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Four endemic human coronaviruses (HCoV), HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43, are closely related to SARS-CoV-2. These coronaviruses are known to infect humans living in temperate areas, including children under 5 years old; however, the seroprevalence of four HCoVs among children in tropical areas, including the Philippines, remains unclear. This study aimed to assess the prevalence of antibodies against four HCoVs and to determine the reactivity and neutralization of these antibodies against SARS-CoV-2 among children in the Philippines. A total of 315 serum samples collected from 2015 to 2018, before the emergence of SARS-CoV-2, in Biliran island, Philippines, were tested for the presence of antibodies against four HCoVs and SARS-CoV-2 using recombinant spike ectodomain proteins by IgG-enzyme-linked immunosorbent assay (ELISA). Reactivity to and neutralization of SARS-CoV-2 were also investigated. The seroprevalence of the four HCoVs was 63.8% for HCoV-229E, 71.4% for HCoV-NL63, 76.5% for HCoV-HKU1, and 83.5% for HCoV-OC43 by ELISA. Age group analysis indicated that seropositivity to all HCoVs reached 80% by 2-3 years of age. While 69/315 (21.9%) of the samples showed reactive to SARS-CoV-2, almost no neutralization against SARS-CoV-2 was detected using neutralization assay. Reactivity of antibodies against SARS-CoV-2 spike protein obtained by ELISA may not correlate with neutralization capability., (© 2023. The Author(s).)

Objectives: Human adenoviruses (HAdV) are known to cause a wide range of diseases including acute respiratory infections, conjunctivitis, and acute gastroenteritis. In this study, we aimed to determine the serotypes of HAdV in patients with influenza-like illness (ILI) in the Philippines from 2006-2012 and to describe the demographic and epidemiological characteristics of patients who tested positive for HAdV., Methods: Between 2006 and 2012, the Philippine National Influenza Centre detected HAdV in 1294 samples of patients with ILI. Serotype determination was done in select samples using microneutralization, polymerase chain reaction (PCR), and sequencing methods., Results: A total of 8 serotypes were identified (HAdV 1-7 and 11), with HAdV-2 (27.8%), and HAdV-3 (27.8%) being the most prevalent. The majority of HAdV infections were found in children below 5 years of age (79.9%)., Conclusions: The identification of HAdV circulating serotypes may serve as guide for designing disease intervention and control strategies and will provide important information regarding the contribution of this virus to respiratory infections, particularly in children, which remain a public health burden in the Philippines., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Lower respiratory tract infection (LRTI) is an important cause of morbidity and mortality in infants and young children. However, the etiological role of viruses and the timing of developing LRTI are not well defined., Methods: We analyzed the data of a prospective cohort study in the Philippines as a birth cohort. We detected LRTI among children who visited healthcare facilities with respiratory symptom, and collected nasopharyngeal swabs for virus detection. We analyzed the incidence rates (IRs) and cumulative proportion of LRTI and severe LRTI by age group and each virus detected., Results: A total of 350 LRTI episodes were observed from 473 child-years yielded from 419 children. The IRs of LRTI were 70.8, 70.7, and 80.8 per 100 child-years for 0-5, 6-11, and 12-23 months of age, respectively. By 12 months of age, 45% of children developed LRTI at least once. Rhinovirus and respiratory syncytial virus were the most frequently detected viruses in all age groups. However, the IRs of influenza virus were low especially at 0-5 months of age., Conclusions: We identified various patterns of age-specific IRs of LRTI and severe LRTI for different viruses, which should be considered to establish more effective interventions including vaccinations., (© 2022. The Author(s).)

Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infection worldwide, but reports of temporal changes in the risk of transmission among close contacts has been scarce. This study aimed to examine an association between the viral load trajectory and transmission risk to develop a better control strategy for the disease spread. We conducted a household-based prospective cohort study in Biliran Province, the Philippines, and enrolled 451 participants to observe the development of acute respiratory infection. Including the cases found at the health-care facility, we analyzed the data of viral loads with symptom records obtained from 172 followed participants who had household member positive for RSV with a rapid test during an RSV outbreak in 2018-2019. We developed a model estimating a temporal change in the viral shedding from the infection and evaluated transmission dynamics. We found that most transmission events occurred within approximately 7 days of the household exposure, including potential presymptomatic transmissions. The inferred risk of infection among those younger than 5 years was 3.5 times higher than that of those older than 5 years. This finding suggested that the initial week after the household exposure is particularly important for preventing RSV spread., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health.)

Introduction. The Philippines, comprising three island groups, namely, Luzon, Visayas and Mindanao, experienced an increase in cholera outbreaks in 2016. Previous studies have shown that Vibrio cholerae isolates obtained from the Philippines are novel hybrid El Tor strains that have evolved in the country and are clearly distinct from those found in Mozambique and Cameroon. Gap statement. The characterization of the strains isolated from outbreaks has been limited to phenotypic characteristics, such as biochemical and serological characteristics, in most previous studies. Aim. We performed multilocus variable-number tandem repeat (VNTR) analysis (MLVA) for V. cholerae isolates obtained from 2015 to 2016 to further characterize and understand the emergence and dissemination of the strains in the Philippines. Methodology. A total of 139 V . cholerae O1 Ogawa biotype El Tor isolates were obtained from the Philippines during diarrhoeal outbreaks in 18 provinces between 2015 and 2016. VNTR data were analysed to classify the MLVA profiles where the large-chromosome types (LCTs) were applied for grouping. Results. We identified 50 MLVA types among 139 isolates originating from 18 provinces, and 14 LCTs. The distribution of the LCTs was variable, and a few were located in specific areas or even in specific provinces. Based on eBURST analysis, 99 isolates with 7 LCTs and 32 MLVA types belonged to 1 group, suggesting that they were related to each other. LCT A was predominant ( n =67) and was isolated from Luzon and Visayas. LCT A had 14 MLVA types; however, it mostly emerged during a single quarter of a year. Eight clusters were identified, each of which involved specific MLVA type(s). The largest cluster involved 23 isolates showing 3 MLVA types, 21 of which were MLVA type A-14 isolated from Negros Occidental during quarter 4 of 2016. Comparative analysis showed that almost all isolates from the Philippines were distinct from those in other countries. Conclusions. The genotypic relationship of the V. cholerae isolates obtained during outbreaks in the Philippines was studied, and their emergence and dissemination were elucidated. MLVA revealed the short-term dynamics of V. cholerae genotypes in the Philippines.

Objectives: Viral acute respiratory infection (ARI) remains a major global health problem, especially among children in low- and middle-income countries. The study was conducted to reveal aetiological significance of respiratory viruses among both non-hospitalized and hospitalized children., Methods: A cohort study of children with ARI at the household, primary healthcare facility, and hospital levels was conducted alongside a hospital-based study including non-cohort children from 2014 to 2016 in the Philippines. The ARI cases were recorded at households and healthcare facilities, and a clinical investigation was performed. Nasopharyngeal swabs were collected from the symptomatic children and tested for respiratory viruses via polymerase chain reaction. Then, the association between healthcare facility utilization and viral detection was investigated., Results: Overall, 18,514 ARI cases were enrolled in the cohort study, and samples were collected from 4735 of these cases. The hospital-based study detected 648 ARI cases, all of which were sampled. Rhinovirus (22.2%; 1052/4735) was most frequently detected followed by respiratory syncytial virus (12.0%; 566/4735). Enterovirus (adjusted odds ratio, 1.8; 95% confidence interval, 1.1-2.8), human metapneumovirus (2.1, 1.4-3.2), rhinovirus (2.1, 1.8-2.6), and respiratory syncytial virus (1.6, 1.2-1.9) were significantly more prevalent in the ARI cases at healthcare facilities than in those in households. Of all ARI cases, 0.6% required hospitalization while 1.8% were hospitalized among the respiratory syncytial virus-positive cases (3.8, 3.0-4.9)., Conclusions: We determined the prevalence of respiratory viruses among children with ARIs at the household, primary healthcare facility, and hospital levels and the association with clinical characteristics. In particular, we discovered a significant disease burden and impact of respiratory syncytial virus infections as well as a considerable aetiological implication of rhinovirus infections., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Vector control measures are critical for the prevention and reduction of dengue virus (DENV) transmission. Effective vector control is reliant not only on knowledge of mosquito abundance, but also on the timely and accurate detection of mosquito-borne infection. Mosquito-based virus surveillance programs typically rely on pool-based mosquito testing, although whether individual-based mosquito testing is a feasible alternative to this has not been widely studied. Applying an individual-based mosquito testing approach, we conducted a 1-month surveillance study of DENV in adult Aedes aegypti mosquitoes in homes of suspected dengue patients during the 2015 peak dengue season in Tarlac City, Philippines to more accurately assess the mosquito infection rate and identify the DENV serotypes and genotypes concurrently co-circulating in mosquitoes and patients there., Methods: We performed a one-step multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection and serotyping of DENV in patients and individual female Ae. aegypti mosquitoes. Additionally, we performed sequencing and phylogenetic analyses to further characterize the detected DENV serotypes in mosquitoes and patients at the genotype level., Results: We collected a total of 583 adult Ae. aegypti mosquitoes, of which we individually tested 359 female mosquitoes for the presence of DENV. Ten (2.8%) of the 359 female mosquitoes were positive for the presence of DENV. We detected DENV-1, DENV-2, and DENV-4 in the field-collected mosquitoes, which was consistent with the serotypes concurrently found in infected patients. Sequencing and phylogenetic analyses of the detected DENV serotypes based on the partial sequence of the evelope (E) gene revealed three genotypes concurrently present in the sampled mosquitoes and patients during the study period, namely DENV-1 genotype IV, DENV-2 Cosmopolitan genotype, and DENV-4 genotype II., Conclusions: We demonstrated the utility of a one-step multiplex real-time RT-PCR assay for the individual-based DENV surveillance of mosquitoes. Our findings reinforce the importance of detecting and monitoring virus activity in local mosquito populations, which are critical for dengue prevention and control.

Rotaviruses are the major cause of severe acute diarrhea in infants and young children. Rotaviruses exhibit zoonosis and thereby infect both humans and animals. Viruses detected in urban rivers possibly reflect the presence of circulating viruses in the catchment. The present study investigates the genetic diversity of species A rotaviruses detected from river water and stool of hospitalized children with acute diarrhea in Tacloban City, the Philippines. Species A rotaviruses were detected by real-time RT-PCR and their genotypes were identified by multiplex PCR and sequencing of partial regions of VP7 and VP4. Rotaviruses were detected in 85.7% (30/35) of the river water samples and 62.7% (151/241) of the clinical samples. Genotypes of VP7 in the river water samples were G1, G2, G3, G4, G5, and G9, and those of VP4 were P[3], P[4], P[6], P[8], and P[13]. Genotypes of viruses from the clinical samples were G2P[4], G1P[8], G3P[8], G4P[6], G5P[6], and G9P[8]. Among those, G2P[4] in clinical samples (77.9%, 81/104) and P[4] of VP4 in river water samples (67.5%, 56/83)) were the most frequently detected rotavirus genotypes. However, G5 was the more frequently detected than G2 in the river water samples (42% vs. 13%) which may be originated from porcine rotavirus. Sequence analyses of eleven gene segments revealed one G5P[6] and two G4P[6] rotaviruses in the clinical samples, wherein, several gene segments were closely related to porcine rotaviruses. The constellation of these rotavirus genes suggests the emergence of reassortment between human and porcine rotavirus due to interspecies transmission. Although two commercial rotavirus vaccines are available now, these vaccines are designed to confer immunity against the major human rotaviruses. Constant monitoring of viral variety in populated areas where humans and domestic animals live in close proximity provides vital information related to the diversity of rotaviruses in a human population., (Copyright © 2020. Published by Elsevier B.V.)

This study aimed to evaluate the prevalence and genetic characteristics of carbapenemase-producing Enterobacteriaceae (CPE) in hospital sewage and river water in the Philippines, which has a typical tropical maritime climate. We collected 83 water samples from 7 hospital sewage and 10 river water sites. CPE were identified using CHROMagar mSuperCARBA, and Gram-negative strains were identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) or 16S rRNA gene sequencing. Resistance genes in Enterobacteriaceae strains were identified using PCR and DNA sequencing, and transferability of carbapenemase genes from the CPE was investigated with conjugation experiments. Genotyping was performed using multilocus sequence typing (MLST) for Escherichia coli and Klebsiella pneumoniae Out of 124 Enterobacteriaceae isolates, we identified 51 strains as CPE and divided these into 7 species, 11 E. coli , 14 Klebsiella spp., 15 Enterobacter spp., and 11 others, including 4 additional species. Conjugation experiments via broth mating and using E. coli J53 revealed that 24 isolates can transfer carbapenemase-encoding plasmids. MLST analysis showed that 6 of 11 E. coli isolates belonged to clonal complex 10 (CC10). Of 11 K. pneumoniae strains, 9 unique sequence types (STs) were identified, including ST147. Five types of carbapenemase genes were identified, with the most prevalent being NDM ( n  = 39), which is epidemic in clinical settings in the Philippines. E. coli CC10 and K. pneumoniae ST147, which are often detected in clinical settings, were the dominant strains. In summary, our results indicate that hospital sewage and river water are contaminated by CPE strains belonging to clinically important clonal groups. IMPORTANCE Carbapenemase-producing Enterobacteriaceae (CPE) cause severe health care-associated infections, and their increasing prevalence is a serious concern. Recently, natural ecosystems have been recognized as important reservoirs of antibiotic resistance genes. We investigated the prevalence and genetic characteristics of CPE isolated from the environment (hospital sewage and river water) in the Philippines and found several CPE, including Escherichia coli and other species, with different carbapenemases. The most prevalent carbapenemase gene type was NDM, which is endemic in clinical settings. This study revealed that isolates belonging to carbapenemase-producing E. coli CC10 and K. pneumoniae sequence type 147 (ST147), which are often detected in clinical settings, were dominant in the natural environment. Our work here provides a report on the presence and characteristics of CPE in the environment in the Philippines and demonstrates that both hospital sewage and river water are contaminated by CPE strains belonging to clinically important clonal groups., (Copyright © 2020 Suzuki et al.)

Background: Respiratory syncytial virus (RSV) is one of the main viral causes of lower respiratory tract illness (LRTI), especially in young children. RSV vaccines, including maternal and infant vaccines, are under development; however, more epidemiological studies are needed to develop effective vaccination strategies., Objectives: To estimate detailed age-specific incidence rates and severity of RSV-associated LRTI (RSV-LRTI) using data from a community-based prospective cohort study in the Philippines., Patients/methods: Cohort children who visited health facilities due to acute respiratory symptoms were identified, and nasopharyngeal swabs were collected to detect RSV. The severity of RSV-LRTI was assessed using the severity definition proposed by the World Health Organization. Risk factors for developing RSV-LRTI and contribution of SpO 2 measurement were also evaluated., Results: A total of 395 RSV episodes which occurred in children aged 2-59 months were categorised as 183 RSV-LRTI, 72 as severe RSV-LRTI and 29 as very severe RSV-LRTI. Children aged 3-5 months had the highest incidence rate of RSV-LRTI, at 207.4 per 1000 child-years (95% CI: 149.0-279.5). Younger age group, place of living and low educational level of caregivers were associated with developing RSV-LRTI. Clinical manifestations had low levels of agreement with hypoxaemia as measured by pulse oximeter., Conclusion: The highest burden of RSV was observed in young infants aged 3-5 months, whereas the burden was also high in those aged 12-20 months. Future vaccination strategies should consider the protection of older children, especially those aged one year, as well as young infants., (© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Background: Several inactivated enterovirus-A71 (EV-A71) vaccines are currently licensed in China; however, the development of additional EV-A71 vaccines is ongoing, necessitating extensive analysis of the molecular epidemiology of the virus worldwide. Until 2012, laboratory confirmation of EV-A71 for hand, foot, and mouth disease (HFMD) and other associated diseases had not occurred in the Philippines. Because EV-A71 has been linked with cases of acute flaccid paralysis (AFP), AFP surveillance is one strategy for documenting its possible circulation in the country. To expand current knowledge on EV-A71, molecular epidemiologic analysis and genetic characterization of EV-A71 isolates were performed in this study., Methods: A retrospective study was performed to identify and characterize nonpolio enteroviruses (NPEVs) associated with AFP in the Philippines, and nine samples were found to be EV-A71-positive. Following characterization of these EV-A71 isolates, the complete viral protein 1 (VP1) gene was targeted for phylogenetic analysis., Results: Nine EV-A71 isolates detected in 2000 (n = 2), 2002 (n = 4), 2005 (n = 2), and 2010 (n = 1) were characterized using molecular methods. Genomic regions spanning the complete VP1 region were amplified and sequenced using specific primers. Phylogenetic analysis of the full-length VP1 region identified all nine EV-A71 Philippine isolates as belonging to the genogroup C lineage, specifically the C2 cluster. The result indicated a genetic linkage with several strains isolated in Japan and Taiwan, suggesting that strains in the C2 cluster identified in the Asia-Pacific region were circulating in the Philippines., Conclusion: The study presents the genetic analysis of EV-A71 in the Philippines. Despite some limitations, the study provides additional genetic data on the circulating EV-A71 strains in the Asia-Pacific region, in which information on EV-A71 molecular epidemiology is incomplete. Considering that EV-A71 has a significant public health impact in the region, knowledge of its circulation in each country is important, especially for formulating vaccines covering a wide variety of strains.

Objective: Pneumonia remains the leading cause of hospitalisations and deaths among children aged <5 years. Diverse respiratory pathogens cause acute respiratory infections, including pneumonia. Here, we analysed viral and bacterial pathogens and risk factors associated with death of hospitalised children., Design: A 9-year case series study., Setting: Two secondary-care hospitals, one tertiary-care hospital and one research centre in the Philippines., Participants: 5054 children aged <5 years hospitalised with severe pneumonia., Methods: Nasopharyngeal swabs for virus identification, and venous blood samples for bacterial culture were collected. Demographic, clinical data and laboratory findings were collected at admission time. Logistic regression analyses were performed to identify the factors associated with death., Results: Of the enrolled patients, 57% (2876/5054) were males. The case fatality rate was 4.7% (238/5054), showing a decreasing trend during the study period (p<0.001). 55.0% of the patients who died were either moderately or severely underweight. Viruses were detected in 61.0% of the patients, with respiratory syncytial virus (27.0%) and rhinovirus (23.0%) being the most commonly detected viruses. In children aged 2-59 months, the risk factors significantly associated with death included age of 2-5 months, sensorial changes, severe malnutrition, grunting, central cyanosis, decreased breath sounds, tachypnoea, fever (≥38.5°C), saturation of peripheral oxygen <90%, infiltration, consolidation and pleural effusion on chest radiograph.Among the pathogens, adenovirus type 7, seasonal influenza A (H1N1) and positive blood culture for bacteria were significantly associated with death. Similar patterns were observed between the death cases and the aforementioned factors in children aged <2 months., Conclusion: Malnutrition was the most common factor associated with death and addressing this issue may decrease the case fatality rate. In addition, chest radiographic examination and oxygen saturation measurement should be promoted in all hospitalised patients with pneumonia as well as bacteria detection to identify patients who are at risk of death., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Hepatitis A virus (HAV) is a common infectious etiology of acute hepatitis worldwide. The Philippines remains highly endemic for hepatitis A, but there is still a lack of information about HAV in the country. To evaluate the HAV contamination in environmental water in the Philippines, we conducted the detection and genetic analyses of HAV RNA in samples from river water. Twelve water samples were collected at 6 sampling sites of 3 rivers in Metro Manila, in both the dry and wet seasons in 2012 and 2013. The HAV RNA was detected in all the 6 samples collected in the dry season, and in one sample from the wet season. Phylogenetic analysis confirmed that the HAV strains detected in the river water included multiple sequences belonging to subgenotypes IA and IIIA. This indicates that at least 2 genotypes of the HAV strains are circulating in the environment in the Philippines, posing a risk of HAV infection to not only residents, but also tourists, especially in the dry season.

Background: Acute respiratory infection (ARI) is of great concern in public health. It remains unclear whether viral infections can affect the host's susceptibility to subsequent ARIs., Methods: A prospective cohort study on ARIs of children below 5 years old was conducted in the Philippines from 2014 to 2016. The respiratory symptoms were recorded daily, and nasopharyngeal swabs were collected at both household and health facilities. The specimens were tested for respiratory viruses. We then determined whether viral etiology was associated with the severity of the present ARI and whether previous viral infections was associated with subsequent ARIs., Results: A total of 3851 children and 16337 ARI episodes were enrolled and recorded, respectively. Samples were collected from 24% of all ARI episodes; collection rate at the healthcare facilities was 95%. Enterovirus D68, rhinovirus species C, and respiratory syncytial virus were significantly associated with severe ARIs. The risk for subsequent ARIs was significantly enhanced after infections with adenovirus, influenza A virus, parainfluenza virus type 4, and rhinovirus species C., Conclusions: This study revealed that viral etiology plays a significant role in the severity of the present ARI and that viral infection affects the host's susceptibility to subsequent ARIs.

Background: Human respiratory syncytial virus (RSV) is the leading cause of severe acute respiratory infection in infants and young children, which is characterized by repeated infections. However, the role of amino acid substitutions in repeated infections remains unclear. Hence, this study aimed to elucidate the genetic characteristics of RSV in children with repeated infections using molecular analyses of F and G genes., Methods: We conducted a cohort study of children younger than 5 years in the Philippines. We collected nasopharyngeal swabs from children with acute respiratory symptoms and compared F and G sequences between initial and subsequent RSV infections., Results: We examined 1802 children from May 2014 to January 2016 and collected 3471 samples. Repeated infections were observed in 25 children, including 4 with homologous RSV-B reinfections. Viruses from the 4 pairs of homologous reinfections had amino acid substitutions in the G protein mostly at O-glycosylation sites, whereas changes in the F protein were identified at antigenic sites V (L173S) and θ (Q209K), considered essential epitopes for the prefusion conformation of the F protein., Conclusions: Amino acid substitutions in G and F proteins of RSV-B might have led to antigenic changes, potentially contributing to homologous reinfections observed in this study.

A case-comparison study was conducted based on an observational study of severe pneumonia among hospitalized children in the Philippines. The children, from 8days to 13years old and hospitalized with clinical diagnosis of severe or very severe pneumonia from August 2012 to February 2015, were recruited. Nasopharyngeal swabs were collected from 1152 cases and B. pertussis were detected from 34 cases by PCR. Pertussis-positive cases were more likely to have no fever, more than one week of coughing and breathing difficulty, decreased breathing sounds, and central cyanosis than pertussis- negative cases. The percentage of underweight was significantly higher in pertussis-positive cases than pertussis-negative cases. Pertussis-positive cases showed remarkably higher fatality rate than pertussis-negative cases. All of the fatal cases among pertussis-positive cases were less than 6months old. More attention should be given to protect young infants from pertussis., (Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The local and global transmission dynamics of influenza B virus is not completely understood mainly because of limited epidemiological and sequence data for influenza B virus. Here we report epidemiological and molecular characteristics of influenza B viruses from 2010 to 2013 in Leyte Island, Philippines. Phylogenetic analyses showed global dissemination of the virus among both neighboring and distant areas. The analyses also suggest that southeast Asia is not a distributor of influenza B virus and can introduce the virus from other areas. Furthermore, we found evidence on the local persistence of the virus over years in the Philippines. Taken together, both local persistence and global dissemination play a significant role in the circulation of influenza B virus., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Human respiratory syncytial virus (HRSV) is a major cause of acute lower respiratory tract infections in infants and children worldwide. We performed molecular analysis of HRSV among infants and children with clinical diagnosis of severe pneumonia in four study sites in the Philippines, including Biliran, Leyte, Palawan, and Metro Manila from June 2012 to July 2013. Nasopharyngeal swabs were collected and screened for HRSV using real-time polymerase chain reaction (PCR). Positive samples were tested by conventional PCR and sequenced for the second hypervariable region (2nd HVR) of the G gene. Among a total of 1,505 samples, 423 samples were positive for HRSV (28.1%), of which 305 (72.1%) and 118 (27.9%) were identified as HRSV-A and HRSV-B, respectively. Two genotypes of HRSV-A, NA1 and ON1, were identified during the study period. The novel ON1 genotype with a 72-nucleotide duplication in 2nd HVR of the G gene increased rapidly and finally became the predominant genotype in 2013 with an evolutionary rate higher than the NA1 genotype. Moreover, in the ON1 genotype, we found positive selection at amino acid position 274 (p<0.05) and massive O- and N-glycosylation in the 2nd HVR of the G gene. Among HRSV-B, BA9 was the predominant genotype circulating in the Philippines. However, two sporadic cases of GB2 genotype were found, which might share a common ancestor with other Asian strains. These findings suggest that HRSV is an important cause of severe acute respiratory infection among children in the Philippines and revealed the emergence and subsequent predominance of the ON1 genotype and the sporadic detection of the GB2 genotype. Both genotypes were detected for the first time in the Philippines.

Background: Human sapovirus (SaV) is a causative agent of acute gastroenteritis. Recently, SaV detection has been increasing worldwide due to the emerging SaV genotype I.2. However, SaV infection has not been reported in the Philippines., Objectives: To evaluate the prevalence and genetic diversity of SaV in hospitalized children aged less than 5 years with acute gastroenteritis., Study Design: Stool samples were collected from children with acute gastroenteritis at three hospitals in the Philippines from June 2012 to August 2013. SaV was detected by reverse transcription real-time PCR, and the polymerase and capsid gene sequences were analyzed. Full genome sequencing and recombination analysis were performed on possible recombinant viruses., Results: SaV was detected in 7.0% of the tested stool samples (29/417). In 10 SaV-positive cases, other viruses were also detected, including rotavirus (n=6), norovirus (n=2), and human astrovirus (n=2). Four known SaV genotypes (GI.1 [7], GI.2 [2], GII.1 [12], and GV [2]) and one novel recombinant (n=3) were identified by polymerase and capsid gene sequence analysis. Full genome sequencing revealed that the 5' nontranslated region (NTR) and nonstructural protein region of the novel recombinant were closely related to the GII.1 Bristol/98/UK variant, whereas the structural protein region and 3' NTR were closely related to the GII.4 Kumamoto6/Mar2003/JPN variant., Discussion and Conclusions: SaV was regularly detected in hospitalized children due to acute gastroenteritis during the study period. A novel recombinant, SaV GII.1/GII.4, was identified in three cases at two different study sites., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Large outbreaks of measles occurred in the Philippines in 2010 and 2011. Genetic analysis was performed to identify the genotype of measles virus (MeV) that was responsible for the large outbreaks., Methods: A total of 114 representative MeVs that were detected in the Philippines from 2008 to 2011 were analyzed by sequencing the C-terminal region of nucleocapsid (N) gene and partial hemagglutinin (H) gene and by inferring the phylogenetic trees., Results: Genetic analysis showed that genotype D9 was the predominant circulating strain during the 4-year study period. Genotype D9 was detected in 23 samples (92%) by N gene sequencing and 93 samples (94%) by H gene analysis. Sporadic cases of genotype G3 MeV were identified in 2 samples (8%) by N gene sequencing and 6 samples (6%) by H gene analysis. Genotype G3 MeV was detected mainly in Panay Island in 2009 and 2010. Molecular clock analysis of N gene showed that the recent genotype D9 viruses that caused the big outbreaks in 2010 and 2011 diverged from a common ancestor in 2005 in one of the neighboring Southeast Asian countries, where D9 was endemic. These big outbreaks of measles resulted in a spillover and were associated with genotype D9 MeV importation to Japan and the USA., Conclusion: Genotype D9 MeV became endemic and caused two big outbreaks in the Philippines in 2010 and 2011. Genotype G3 MeV was detected sporadically with limited geographic distribution. This study highlights the importance of genetic analysis not only in helping with the assessment of measles elimination program in the country but also in elucidating the transmission dynamics of measles virus.

Objectives: The present study was designed to determine the genotypes of circulating Bordetella pertussis in the Philippines by direct molecular typing of clinical specimens., Methods: Nasopharyngeal swabs (NPSs) were collected from 50 children hospitalized with pertussis in three hospitals during 2012-2014. Multilocus variable-number tandem repeat analysis (MLVA) was performed on the DNA extracts from NPSs. B. pertussis virulence-associated allelic genes (ptxA, prn, and fim3) and the pertussis toxin promoter, ptxP, were also investigated by DNA sequence-based typing., Results: Twenty-six DNA extracts yielded a complete MLVA profile, which were sorted into 10 MLVA types. MLVA type 34 (MT34), which is rare in Australia, Europe, Japan, and the USA, was the predominant strain (50%). Seven MTs (MT29, MT32, MT33, and MT283-286, total 42%) were single-locus variants of MT34, while two (MT141 and MT287, total 8%) were double-locus variants of MT34. All MTs had the combination of virulence-associated allelic genes, ptxP1-ptxA1-prn1-fim3A., Conclusions: The B. pertussis population in the Philippines comprises genetically related strains. These strains are markedly different from those found in patients from other countries where acellular pertussis vaccines are used. The differences in vaccine types between these other countries and the Philippines, where the whole-cell vaccine is still used, may select for distinct populations of B. pertussis., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This study aimed to determine the role of influenza-like illness (ILI) surveillance conducted on Leyte Island, the Philippines, including involvement of other respiratory viruses, from 2010 to 2013. ILI surveillance was conducted from January 2010 to March 2013 with 3 sentinel sites located in Tacloban city, Palo and Tanauan of Leyte Island. ILI was defined as fever ≥38°C or feverish feeling and either cough or running nose in a patient of any age. Influenza virus and other 5 respiratory viruses were searched. A total of 5,550 ILI cases visited the 3 sites and specimens were collected from 2,031 (36.6%) cases. Among the cases sampled, 1,637 (75.6%) were children aged <5 years. 874 (43.0%) cases were positive for at least one of the respiratory viruses tested. Influenza virus and respiratory syncytial virus (RSV) were predominantly detected (both were 25.7%) followed by human rhinovirus (HRV) (17.5%). The age distributions were significantly different between those who were positive for influenza, HRV, and RSV. ILI cases were reported throughout the year and influenza virus was co-detected with those viruses on approximately half of the weeks of study period (RSV in 60.5% and HRV 47.4%). In terms of clinical manifestations, only the rates of headache and sore throat were significantly higher in influenza positive cases than cases positive to other viruses. In conclusion, syndromic ILI surveillance in this area is difficult to detect the start of influenza epidemic without laboratory confirmation which requires huge resources. Age was an important factor that affected positive rates of influenza and other respiratory viruses. Involvement of older age children may be useful to detect influenza more effectively.

From November 2009 to December 2013 in the Philippines, 15 influenza C viruses were isolated, using MDCK cells, from specimens obtained from children with severe pneumonia and influenza-like illness (ILI). This is the first report of influenza C virus isolation in the Philippines. In addition, from January 2008 to December 2013, 7 influenza C viruses were isolated from specimens that were obtained from children with acute respiratory illness (ARI) in Sendai city, Japan. Antigenic analysis with monoclonal antibodies to the hemagglutinin-esterase (HE) glycoprotein showed that 19 strains (12 from the Philippines and 7 from Japan) were similar to the influenza C virus reference strain C/Sao Paulo/378/82 (SP82). Phylogenetic analysis of the HE gene showed that the strains from the Philippines and Japan formed distinct clusters within an SP82-related lineage. The clusters that included the Philippine and Japanese strains were shown to have diverged from a common ancestor around 1993. In addition, phylogenetic analysis of the internal genes showed that all strains isolated in the Philippines and Japan had emerged through reassortment events. The composition of the internal genes of the Philippine strains was different from that of the Japanese strains, although all strains were classified into an SP82-related lineage by HE gene sequence analysis. These observations suggest that the influenza C viruses analyzed here had emerged through different reassortment events; however, the time and place at which the reassortment events occurred were not determined., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Enterovirus D68 (EV-D68) has been recognized as an important cause of acute respiratory infections. Here we report the molecular epidemiology of EV-D68 in Philippines from 2013 to 2014; we found cases in areas affected by Typhoon Haiyan and found new strains in the country., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: Hepatitis E virus (HEV) infection is a significant public health concern in Asia, and swine is an important source of sporadic HEV infection in human. However, no epidemiological data are available regarding HEV infection among the swine or human population in the Philippines. To assess the HEV infection status among pigs in rural areas, we investigated the molecular characteristics and seroprevalence of HEV among household-raised pigs in San Jose, Tarlac Province, the Philippines., Result: Serum and rectal swab samples were collected from 299 pigs aged 2-24 months from 155 households in four barangays (villages) between July 2010 and June 2011. Enzyme-linked immunosorbent assay (ELISA) revealed that 50.3% [95% confidence interval (CI) 44.5-56.2%] and 22.9% (95% CI 18.2-28.1%) of pigs tested positive for anti-HEV IgG and IgM, respectively. HEV RNA was detected in the feces of 22 pigs (7.4%, 95% CI 4.7-10.9%). A total of 103 households (66.5%, 95% CI 58.4-73.8%) had at least one pig that tested positive for anti-HEV IgG or IgM or HEV RNA. The prevalence of anti-HEV IgG and IgM in breeding pig (8-24 months) were higher than that in growing pigs (2-4 months) (p < 0.0001 and p = 0.008, respectively). HEV RNA was more frequently detected in 2-4-month-old pigs (9.2%, 95% CI 5.4-14.6%) than in ≥5-month-old pigs (4.8%, 95% CI 1.1-8.5%) without statistical significance (p = 0.142). HEV RNA showed 0-27.6% nucleotide difference at the partial ORF2 gene among the detected viruses, and a majority of them belonged to subtype 3a (20/22, 90.9%)., Conclusion: We found a high prevalence of HEV antibodies in the household-raised pig population in rural areas of the Philippines, which indicates the potential risk of HEV infection among local residents. Only genotype 3 of HEV was observed, and genetically diverse strains of HEV were found to be circulating in pigs in this study.

Rabies still remains a public health threat in the Philippines. A significant number of human rabies cases, about 200-300 cases annually, have been reported, and the country needs an effective strategy for rabies control. To develop an effective control strategy, it is important to understand the transmission patterns of the rabies viruses. We conducted phylogenetic analyses by considering the temporal and spatial evolution of rabies viruses to reveal the transmission dynamics in the Philippines. After evaluating the molecular clock and phylogeographic analysis, we estimated that the Philippine strains were introduced from China around the beginning of 20th century. Upon this introduction, the rabies viruses evolved within the Philippines to form three major clades, and there was no indication of introduction of other rabies viruses from any other country. However, within the Philippines, island-to-island migrations were observed. Since then, the rabies viruses have diffused and only evolved within each island group. The evolutionary pattern of these viruses was strongly shaped by geographical boundaries. The association index statistics demonstrated a strong spatial structure within the island group, indicating that the seas were a significant geographical barrier for viral dispersal. Strong spatial structure was also observed even at a regional level, and most of the viral migrations (79.7% of the total median number) in Luzon were observed between neighboring regions. Rabies viruses were genetically clustered at a regional level, and this strong spatial structure suggests a geographical clustering of transmission chains and the potential effectiveness of rabies control that targets geographical clustering. Dog vaccination campaigns have been conducted independently by local governments in the Philippines, but it could be more effective to implement a coordinated vaccination campaign among neighboring areas to eliminate geographically-clustered rabies transmission chains., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

To understand the hepatitis E virus (HEV)-pollution status in the environment in the Philippines, a total of 12 water samples were collected from rivers in Manila City for detection of HEV RNA. Three of 12 samples were positive for HEV RNA indicating that HEV is circulating in the Philippines. Phylogenetic analysis classified all of the HEV sequences into genotype 3.

Human adenovirus (HAdV) serotype 7 is an important etiological agent of severe childhood pneumonia. The aim of this study was to define the role of HAdV7 and to describe its clinical and molecular epidemiological characteristics in the Philippines in 2011. HAdVs were detected by viral culture, and a partial region of hexon gene was sequenced. A total of 700 patients were enrolled, of which 22 (3.1%) died. Nine (1.3%) HAdV cases were confirmed, of which 7 were positive for HAdV7, 1 for HAdV3, and 1 for HAdV5. Among the 9 HAdV-positive cases, 4 (44%) with HAdV7 died. Molecular analysis revealed that all HAdV7 isolates were closely related to genome type h strains. This study demonstrated the significance of HAdV7 as an etiological agent of severe pediatric pneumonia with a high fatality rate. Hence, continuous monitoring is required to define the clinical and public health significance of HAdV7 infection.

Background: Detection of Enterovirus 68 (EV68) has recently been increased. However, underlying evolutionary mechanism of this increasing trend is not fully understood., Methods: Nasopharyngeal swabs were collected from 5,240 patients with acute respiratory infections in the Philippines from June 2009 to December 2011. EV68 was detected by polymerase chain reaction (PCR) targeting for 5' untranslated region (5'UTR), viral protein 1 (VP1), and VP4/VP2. Phylogenetic trees were generated using the obtained sequences., Results: Of the 5,240 tested samples, 12 EV68 positive cases were detected between August and December in 2011 (detection rate, 0.23%). The detection rate was higher among inpatients than outpatients (p<0.0001). Among VP1 sequences detected from 7 patients in 2011, 5 in lineage 2 were diverged from those detected in the Philippines in 2008, however, 2 in lineage 3 were not diverged from strains detected in the Philippines in 2008 but closely associated with strains detected in the United States. Combined with our previous report, EV68 occurrences were observed twice in the Philippines within the last four years., Conclusions: EV68 detections might be occurring in cyclic patterns, and viruses might have been maintained in the community while some strains might have been newly introduced.

Background: Human respiratory syncytial virus (HRSV) is the leading cause of acute lower respiratory tract infection in infants and young children. However, molecular characteristic of HRSV is still unknown in the Philippines., Objective: To describe the molecular epidemiology of circulating HRSV detected in the Philippines., Study Design: From May 2008 to April 2012, nasopharyngeal swabs were collected from infants and children aged between 7 days and 14 years who were hospitalized with severe pneumonia. HRSV was detected by nested PCR targeting M2 gene, and C-terminus of the G gene was sequenced for phylogenetic analysis., Result: Out of total 2150 samples, 19.3% (n = 415) were positive for HRSV, and 65.0% of them (n = 270) were identified as HRSV-A and 35.0% (n = 145) as HRSV-B. There were two major HRSV outbreaks: between June 2008 and February 2009, and between June and March 2012. Majority of HRSV strains detected during the former outbreak were HRSV-A (97.5%, 203/208) whereas during the later outbreak, both HRSV-A (54/158, 34.2%) and HRSV-B (104/158, 65.8%) were detected. All HRSV-A strains were classified as genotype NA1 and all HRSV-B as genotype BA, which had 60-nucleotide duplication in secondary hypervariable region of the G gene. Among HRSV-B positive samples, there were 2 distinct clusters with unique amino acid changes and low homology in compared to other strains in BA, suggesting emergence of new variant of HRSV-B., Conclusion: The study provides an overview of the genetic variation in circulating HRSV viruses in the Philippines along with identification of possibly a novel variant of HRSV-B., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Rabies continues to be a major public health problem in the Philippines, where 200-300 human cases were reported annually between 2001 and 2011. Understanding the phylogeography of rabies viruses is important for establishing a more effective and feasible control strategy., Methods: We performed a molecular analysis of rabies viruses in the Philippines using rabied animal brain samples. The samples were collected from 11 of 17 regions, which covered three island groups (Luzon, Visayas, and Mindanao). Partial nucleoprotein (N) gene sequencing was performed on 57 samples and complete glycoprotein (G) gene sequencing was performed on 235 samples collected between 2004 and 2010., Results: The Philippine strains of rabies viruses were included in a distinct phylogenetic cluster, previously named Asian 2b, which appeared to have diverged from the Chinese strain named Asian 2a. The Philippine strains were further divided into three major clades, which were found exclusively in different island groups: clades L, V, and M in Luzon, Visayas, and Mindanao, respectively. Clade L was subdivided into nine subclades (L1-L9) and clade V was subdivided into two subclades (V1 and V2). With a few exceptions, most strains in each subclade were distributed in specific geographic areas. There were also four strains that were divided into two genogroups but were not classified into any of the three major clades, and all four strains were found in the island group of Luzon., Conclusion: We detected three major clades and two distinct genogroups of rabies viruses in the Philippines. Our data suggest that viruses of each clade and subclade evolved independently in each area without frequent introduction into other areas. An important implication of these data is that geographically targeted dog vaccination using the island group approach may effectively control rabies in the Philippines.

Tohoku University Graduate School of Medicine has established the Tohoku-RITM Collaborative Research Center on Emerging and Re-emerging Diseases at Research Institute for Tropical Medicine (RITM) in the Philippines in 2008. Our aim of the center is to conduct operational researches, which can contribute to control of infectious diseases in the Philippines. Therefore most of our researches in the Philippines are being conducted in the fields. Main research themes include severe acute respiratory infections in children, influenza disease burden study, molecular epidemiology of rabies, and viral etiology of acute diarrhea. The study on severe acute respiratory infections in children in Leyte Island has recruited hospitalized cases with severe pneumonia. We showed that enterovirus 68 was one of important causative agents in severe pneumonia cases. We also conducted other analyses including molecular epidemiology of respiratory syncytial virus (RSV) and pathogenesis of human rhinoviruses (HRV). Based on these studies, we initiated more comprehensive researches in the Philippines since 2010.

Background: Pneumonia remains a leading cause of child death in developing countries. The viruses in severe pneumonia remain poorly defined., Methods: The study was conducted at the Eastern Visayas Regional Medical Center in Tacloban City, Philippines from May 2008 to May 2009. Patients aged 8 days to 13 years old who were admitted to the Department of Pediatrics with severe pneumonia were enrolled for the study. Upon admission, polymerase chain reaction was performed using nasopharyngeal swabs and blood cultures to detect respiratory viruses and bacteria, respectively., Result: Among the 819 patients enrolled, at least one virus was detected in 501 cases (61.2%). In addition, 423 cases were positive for a single virus while bacteria were detected in the blood culture sample of 31 cases. The most commonly detected viruses were human rhinoviruses (n = 189), including types A (n = 103), B (n = 17), and C (n = 69), and respiratory syncytial virus (RSV) (n = 165). Novel viruses such as human metapneumovirus, human coronavirus NL63, human bocavirus, and human polyomaviruses WU and KI were also detected. There were 70 deaths, and one or more viruses were detected in 35 (50%) of these cases. Positivity only for influenza A virus (OR = 4.3, 95% CI = 1.3-14.6) was significantly associated with fatal outcome. From the blood culture, Burkholderia cepacia group (n = 9), Streptococcus pneumoniae (n = 4), Staphylococcus aureus (n = 4), Haemophilus influenzae (n = 1), and Salmonella C1 (n = 1) were also isolated., Conclusion: Viruses were commonly detected in children with severe pneumonia in the Philippines. Hence, viral etiologies should be considered while developing better effective strategies to reduce child pneumonia-related deaths in developing countries.

Despite the vast distribution and expansive diversity of enteroviruses reported globally, indicators defining a complete view of the epidemiology of enteroviruses in tropical countries such as the Philippines are yet to be established. Detection of enteroviruses in the environment has been one of the markers of circulating viruses in a community. This study aimed to bridge the gap in the epidemiology of enteroviruses in the Philippines by providing an overview of the occurrence of enteroviruses in both urban and rural rivers. Molecular detection directed at the VP1 region of the enterovirus genome was performed on 44 grab river water samples collected from April to December 2009. The majority of the enterovirus serotypes detected were clustered with human enterovirus C species (HEV-C; 21/42), followed by HEV-B (12/42) and HEV-A (9/42). Porcine enterovirus 9 was also found in 12 out of 44 water samples. Phylogenetic analysis indicated that the viruses detected were closely related, if not all forming a monophyletic clade, with those enteroviruses detected previously from acute flaccid paralysis cases in the country. The clustering of environmental and human enterovirus strains implies that the circulation of these strains were associated with river contamination. This study gives further evidence of the environmental persistence of enteroviruses once they are shed in feces and likewise, provides additional data which may help in understanding the epidemiology of enteroviruses in humans, highlighting the need for more studies on the potential public health risks linked with enteroviruses found in the environment and their eventual clinical consequences in the country.

Background: Ebola viruses cause viral hemorrhagic fever in humans and non-human primates and are endemic in Africa. Reston ebolavirus (REBOV) has caused several epizootics in cynomolgus monkeys (Macaca fascicularis) but is not associated with any human disease. In late 2008, REBOV infections were identified in swine for the first time in the Philippines., Methods: A total of 215 swine sera collected at two REBOV-affected farms in 2008, in Pangasinan and Bulacan, were tested for the presence of REBOV-specific antibodies using multiple serodiagnosis systems. A total of 98 swine sera collected in a non-epizootic region, Tarlac, were also tested to clarify the prevalence of REBOV infection in the general swine population in the Philippines., Results: Some 70 % of swine sera at the affected farms were positive for REBOV antibodies in the multiple serodiagnosis systems. On the other hand, none of the swine sera collected in Tarlac showed positive reactions in any of the diagnosis systems., Conclusions: The high prevalence of REBOV infection in swine in the affected farms in 2008 suggests that swine is susceptible for REBOV infection. The multiple serological assays used in the study are thought to be useful for future surveillance of REOBV infection in swine in the Philippines.

Enterovirus 68 (EV68) is a rare enterovirus associated with respiratory illness that, unlike other enteroviruses, has been identified only from respiratory specimens. We identified EV68 from respiratory specimens of children hospitalized with a diagnosis of severe pneumonia in Leyte, Republic of the Philippines. Twenty-one samples showed high similarity with EV68 by sequencing of 5' nontranslated region; 17 of these samples were confirmed as EV68 by sequencing of viral protein 1 capsid coding region. Most previously reported EV68 cases had been identified as sporadic cases. All 21 patients we identified had severe illness, and 2 died, possibly the first reported fatal cases associated with EV68 infection. Our study suggests that EV68 may be a possible causative agent of severe respiratory illnesses.

Molecular analysis of measles viruses in the Philippines was conducted from 2000 to 2008. No confirmed measles cases were detected in the surveillance in 2005 after the mass vaccination campaign in 2004. However, a re-emergence of measles cases occurred in 2007, which was caused by other genotypes and the previous circulating genotype had disappeared., (Copyright © 2011 Wiley-Liss, Inc.)

Human rhinovirus (HRV) C was recently identified as the third species of HRV using a molecular technique. Infections caused by previously identified HRVs (A and B) are thought to be limited to the respiratory tract; however, pathogenesis of HRVC is still largely unknown. A total of 816 nasopharyngeal swabs from hospitalized children with severe respiratory infections in the Philippines (May 2008-May 2009) were tested for HRV by reverse transcription polymerase chain reaction (RT-PCR), and 243 samples (29.8%) were positive for HRV. Among these patients, serum samples were also tested to determine whether specific HRV species were associated with viremia. Only 30 serum samples (12.3%) were positive for HRV. However, the HRV positive rates were different among HRV species, 3% (4/135) for HRVA, 0% (0/25) for HRVB, and 31% (26/83) for HRVC, and were the highest on 2 days after the onset of symptoms. These results suggest that HRVC may have a different pathogenicity and can more commonly cause viremia than HRVA and HRVB. Serum positive rates for HRV are affected by age, i.e., higher positive rates for those aged 1 year or more. HRVC that were detected from serum exhibited the same level of sequence diversity as those positive only for nasopharyngeal samples in phylogenetic analysis. However, all HRVA which were detected from serum were clustered in a monophyletic clade based on their 5' non-coding region (NCR) sequences, which is closely related with a certain HRVC genotype (A2) in 5'-NCR. This finding suggests that the 5'NCR region may be associated with viremia.

A new alkaloid (1a), named pandamarilactonine-H, which possesses a methyl-2-(pyrrolidin-2-yl)acetate function, was isolated from the roots of Pandanus amaryllifolius. Eleven known alkaloids were also isolated. Unambiguous assignment of the structure of 1a, including the absolute configuration, was accomplished by spectroscopic analysis and total synthesis of its enantiomer.

Because most cases of secondary dengue virus infection are associated with an increased level of platelet-associated IgG, a high dose of intravenous immunoglobulin (IVIG) may have an effect on the development of severe thrombocytopenia in this disease. A randomized, controlled study was conducted with two treatment groups consisting of a treatment (IVIG) group (n = 15) and a non-treatment (non-IVIG) group (n = 16) to determine whether a high dose of IVIG is effective in hastening the recovery from thrombocytopenia in patients with secondary dengue virus infection. No significant difference was found in the baseline demographic data between the two groups. No adverse effect of IVIG was observed, but no effect in hastening the recovery of platelet counts was found in patients with secondary dengue infections. The lack of efficacy of IVIG suggests that platelet clearance by macrophages through Fc gamma receptors is not a primary mechanism in this disease.

Two new pyrrolidine alkaloids, pandamarilactonine-C and -D, were isolated from Pandanus amaryllifolius. Based on the total synthesis of pandamarilactonine-C and its related alkaloid, pandamarilactonine-A, the relative stereochemistry of pandamarilactonine-A and -B, which was previously proposed by spectroscopic analysis, was revised.