Envelope-dimer epitope-like broadly protective antibodies against dengue in children following natural infection and vaccination.

Cross-reactive antibodies (Abs) to epitopes that span envelope proteins on the virion surface are hypothesized to protect against dengue. Here, we measured Abs targeting the quaternary envelope dimer epitope (EDE) as well as neutralizing and binding Abs and evaluate their association with dengue virus (DENV) infection, vaccine response, and disease outcome in dengue vaccinated and unvaccinated children (n=252) within a longitudinal cohort in Cebu, Philippines (n=2,996). Abs targeting EDE were prevalent and strongly associated with broad neutralization of DENV1-4 in those with baseline multitypic immunity. Subsequent natural infection and vaccination boosted EDE-like, neutralizing, and binding Abs. EDE-like Abs were associated with reduced dengue risk and mediated the protective effect of binding and neutralizing Abs on symptomatic and severe dengue. Thus, Abs targeting quaternary epitopes help explain broad cross protection in those with multiple prior DENV exposures, making them useful for evaluation and development of future vaccines and therapeutics.

Effect of single-dose, live, attenuated dengue vaccine in children with or without previous dengue on risk of subsequent, virologically confirmed dengue in Cebu, the Philippines: a longitudinal, prospective, population-based cohort study.

BACKGROUND: A three-dose dengue vaccine (CYD-TDV) was licensed for use in children aged 9 years and older starting in 2015 in several dengue-endemic countries. In 2016, the Philippine Department of Health implemented a dengue vaccination programme, which was discontinued because of safety concerns. We assessed the relative risk of developing virologically confirmed dengue among children who did or did not receive a single dose of CYD-TDV by previous dengue virus (DENV) infections at baseline classified as none, one, and two or more infections. METHODS: In this longitudinal, prospective, population-based cohort study, we enrolled healthy children (aged 9-14 years) residing in Bogo or Balamban, Cebu, Philippines, between May 2, and June 2, 2017, before a mass dengue vaccination campaign, via the Rural Health Unit in Bogo and three Rural Health Units in Balamban. We collected demographic information and sera for baseline DENV serostatus and conducted active surveillance for acute febrile illness. Children who developed acute febrile illness were identified, clinical data were collected, and blood was drawn for confirmation of dengue by RT-PCR. The primary outcome was the relative risk of developing virologically confirmed dengue among children who received or did not receive a single dose of CYD-TDV by DENV serostatus at baseline. FINDINGS: A single dose of CYD-TDV did not confer protection against virologically confirmed dengue in children who had none or one previous DENV infection at baseline. One dose conferred significant protection against hospital admission for virologically confirmed dengue among participants who had two or more previous DENV infections at baseline during the first 3 years (70%, 95% CI 20-88; p=0·017) and the entire follow-up period (67%, 19-87; p=0·016). INTERPRETATION: The risk of developing virologically confirmed dengue after a single dose of CYD-TDV varied by baseline DENV serostatus. Since the study assessed the effect of only a single dose, the findings cannot inform decisions on vaccination by public health officers. However, the findings have implications for children who receive an incomplete vaccination regimen and these results should prompt more detailed analyses in future trials on dengue vaccines. FUNDING: The Philippine Department of Health, Hanako Foundation, WHO, Swedish International Development Cooperation Agency, International Vaccine Institute, University of North Carolina, and US National Institute of Allergy and Infectious Diseases.

High transmission of endemic human coronaviruses before and during the COVID-19 pandemic in adolescents in Cebu, Philippines.

Background: SARS-CoV-2, the causative agent of COVID-19, is a betacoronavirus belonging to the same genus as endemic human coronaviruses (hCoVs) OC43 and HKU1 and is distinct from alpha hCoVs 229E and NL63. In a study of adolescents in the Philippines, we evaluated the seroprevalence to hCoVs, whether pre-pandemic hCoV immunity modulated subsequent risk of SARS-CoV-2 infection, and if SARS-CoV-2 infection affected the transmission of the hCoVs. Methods: From 499 samples collected in 2021 and screened by SARS-CoV-2 receptor binding domain (RBD) enzyme-linked immunosorbent assay (ELISA), we randomly selected 59 SARS-CoV-2 negative and 61 positive individuals for further serological evaluation. We measured RBD and spike antibodies to the four hCoVs and SARS-CoV-2 by ELISA in samples from the same participants collected pre-pandemic (2018-2019) and mid-pandemic (2021), before COVID-19 vaccination. Results: We observed over 72% seropositivity to the four hCoVs pre-pandemic. Binding antibodies increased with age to 229E and OC43, suggesting endemic circulation, while immunity was flat across ages for HKU1 and NL63. During the COVID-19 pandemic, antibody level increased significantly to the RBDs of OC43, NL63, and 229E and spikes of all four hCoVs in both SARS-CoV-2 negative and positive adolescents. Those aged 12-15 years old in 2021 had higher antibodies to RBD and spike of OC43, NL63, and 229E than adolescents the same age in 2019, further demonstrating intense transmission of the hCoVs during the pandemic. Conclusions: We observe a limited impact of the COVID-19 pandemic on endemic hCoV transmission. This study provides insight into co-circulation of hCoVs and SARS-CoV-2.

Effectiveness of a single-dose mass dengue vaccination in Cebu, Philippines: A case-control study.

BACKGROUND: Dengue fever is an important public health problem in the Philippines. In April 2016, the Department of Health launched a three-dose school based dengue vaccination program of nine- to fourteen-year-old children in three regions with the highest number of dengue cases using CYD-TDV (Dengvaxia, Sanofi Pasteur). In July 2017, a community-based dengue vaccination program was implemented in Cebu province. The program was discontinued in December 2017 amidst public controversy, after the first dose had been administered. We assessed the effectiveness of a single dose of CYD-TDV against hospitalized virologically confirmed dengue (VCD). METHODS: We conducted a case-control study in Cebu province following the dengue mass vaccination. Children who were nine to fourteen years of age during the mass vaccination and subsequently admitted to any of four participating public hospitals with suspected dengue were enrolled in the study as cases. Blood for RT-PCR and clinical and socio-demographic information were obtained. To estimate the level of vaccine protection, vaccination status was compared between children with hospitalized virologically confirmed dengue and controls of the same six-year age-group as the cases, matched on sex, neighborhood and time of occurrence of cases. FINDINGS: We enrolled 490 cases and 980 controls. Receipt of one dose of CYD-TDV was associated with 26% (95 % CI, -2 to 47%; p = 0 0675) overall protection against hospitalized virologically confirmed dengue and 51% (95 % CI, 23 to 68; p = 0 0016) protection against dengue with warning signs. INTERPRETATION: A single dose of CYD-TDV given to nine to fourteen-year-old children through a community-based mass vaccination program conferred protection against dengue with warning signs and severe dengue but we were unable to conclude on protection against milder illness.

Determining dengue virus serostatus by indirect IgG ELISA compared with focus reduction neutralisation test in children in Cebu, Philippines: a prospective population-based study.

BACKGROUND: Detection of dengue virus antibodies is important for understanding future dengue virus risk and for prevaccination screening. We aimed to evaluate the performance of a dengue IgG indirect ELISA in determining dengue seroprevalence in a cohort of children in the Philippines, using a focus reduction neutralisation test (FRNT) as the reference test. METHODS: In this prospective population-based cohort study, we enrolled healthy children residing in Bogo or Balamban, Cebu, Philippines, who were to be aged 9-14 years at the time of a mass dengue vaccination campaign. Sera were collected from participants and batch tested by indirect IgG ELISA and FRNT. The primary endpoint was dengue seroprevalence in the cohort, detected by ELISA, and validated by that detected by reference FRNT. This study is registered with ClinicalTrials.gov, NCT03465254. FINDINGS: We collected 2996 serum samples between May 2, and June 2, 2017, and we tested each sample with IgG ELISA. Using 1961 samples (65·5%) that were tested with FRNT, and 1035 samples (34·5%) with imputed results, we found that 320 (10·7%) of 2996 children were dengue naive and 2676 (89·3%) were seropositive for previous dengue virus infection. Based on the 1961 non-imputed FRNT results classified as dengue seronegative or seropositive, the ELISA (with a 0·9 index value cutoff) showed 95·2% sensitivity, 93·4% specificity, 6·6% false positivity, and 4·8% false negativity. However, sensitivity of the ELISA was poor (77·1%) among children with immunity to just one dengue virus serotype. Of the 11 sera that were false positive with ELISA, seven samples (63·6%) were seropositive for Zika virus or Japanese encephalitis virus with FRNT. INTERPRETATION: Most children (89·3%) assessed in our study and eligible to participate in the mass dengue vaccination campaign were seropositive for previous dengue virus infection. Compared with FRNT, ELISA had high sensitivity and specificity (>90%), but the false-negative and false-positive rates makes the test suboptimal for prevaccination screening. Individuals who are falsely identified as seropositive by dengue IgG ELISA and then vaccinated might be at risk of developing severe disease during a subsequent exposure to wild-type dengue virus. Those with a monotypic profile would benefit the most from vaccination, but the sensitivity of the IgG ELISA was much lower in this group than in those with a multitypic profile. FUNDING: Philippine Department of Health, Hanako Foundation, WHO, Swedish International Development Cooperation Agency through the International Vaccine Institute, and University of North Carolina, Chapel Hill, NC, USA.

Evaluation of a new point-of-care test to determine prior dengue infection for potential use in pre-vaccination screening.

OBJECTIVE: Vaccination with the first licensed dengue vaccine is recommended only for those who have had previous infection with dengue virus (DENV). A point-of-care test with the desired sensitivity of 95% and specificity of 98% could facilitate pre-vaccination screening. We evaluated a newly developed, automated dengue immunoglobulin fluorescence immunoassay for determining dengue serostatus. METHODS: We used serum samples collected just prior to a mass dengue vaccination in Cebu, Philippines. Healthy children residing in Bogo and Balamban who would be 9-14 years old at the time of the mass dengue vaccination were eligible to participate. We evaluated the ichroma™ II dengue fluorescence immunoassay (Boditech Med Incorporated, Gang-won-do, Republic of Korea) using a neutralization test (NT) as the reference assay. RESULTS: We enrolled 2996 children (mean age 10.39 years, 51.7% female) in the cohort and included a subsample of 1000 (mean age 10.56 years, 54.4% female) in this study. Of the 1000 children, 86/1000 (8.6%) tested seronegative and 914/1000 (91.4%) seropositive for DENV antibodies by neutralization testing. Compared with the NT, the dengue IgG fluorescence immunoassay had an overall specificity of 90.7% (95%CI: 82.5-95.9%) and a sensitivity of 91.8% (95%CI: 89.8-93.5%) for determining dengue seropositivity. The sensitivity declined to 51.2% (42.3-61.0%) for the detection of the subset with a monotypic dengue profile. CONCLUSION: The insufficient specificity and sensitivity (particularly in the detection of a previous monotypic dengue infection) would render the test, in its current state, inadequate for pre-vaccination screening. Considering its user-friendly interphase and possibility of point-of-care use, the test could be further developed and validated to improve its performance characteristics.

Performance of Dried Blood Spots Compared with Serum Samples for Measuring Dengue Seroprevalence in a Cohort of Children in Cebu, Philippines.

Dengue seroprevalence data are useful for understanding epidemiologic trends and transmission dynamics, and for making decisions about implementation of dengue control programs. A logistical challenge to seroprevalence surveys is the collection and transport of serum samples. For conducting large and repeated dengue serosurveys, dried blood spots (DBS) would allow easier sample collection, shipment, transport, and storage than standard serum collection methods. Further evidence is needed to understand how well DBS performs compared with standard serum collection methods in laboratory assays. We evaluated the detection of anti-dengue antibodies by IgG indirect ELISA when using DBS compared with sera. Specimens were collected from healthy children in Cebu, Philippines, who would be 9-14 years of age at the time of a mass dengue vaccination program. Using an ELISA index value cutoff of 0.9, 1,285/1,488 (86.4%) of the DBS were seropositive and 203 (13.6%) were seronegative, compared with 1,292/1,488 (86.8%) seropositive and 196 (13.2%) seronegative serum samples. Compared with sera, the DBS method had a 98.3% sensitivity, 92.4% specificity, 98.9% positive predictive value, and 89.2% negative predictive value. Considering the advantages in terms of sample collection, shipment, and storage, DBS sampling may be appropriate for dengue population serosurveys.

Urine Xpert MTB/RIF for the diagnosis of childhood tuberculosis.

INTRODUCTION: Xpert MTB/RIF is recommended for the simultaneous detection of tuberculosis (TB) and rifampicin resistance directly from sputum specimens. Since young children cannot always expectorate, we assessed urine as a possible specimen source to diagnose TB in children using Xpert MTB/RIF. METHODS: During a field study to enhance childhood TB identification, spot urine samples were prospectively collected from consecutive ambulatory children aged 0 to 14 years presenting with presumptive pulmonary TB in community health centers. Urine Xpert MTB/RIF was performed by blinded technicians in 182 samples using 2ml of unprocessed urine. RESULTS: The mean age of presumptive TB cases was 5.9 years (median 5.4, range 0.1 to 14.7) with more males (113, 62%) compared to females. All urine samples tested negative for Xpert MTB/RIF, regardless of whether concentration was performed or not. Out of these 182 presumptive TB cases, 50 (28%) were clinically diagnosed and 5 (3%) were bacteriologically diagnosed to have TB disease using either sputum or nasopharyngeal aspirate specimens. CONCLUSIONS: In this community-based study, urine Xpert MTB/RIF does not appear to contribute to the diagnosis of childhood TB.

Effectiveness of monovalent rotavirus vaccine in the Philippines.

Rotavirus (RV) is an important cause of diarrheal disease particularly in children aged under 5 years. Monovalent RV vaccine (RVV) was selectively introduced in 2012 in the Philippines and in July 2014 was introduced in the public health program of a province. Two RVV doses are recommended at 6 and 10 weeks of age. We conducted a test negative case-control evaluation to assess the effectiveness of RVV when given in a routine public health program in the Philippines. From September 2014 to August 2017, 967 children aged <5 years were hospitalized with diarrhea and of these, we enrolled 600 who were eligible to have received RVV and provided stool specimens for testing. Among children ≥8 months of age who were age-eligible to have received RVV, at least one dose of RVV had an adjusted vaccine effectiveness (VE) against RV hospitalization of 60% (95% confidence interval, CI: 24%, 79%), and against severe rotavirus diarrhea, VE was 64% (95% CI: 11%, 85%). These findings support the introduction of RVV into routine public health use in the Philippines. However, other factors such as costs, cost-effectiveness and operational issues must be considered prior to adoption of the vaccine into the countries' public immunization program.

Hepatitis B seroprevalence among 5 to 6 years old children in the Philippines born prior to routine hepatitis B vaccination at birth.

To assess the prevalence of hepatitis B in the Philippines, we conducted a cross-sectional study among 5 to 6 year old children born in 2007-2008, when the birth dose started to be implemented in the country. The study was conducted from 25 July to 22 October 2013 in 24 provinces and used a 3-stage cluster design and probability-proportional to size sampling. Blood was obtained and sera were tested for hepatitis B surface antigen (HBsAg). The survey included 2,769 children, of whom 26% received a timely birth dose (within 24 hours of birth) and 89% received 3 doses of the hepatitis B vaccine. Due to problems in the initial testing algorithm, only 2,407 sera were available for HBsAg testing, 20 (weighted%, 0.86%) were HBsAg positive. By immunization card and recall, among HBsAg positive children, 2 (weighted%, 20%) received a timely birth dose while 17 (weighted%, 85%) received 3 doses of the hepatitis B vaccine. The seroprevalence of HBsAg that we detected was lower than expected. However, there were several limitations in the field and in the laboratory that may have affected the representativeness of the results. Follow up studies need to be conducted to validate these results.