immunological advantages revealed that JE-CV-loaded mucoadhesive NPs are a promising approach for IN vaccination as an alternative route for JE protection due to the stimulatory effects on both mucosal and systemic immune responses.

Persistence of neutralizing antibody and its protective efficacy induced by a live attenuated tetravalent dengue vaccine, KD-382, in cynomolgus monkeys.

An effective dengue vaccine should induce a long-lasting immune response against all four serotypes simultaneously with a minimum number of immunizations. Our live attenuated tetravalent dengue vaccine candidate, KD-382, was developed using a classical host range mutation strategy (no addition of artificial genetic modification). In our previous study, cynomolgus monkeys immunized with a single dose of KD-382 seroconverted to all four serotypes. However, it is important to determine if neutralizing antibodies (NAbs) induced by KD-382 can work as a long-lasting immune response to prevent dengue. In this study, a single dose of KD-382 induced a strong NAb response against all four serotypes in cynomolgus monkeys. We also confirmed that NAb titers against all four serotypes persist for at least five years, indicating its high potential as a dengue vaccine candidate. Next, we evaluated the effect of pre-existing dengue immunity on NAb responses induced by KD-382. We administered KD-382 to cynomolgus monkeys pre-administered one of the monovalent parental wild-type strains 60 days before vaccination. Regardless of the pre-immunized serotype, all the monkeys showed sufficient tetravalent NAb responses, which lasted for over two years. All the KD-382 vaccinated monkeys were then challenged with different parental wild-type viruses than that used for pre-administration; viral RNA in the serum was less than the lower limit of quantification, indicating complete protection against secondary heterologous dengue infection without any harmful disease enhancement. Consequently, KD-382 successfully induced a long-lasting and protective tetravalent NAb response in monkeys, suggesting that KD-382 is a promising vaccine candidate usable for both dengue seronegative and seropositive individuals.

Immunogenicity of Vero Cell Culture-derived Japanese Encephalitis Vaccine in Pediatric and Young Hematopoietic Stem Cell Transplantation Recipients.

BACKGROUND: Children and young adults undergoing hematopoietic stem cell transplantation (HSCT) typically lose their immunity to vaccine-preventable diseases, including Japanese encephalitis (JE). Revaccination against JE in this population has not been well characterized. METHODS: This prospective study evaluated the immunogenicity of inactivated Vero cell culture-derived JE vaccine in children and young adults (<25 years of age) who had completed HSCT >1 year prior. Each patient received inactivated Vero cell culture-derived JE vaccine at enrollment and 1 month after enrollment, as well as a booster dose 13 months after enrollment. Serum JE plaque reduction neutralization test and JE-specific T lymphocyte count assay were performed at baseline, 1 month after the second dose, on the day of the booster dose, and 1 month after the booster dose. RESULTS: Thirty-seven patients were enrolled. At baseline, 15 patients (40.5%) had plaque reduction neutralization titer >10, which is considered protective. Among 22 seronegative patients, 15 (68.2%) and 19 (86.4%) exhibited seroconversion after revaccination and booster dose, respectively. Median JE-specific T lymphocyte counts also increased. Twenty of 111 (18.0%) vaccination doses resulted in self-limiting side effects. CONCLUSIONS: The inactivated Vero cell culture-derived JE vaccine may be safe and effective for immunization against JE virus in children and young adults who have undergone HSCT.

A modified IgG avidity assay for reliability improvement of an in-house capture ELISA to discriminate primary from secondary dengue virus infections.

Although discrimination between primary and secondary dengue infections can be performed using commercially available immunoassays or in-house tests, the evaluation of these methods is important, but is often problematic due to incomplete clinical data. In many cases, patients' sera submitted to the laboratory may not include the date of onset of illness which is necessary to discriminate primary and secondary dengue infections. This study reports improvement of an in-house capture ELISA using IgG avidity to discriminate primary and secondary dengue virus infection. Modified definition criteria were applied to characterize 99 single sera based on their IgM/IgG ratios. Regressive analysis indicated that the avidity test results (avidity index of 60 % as cutoff) for the discrimination showed good agreement (96 %) and a high correlation (r = -0.81) with those of the in-house capture ELISA (IgM/IgG ratio at 1.2 as cutoff). To further evaluate the in-house tests, 318 convalescent sera were compared with a Focus Diagnostics' anti-dengue IgM ELISA. Compared with the Focus Diagnostics system, the sensitivity of an in-house IgM determination was 83 %, whereas using both IgM and IgG capture ELISAs the sensitivity increased to 95 %.

Survey on neutralizing antibodies against Zika virus eighteen months post-outbreak in two southern Thailand communities.

BACKGROUND: In 2016 and 2017, Zika virus (ZIKV) infection outbreaks occurred in two communities in southern Thailand. This re-immerging infection can widely spread by mosquito bites and cause serious complications in a central nervous system among children born to infected mothers. Thus, they should be protected. This study aims to (1) To determine the prevalence of neutralizing ZIKV antibodies in the post-outbreak areas among the general population and pregnancy women residing at various distances from the houses of the nearest index patients; (2) To examine the cross-neutralizing capacity of antibodies against ZIKV on other flaviviruses commonly found in the study areas; (3) To identify factors associated with the presence of neutralizing ZIKV antibodies. METHODS: The two post-outbreak communities were visited at 18 months after the outbreaks. We enrolled (1) 18 confirmed ZIKV infected (index) cases, (2) sample of 554 neighbors in the outbreak areas who lived at various distances from the index patients' houses, (3) 190 residents of non-outbreak areas, and (4) all pregnant women regardless of gestational age residing in the study areas (n = 805). All serum specimens underwent the plaque reduction neutralization test (PRNT). Ten randomly selected ZIKV seropositive and ten randomly selected seronegative specimens were tested for dengue virus serotypes 1-4 (DENV1-4) and Japanese encephalitis virus (JEV) antibodies using PRNT90. Serum titer above 1:10 was considered positive. Multiple logistic regression was used to assess factors associated with seropositivity. RESULTS: Out of all 18 index cases, 9 remained seropositive. The seroprevalence (95% CI) in the two outbreak areas were 43.7% (35.9-51.6%) and 29.7% (23.3-36.0%) in general population, and 24.3% (20.1-28.8%) and 12.8% (9.7-16.5%) in pregnant women. Multivariate analysis showed that seropositivity was independent of the distance gradient from the index's houses. However, being elderly was associated with seropositivity. DENV1-4 and JEV neutralizing antibodies were present in most ZIKV-positive and negative subsamples. CONCLUSION: Protective herd immunity for ZIKV infection is inadequate, especially among pregnant women in the two post-outbreak areas in southern Thailand.

Well-balanced immune response and protective efficacy induced by a single dose of live attenuated tetravalent dengue vaccine (KD-382) in monkeys.

One of the challenges developing a live attenuated tetravalent dengue vaccine (TDV) is to overcome the presumed viral interference that may be preventing the induction of a balanced immune response to all 4 serotypes of the dengue virus (DENV1-4). Our live attenuated TDV candidate was developed from wild-type (wt) parental strains (DENV1/03135, DENV2/99345, DENV3/16562, and DENV4/1036, respectively) using a classical host range mutation strategy: the same strategy used for the approved live attenuated smallpox, polio, and MMR vaccines. Our vaccine candidate is expected to mimic natural dengue virus infection, as it provides all the components of dengue virus, including both structural and nonstructural proteins. Therefore, induction of more solid and comprehensive immune responses against pathogenic dengue viruses is also expected. In this study, we evaluated the neutralizing antibody responses for each serotype induced by a single subcutaneous administration of 6 formulations, which were composed of different combinations of vaccine strains and were all of different dosages. These formulations were tested in dengue-naïve cynomolgus macaques. As a result, regardless of the TDV formulation, all the monkeys immunized with TDVs seroconverted to all the 4 serotypes at day 30. Next, we evaluated protection ability of the selected formulations of TDV candidate, no RNAemia was detected from any of the immunized monkeys upon s.c. challenge with wtDENV. The findings of this non-human primate study indicate that our vaccine candidate is very promising; it can be further evaluated for safety and efficacy in human clinical studies.

Immunogenicity and safety of two-visit, intradermal pre-exposure rabies prophylaxis simultaneously administrated with chimeric live-attenuated Japanese encephalitis vaccine in children living in rabies and Japanese encephalitis endemic country.

BACKGROUND: Reducing the number of doses required for pre-exposure prophylaxis (PrEP) would make it more feasible and cost-effective to implement in children at the highest risk of rabies exposure in Asia. We studied immune response of 2-site intradermal (ID) injection of rabies vaccine on days 0 and 28 for rabies PrEP simultaneously administrated with live-attenuated Japanese encephalitis chimeric virus vaccine (JE-CV) for children living in endemic area. RESEARCH DESIGN AND METHODS: Seronegative children (n = 49) aged 12-16 months were randomized 2:1 into two groups: Group A subjects were vaccinated with 0.1-mL ID injection of purified Vero cell rabies vaccine (PVRV), each at two sites on day (D) 0 and D28; Group B subjects were vaccinated with conventional 0.5-mL intramuscular PVRV on D0, D7 and D28. Both groups received one dose of JE-CV subcutaneously on D0 and D365. Rabies virus neutralizing antibody (RVNA) titers were measured on D0, D42 and D365 after vaccination; Japanese Encephalitis (JE) neutralizing antibody titers were determined on D0, D42, D365 and D379. RESULTS: All children had RVNA ≥ 0.5 IU/mL on D42 (geometric mean titers [GMTs] of RVNA 14.35 IU/mL [Group A] and 14.83 IU/mL [Group B], p > 0.05]). On D365, RVNA GMTs of subjects in group A and B were 1.50 IU/mL and 2.00 IU/mL (p > 0.05), respectively. All children had seroprotection following booster dose of JE-CV. There were no vaccine-related SAEs observed. CONCLUSION: The 2-site ID PrEP with PVRV on days 0 and 28 co-administrated with JE-CV are safe and immunogenic.

Evaluation of the antiviral activity of orlistat (tetrahydrolipstatin) against dengue virus, Japanese encephalitis virus, Zika virus and chikungunya virus.

Many mosquito transmitted viruses of the genera Alphavirus and Flavivirus are human pathogens of significant concern, and there is currently no specific antiviral for any member of these two genera. This study sought to investigate the broad utility of orlistat (tetrahydrolipstatin) in reducing virus infection for several mosquito borne viruses including flaviviruses (dengue virus (DENV; nine isolates analyzed), Japanese encephalitis virus (JEV; one isolate analyzed) and Zika virus (ZIKV; 2 isolates analyzed)) as well as an alphavirus (chikungunya virus; CHIKV; 2 isolates analyzed). Three different treatment regimens were evaluated, namely pre-treatment (only), post-treatment (only) and pre- and post-treatment, and three factors were evaluated, namely level of infection, virus titer and genome copy number. Results showed that all three treatment modalities were able to significantly reduce virus titer for all viruses investigated, with the exception of three isolates of DENV in the pre-treatment only regimen. Pre- and post-treatment was more effective in reducing the level of infection and genome copy number of all viruses investigated than either pre-treatment or post-treatment alone. Collectively, these results suggest orlistat has potential as a broad-spectrum agent against multiple mosquito transmitted viruses.

The use of immature platelet fraction to predict time to platelet recovery in patients with dengue infection.

Background: In dengue infection, knowing time to platelet recovery is essential for optimal management.Aims: To determine a predictor for platelet recovery in patients with dengue infection.Methods: Platelet count and immature platelet fraction (IPF) from daily blood samples of patients with dengue infection during hospitalisation and 1-4 weeks after discharge were retrospectively analysed. The levels of patients' IPF were compared with normal controls recruited from healthy children with normal platelet counts.Results: A total of 244 EDTA blood samples were collected daily from 64 patients (45 males) with dengue infection (36 dengue fever, 28 dengue haemorrhagic fever) during hospitalisation and after discharge from the hospital. They did not receive any platelet concentrate transfusion. The median IPF among normal children was 3.6% with a 95 percentile of 9.9%. In dengue patients, an IPF of ≥10.0% after defervescence was associated with a subsequent platelet count of ≥60 × 109/L within 72 hours.Conclusion: In patients with dengue infection, IPF ≥10.0% after defervescence is a predictor of subsequent platelet recovery to a haemostatic level ≥60 × 109/L within 72 hours.

Daily Dengue Severity Score to Assess Severe Manifestations.

BACKGROUND: The study aimed to develop dengue severity score to assess severe manifestations among hospitalized patients with dengue infection. METHOD: Children and adolescents with serologically confirmed dengue infection admitted at Ramathibodi Hospital from 2004 to 2018 and treated by an expert multidisciplinary team were recruited. Medical records were retrospectively reviewed and 14 items, related to clinical parameters and managements during hospitalization, were obtained daily as dengue severity score. RESULTS: A total of 191 patients with a mean age of 10.7 years from 2004 to 2013 were recruited. They were classified as dengue fever (35), dengue hemorrhagic fever (DHF) I (53), II (50), III (37) and IV (16). The analysis of 593 daily records revealed the range of daily severity score among patients with DHF grades III (10-20) and IV (31-47) were significantly higher than those of other groups (dengue fever, 5-13; DHF I, 2-10; DHF II, 6-11) with P-values of 0.0001. Using a validity test, a total daily score of ≥12 was an assessment tool for dengue shock syndrome with sensitivity, 86% and specificity, 84%. An additional 51 hospitalized patients with DHF grades II, III and IV with similar ages from 2014 to 2018 were recruited. The number of patients with severe manifestations, having daily score of ≥12, was significantly higher than those without severe manifestations starting from Day -3 to Day +1 of illness. CONCLUSIONS: Daily dengue severity score of ≥12 was an accurate assessment tool for severe manifestations.