Koya Ariyoshi

Respiratory syncytial virus: co-infection and paediatric lower respiratory tract infections

Comprehensive population-based data on the role of respiratory viruses in the development of lower respiratory tract infections (LRTIs) remain unclear. We investigated the incidence and effect of single and multiple infections with respiratory viruses on the risk of LRTIs in Vietnam. Population-based prospective surveillance and a case–control study of hospitalised paediatric patients with acute respiratory infection (ARI) were conducted from April 2007 through to March 2010. Healthy controls were randomly recruited from the same community. Nasopharyngeal samples were collected and tested for 13 respiratory viruses using multiplex PCRs. 1992 hospitalised ARI episodes, including 397 (19.9%) with LRTIs, were enrolled. Incidence of hospitalised LRTIs among children aged <24 months was 2171.9 per 100 000 (95% CI 1947.9–2419.7). The majority of ARI cases (60.9%) were positive for at least one virus. Human rhinovirus (24.2%), respiratory syncytial virus (20.1%) and influenza A virus (12.0%) were the most common and 9.5% had multiple-viral infections. Respiratory syncytial virus and human metapneumovirus infections independently increased the risk of LRTIs. Respiratory syncytial virus further increased the risk, when co-infected with human rhinovirus, human metapneumovirus and parainfluenza virus-3 but not with influenza A virus. The case–control analysis revealed that respiratory syncytial virus and influenza A virus increased the risk of ARI hospitalisation but not human rhinovirus. Respiratory syncytial virus is the leading pathogen associated with risk of ARI hospitalisation and LRTIs in Vietnam.

A novel high-throughput method for molecular serotyping and serotype-specific quantification of Streptococcus pneumoniae using a nanofluidic real-time PCR system

Serotype-specific quantification data are essential for elucidating the complex epidemiology of Streptococcus pneumoniae and evaluating pneumococcal vaccine efficacy. Various PCR-based assays have been developed to circumvent the drawback of labour-intensive and time-consuming culture-based procedures for serotype determination and quantification of pneumococcus. Here, we applied a nanofluidic real-time PCR system to establish a novel assay. Twenty-nine primer pairs, 13 of which were newly designed, were selected for the assay to cover 50 serotypes including all currently available conjugate and polysaccharide vaccine serotypes. All primer pairs were evaluated for their sensitivity, specificity, efficiency, repeatability, accuracy and reproducibility on the Fluidigm Biomark HD System, a nanofluidic real-time PCR system, by drawing standard curves with a serial dilution of purified DNA. We applied the assay to 52 nasopharyngeal swab samples from patients with pneumonia confirmed by chest X-ray to validate its accuracy. Minimum detection levels of this novel assay using the nanofluidic real-time PCR system were comparable to the conventional PCR-based assays (between 30 and 300 copies per reaction). They were specific to their targets with good repeatability (sd of copy number of 0.1), accuracy (within ±0.1 fold difference in log10 copy number) and reproducibility (sd of copy number of 0.1). When artificially mixed DNA samples consisting of multiple serotypes in various ratios were tested, all the serotypes were detected proportionally, including a minor serotype of one in 1000 copies. In the nasopharyngeal samples, the PCR system detected all the culture-positive samples and 22 out of 23 serotypes identified by the conventional method were matched with PCR results. We conclude that this novel assay, which is able to differentially quantify 29 pneumococcus groups for 45 test samples in a single run, is applicable to the large-scale epidemiological study of pneumococcus. We believe that this assay will facilitate our understanding of the roles of serotype-specific bacterial loads and implications of multiple serotype detections in pneumococcal diseases.

The incidence and aetiology of hospitalised community-acquired pneumonia among Vietnamese adults: a prospective surveillance in Central Vietnam

BackgroundLower respiratory tract infection (LRTI) including Community-acquired pneumonia (CAP) is a common infectious disease that is associated with significant morbidity and mortality. The patterns of aetiological pathogens differ by region and country. Special attention must be paid to CAP in Southeast Asia (SEA), a region facing rapid demographic transition. Estimates burden and aetiological patterns of CAP are essential for the clinical and public health management. The purposes of the study are to determine the incidence, aetiological pathogens, clinical pictures and risk factors of community-acquired pneumonia (CAP) in the Vietnamese adult population.MethodsA prospective surveillance for hospitalised adult CAP was conducted in Khanh Hoa Province, Central Vietnam. All adults aged ≥15 years with lower respiratory tract infections (LRTI) admitted to a provincial hospital from September 2009 to August 2010 were enrolled in the study. Patients were classified into CAP and non-pneumonic LRTI (NPLRTI) according to the radiological findings. Bacterial pathogens were identified from sputum samples by the conventional culture and polymerase chain reaction (PCR) for Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis; 13 respiratory viruses were identified from nasopharyngeal specimens by PCR.ResultsOf all 367 LRTI episodes examined, 174 (47%) were CAP. Older age, the presence of underlying respiratory conditions, and higher index score of smoking were associated with CAP. The one-year estimated incidence of hospitalised adult CAP in our study population was 0.81 per 1,000 person years. The incidence increased considerably with age and was highest among the elderly. The case fatality proportion of hospitalised CAP patients was 9.8%. Among 286 sputum samples tested for bacterial PCR, 79 (28%) were positive for H. influenzae, and 65 (23%) were positive for S. pneumoniae. Among 357 samples tested for viral PCR, 73 (21%) were positive for respiratory viruses; influenza A (n = 32, 9%) was the most common.ConclusionsThe current adult CAP incidence in Vietnam was relatively low; this result was mainly attributed to the young age of our study population.

Association of RSV-A ON1 genotype with Increased Pediatric Acute Lower Respiratory Tract Infection in Vietnam

Since the initial discovery of RSV-A ON1 in Canada in 2010, ON1 has been reported worldwide, yet information regarding its clinical impact and severity has been controversial. To investigate the clinical relevance of RSV-A ON1,acute respiratory infection (ARI) cases enrolled to our population-based prospective pediatric ARI surveillance at Khanh Hoa General Hospital, Central Vietnam from January 2010 through December 2012 were studied. Clinical-epidemiological information and nasopharyngeal samples were collected. Multiplex PCR assays were performed for screening 13 respiratory viruses. RSV-positive samples were further tested for subgroups (A/B) and genotypes information by sequencing the G-glycoprotein 2nd hypervariable region. Statistical analysis was performed to evaluate the clinical-epidemiological characteristics of RSV-A ON1. A total of 1854 ARI cases were enrolled and 426 (23.0%) of them were RSV-positive. During the study period, RSV-A and B had been co-circulating. NA1 was the predominant RSV-A genotype until the appearance of ON1 in 2012. RSV-related ARI hospitalization incidence significantly increased after the emergence of ON1. Moreover, multivariate analysis revealed that risk of lower respiratory tract infection was 2.26 (95% CI: 1.37–3.72) times, and radiologically-confirmed pneumonia was 1.98 (95% CI: 1.01–3.87) times greater in ON1 compared to NA1 cases. Our result suggested that ON1 ARI cases were clinically more severe than NA1.

Mortality Associated With Pulmonary Hypertension in Congenital Rubella Syndrome

OBJECTIVE: Outbreaks of rubella and congenital rubella syndrome (CRS) continue to arise in various countries where a rubella-containing vaccine is not included in the national immunization program. After a large-scale rubella outbreak in 2011, CRS cases emerged in Vietnam. The aim of this study was to clarify the clinical features of these cases with an emphasis on cardiovascular complications and outcomes. METHODS: From October 2011 to September 2012, we conducted a prospective surveillance study of infants <12 months of age who had manifestations suggesting CRS at the only referral hospital in Khanh Hoa Province. These infants underwent standard examinations, echocardiography, cranial ultrasonography, automated auditory brainstem responses, blood cell count measurements, and rubella-specific antibody testing. Detected cardiovascular defects were regularly followed with echocardiography. RESULTS: We enrolled 38 cases of CRS characterized by a low birth weight (71%), cardiovascular defects (72%), cataracts (13%), hearing impairment (93%), purpura (84%), hepatosplenomegaly (68%), and thrombocytopenia (76%). Patent ductus arteriosus, the most common cardiovascular complication, was often associated with progressive pulmonary hypertension (PH). As of January 2013, 13 infants (34%) had died, and PH was significantly more frequent among the fatalities (P = .004); however, therapeutic closure of the ductus reversed the PH in several cases. CONCLUSIONS: PH-associated mortality is high among infants who have CRS in Vietnam. Providing proper assessments, continuous follow-up, and timely intervention for cardiovascular defects is critical for the management of CRS patients. Echocardiography is of diagnostic and prognostic value and can support better clinical management of CRS, even in a developing country setting.

Bacterial Load of Pneumococcal Serotypes Correlates with Their Prevalence and Multiple Serotypes Is Associated with Acute Respiratory Infections among Children Less Than 5 Years of Age

Background Among pneumococcal serotypes, some serotypes are more prevalent in the nasopharynx than others; determining factors for higher prevalence remain to be fully explored. As non-vaccine serotypes have emerged after the introduction of 7-valent conjugate vaccines, study of serotype specific epidemiology is in need. When two or more serotypes co-colonize, they evolve rapidly to defend hosts immune responses; however, a clear association of co-colonization with a clinical outcome is lacking. Methods Children less than 5 years old who were admitted to hospital due to acute respiratory infections (ARI) (n = 595) and healthy children (n = 350) were recruited. Carriage of pneumococcus was determined by culture and lytA PCR in the nasopharyngeal samples. Serotype/serogroup detection and its quantification were done by the nanofluidic real time PCR system. Spearmans correlation and logistic regression were used to examine a correlation of serotype/serogroup specific bacterial load with its prevalence and an association of co-colonization with ARI respectively. Results Serotype/serogroup specific bacterial load was correlated with its prevalence, both in ARI cases (Spearmans rho = 0.44, n = 186; P<0.0001) and healthy children (Spearmans rho = 0.41, n = 115; P<0.0001). The prevalence of multiple serotypes was more common in ARI cases than in healthy children (18.5% vs 7.1%; aOR 2.92, 95% CI: 1.27–6.71; P = 0.01). The dominant serotype in the co-colonization had a 2 log10 higher bacterial load than the subdominant serotype, both in ARI cases (P<0.001) and healthy children (P<0.05). Conclusions High bacterial load in the nasopharynx may help transmit pneumococci among hosts, and increase the chance of successful acquisition and colonization. Co-colonization of multiple serotypes of pneumococci is linked with ARI, which infers the interactions of multiple serotypes may increase their pathogenicity; however, they may compete for growth in number.

Molecular evolution of respiratory syncytial virus subgroup A genotype NA1 and ON1 attachment glycoprotein (G) gene in central Vietnam.

We performed molecular evolutionary analyses of the G gene C-terminal 3rd hypervariable region of RSV-A genotypes NA1 and ON1 strains from the paediatric acute respiratory infection patients in central Vietnam during the 2010-2012 study period. Time-scaled phylogenetic analyses were performed using Bayesian Markov Chain Monte Carlo (MCMC) method, and pairwise distances (p-distances) were calculated. Bayesian Skyline Plot (BSP) was constructed to analyze the time-trend relative genetic diversity of central Vietnam RSV-A strains. We also estimated the N-glycosylation sites within G gene hypervariable region. Amino acid substitutions under positive and negative selection pressure were examined using Conservative Single Likelihood Ancestor Counting (SLAC), Fixed Effects Likelihood (FEL), Internal Fixed Effects Likelihood (IFEL) and Mixed Effects Model for Episodic Diversifying Selection (MEME) models. The majority of central Vietnam ON1 strains detected in 2012 were classified into lineage 1 with few positively selected substitutions. As for the Vietnamese NA1 strains, four lineages were circulating during the study period with a few positive selection sites. Shifting patterns of the predominantly circulating NA1 lineage were observed in each year during the investigation period. Median p-distance of central Vietnam NA1 strains was wider (p-distance=0.028) than that of ON1 (p-distance=0.012). The molecular evolutionary rate of central Vietnam ON1 strains was estimated to be 2.55×10-2 (substitutions/site/year) and was faster than NA1 (7.12×10-3 (substitutions/site/year)). Interestingly, the evolutionary rates of both genotypes ON1 and NA1 strains from central Vietnam were faster than the global strains respectively. Furthermore, the shifts of N-glycosylation pattern within the G gene 3rd hypervariable region of Vietnamese NA1 strains were observed in each year. BSP analysis indicated the rapid growth of RSV-A effective population size in early 2012. These results suggested that the molecular evolution of RSV-A G gene detected in central Vietnam was fast with unique evolutionary dynamics.

Population Based Cohort Study for Pediatric Infectious Diseases Research in Vietnam

A population-based cohort study on pediatric infectious diseases was established at Khanh Hoa Province, central Vietnam in 2006, to determine the etiology and risk factors for severe pediatric infectious diseases (SPID) such as acute respiratory infection (ARI), diarrhea and dengue which are the major causes of under 5 mortality. A population census survey was conducted in Nha-Trang and Ninh-Hoa to collect demographic, social-behavioral data and disease burden on SPID. The study site covered a population of 353,525 residing in 75,826 households with 24,781 children less than 5 years. Hospital databases from two hospitals covering the region were obtained. Linking the census and hospital databases, we were able to investigate on a variety of SPID such as environmental tobacco smoking exposure and increased risked of pediatric pneumonia hospitalization, population density, water supply and risk of dengue fever and animal livestock and risk of hospitalized diarrhea. To determine incidence, viral etiology and risk factors for pediatric ARI/pneumonia, we setup a population based prospective hospitalized Pediatric ARI surveillance at Khanh Hoa General Hospital, Nha-Trang in February 2007. The study has revealed RSV, rhinovirus and influenza A as major viral pathogens, role of multiple viral infection and its interaction with bacteria in the development of pneumonia. In addition, we are also conducting a birth cohort study to investigate the incidence of congenital infection and its impact on physical-neurological development, and role of host genetic polymorphism on SPID hospitalization in Vietnam. Population mobility, high cost of regular census update and low mortality are the challenges.

Modeling the impact of rubella vaccination in Vietnam

Supported by GAVI Alliance, measles-rubella vaccination was introduced in Vietnam in 2014, involving a mass campaign among 1–14 year olds and routine immunization of children aged 9 months. We explore the impact on the incidence of Congenital Rubella Syndrome (CRS) during 2013–2050 of this strategy and variants involving women aged 15–35 years. We use an age and sex-structured dynamic transmission model, set up using recently-collected seroprevalence data from Central Vietnam, and also consider different levels of transmission and contact patterns. If the serological profile resembles that in Central Vietnam, the planned vaccination strategy could potentially prevent 125,000 CRS cases by 2050 in Vietnam, despite outbreaks predicted in the meantime. Targeting the initial campaign at 15–35 year old women with or without children aged 9 months–14 years led to sustained reductions in incidence, unless levels of ongoing transmission were medium-high before vaccination started. Assumptions about contact greatly influenced predictions if the initial campaign just targeted 15–35 year old women and/or levels of ongoing transmission were medium-high. Given increased interest in rubella vaccination, resulting from GAVI Alliance funding, the findings are relevant for many countries.

Early indication for a reduced burden of radiologically confirmed pneumonia in children following the introduction of routine vaccination against Haemophilus influenzae type b in Nha Trang, Vietnam

Abstract Introduction Despite the global success of Hib vaccination in reducing disease and mortality, uncertainty about the disease burden and the potential impact of Hib vaccination in Southeast Asia has delayed the introduction of vaccination in some countries in the region. Hib vaccination was introduced throughout Vietnam in July 2010 without catch-up. In an observational, population based surveillance study we estimated the impact of routine Hib vaccination on all cause radiologically confirmed childhood pneumonia in Nha Trang, Vietnam. Materials and methods In 2007 active hospital based surveillance was established in Khanh Hoa General Hospital, the only hospital in Nha Trang, Khanh Hoa province. Nasopharyngeal samples and chest radiographs are taken routinely from all children diagnosed with acute respiratory illness on admission. For admissions between 02/2007 and 03/2012 chest radiographs were interpreted for the presence of WHO primary endpoint pneumonia and nasopharyngeal swabs were analysed by PCR for the presence of Influenza A or B, RSV and rhinovirus. We employed Poisson regression to estimate the impact of Hib vaccination on radiologically confirmed pneumonia (RCP) while statistically accounting for potential differences in viral circulation in the post vaccination era which could have biased the estimate. Results Of 3151 cases admitted during the study period, 166 had RCP and major viruses were detected in 1601. The adjusted annual incidence of RCP in children younger than 5 years declined by 39% (12–58%) after introduction of Hib vaccination. This decline was most pronounced in children less than 2 years old, adjusted IRR: 0.52 (0.33–0.81), and no significant impact was observed in the 2–4 years old who were not eligible for vaccination, adjusted IRR: 0.96 (0.52–1.72). Discussion We present early evidence that the burden of Hib associated RCP in Nha Trang before vaccination was substantial and that shortly after introduction to the routine childhood immunisation scheme vaccination has substantially reduced that burden.