Incidence of lower respiratory tract infection and associated viruses in a birth cohort in the Philippines.

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.

Risk of Transmission and Viral Shedding From the Time of Infection for Respiratory Syncytial Virus in Households.

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.

Association Between Preceding Viral Respiratory Infection and Subsequent Respiratory Illnesses Among Children: A Prospective Cohort Study in the Philippines.

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.

Molecular Characterization of Chikungunya Virus, Philippines, 2011-2013.

During 2011-2013, a nationwide outbreak of chikungunya virus infection occurred in the Philippines. The Asian genotype was identified as the predominant genotype; sporadic cases of the East/Central/South African genotype were detected in Mindanao. Further monitoring is needed to define the transmission pattern of this virus in the Philippines.

Transmitted drug resistance in recently infected HIV-positive Individuals from four urban locations across Asia (2007-2010) - TASER-S.

BACKGROUND: The availability of HIV antiretroviral therapy (ART) has been associated with the development of transmitted drug resistance-associated mutations (TDRM). TDRM can compromise treatment effectiveness in patients initiating ART and the prevalence can vary in different clinical settings. In this study, we investigated the proportion of TDRM in treatment-naïve, recently infected HIV-positive individuals sampled from four urban locations across Asia between 2007-2010. METHODS: Patients enrolled in the TREAT Asia Studies to Evaluate Resistance - Surveillance Study (TASER-S) were genotyped prior to ART initiation, with resulting resistance mutations analysed according to the WHO 2009 list. RESULTS: Proportions of TDRM from recently infected individuals from TASER-S ranged from 0% to 8.7% - Hong Kong: 3/88 (3.4%, 95% CI (0.71%-9.64%)); Thailand: Bangkok: 13/277 (4.7%, 95% CI (2.5%-7.9%)), Chiang Mai: 0/17 (0%, 97.5% CI (0%-19.5%)); and the Philippines: 6/69 (8.7%, 95% CI (3.3%-18.0%)). There was no significant increase in TDRM over time across all four clinical settings. CONCLUSIONS: The observed proportion of TDRM in TASER-S patients from Hong Kong, Thailand and the Philippines was low to moderate during the study period. Regular monitoring of TDRM should be encouraged, especially with the scale-up of ART at higher CD4 levels.

Epidemiological and clinical characteristics of children with acute respiratory viral infections in the Philippines: a prospective cohort study.

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.

Genetic diversity of species A rotaviruses detected in clinical and environmental samples, including porcine-like rotaviruses from hospitalized children in the Philippines.

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.

A prospective nested case-control study of Dengue in infants: rethinking and refining the antibody-dependent enhancement dengue hemorrhagic fever model.

BACKGROUND: Dengue hemorrhagic fever (DHF) is the severe and life-threatening syndrome that can develop after infection with any one of the four dengue virus (DENV) serotypes. DHF occurs almost exclusively in individuals with secondary heterologous DENV infections and infants with primary DENV infections born to dengue immune mothers. The widely accepted explanation for the pathogenesis of DHF in these settings, particularly during infancy, is antibody-dependent enhancement (ADE) of DENV infection. METHODS AND FINDINGS: We conducted a prospective nested case-control study of DENV infections during infancy. Clinical data and blood samples were collected from 4,441 mothers and infants in up to two pre-illness study visits, and surveillance was performed for symptomatic and inapparent DENV infections. Pre-illness plasma samples were used to measure the associations between maternally derived anti-DENV3 antibody-neutralizing and -enhancing capacities at the time of DENV3 infection and development of infant DHF. The study captured 60 infants with DENV infections across a wide spectrum of disease severity. DENV3 was the predominant serotype among the infants with symptomatic (35/40) and inapparent (15/20) DENV infections, and 59/60 infants had a primary DENV infection. The estimated in vitro anti-DENV3 neutralizing capacity at birth positively correlated with the age of symptomatic primary DENV3 illness in infants. At the time of symptomatic DENV3 infection, essentially all infants had low anti-DENV3 neutralizing activity (50% plaque reduction neutralizing titers [PRNT(50)] 50 is associated with protection from symptomatic DENV3 illness. We did not find a significant association between DENV3 ADE activity at illness onset and the development of DHF compared with less severe symptomatic illness. The results of this study should encourage rethinking or refinement of the current ADE pathogenesis model for infant DHF and stimulate new directions of research into mechanisms responsible for the development of DHF during infancy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00377754.

Age-specific incidence rates and risk factors for respiratory syncytial virus-associated lower respiratory tract illness in cohort children under 5 years old in the Philippines.

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 SpO2 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.

Transmission of Respiratory Syncytial Virus Among Children Under 5 Years in Households of Rural Communities, the Philippines.

BACKGROUND: To develop a more effective vaccination strategy for reducing the impact of respiratory syncytial virus (RSV) infection, especially in young infants (<6 months old), it is necessary to understand the transmission dynamics of RSV. METHODS: We conducted a community-based prospective cohort study from 2014 to 2016 in Biliran Province, the Philippines, on children <5 years old. We collected nasopharyngeal swabs from symptomatic children with acute respiratory infection (ARI) during household visits and at health facilities. In households (n = 181) with RSV-positive ARI cases (RSV-ARI), we also identified ARI episodes among other children <5 years old in the same household. In addition, we determined the serial interval to estimate the basic reproduction number (R 0), the average number of secondary cases generated by a single primary case. RESULTS: In the 181 households analyzed, we found 212 RSV-ARI in 152 households with a single case and 29 households with multiple cases, which included 29 1st RSV-ARI and 31 2nd RSV-ARI. We also found possible index cases among children <5 years old in the same household for 29.0% (18 of 62) of young infants with RSV-ARI. The estimated mean serial interval was 3.2 days, and R 0 was estimated to be 0.92-1.33 for RSV-A and 1.04-1.76 for RSV-B, which varied between different times (2014 and 2015) and places. CONCLUSIONS: Young infants are likely to acquire RSV infection from older children in the same household. Therefore, vaccination targeting older children might protect infants from RSV infection.

Mycobacterium tuberculosis whole genome sequencing provides insights into the Manila strain and drug-resistance mutations in the Philippines.

The Philippines has a high incidence of tuberculosis disease (TB), with an increasing prevalence of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) strains making its control difficult. Although the M. tuberculosis "Manila" ancient lineage 1 strain-type is thought to be prevalent in the country, with evidence of export to others, little is known about the genetic diversity of circulating strains. By whole genome sequencing (WGS) 178 isolates from the Philippines National Drug Resistance Survey, we found the majority (143/178; 80.3%) belonged to the lineage 1 Manila clade, with the minority belonging to lineages 4 (European-American; n = 33) and 2 (East Asian; n = 2). A high proportion were found to be multidrug-resistant (34/178; 19.1%), established through highly concordant laboratory drug susceptibility testing and in silico prediction methods. Some MDR-TB isolates had near identical genomic variation, providing potential evidence of transmission. By placing the Philippine isolates within a phylogeny of global M. tuberculosis (n > 17,000), we established that they are genetically similar to those observed outside the country, including a clade of Manila-like strain-types in Thailand. An analysis of the phylogeny revealed a set of ~200 SNPs that are specific for the Manila strain-type, and a subset can be used within a molecular barcode. Sixty-eight mutations known to be associated with 10 anti-TB drug resistance were identified in the Philippine strains, and all have been observed in other populations. Whilst nine putative streptomycin resistance conferring markers in gid (8) and rrs (1) genes appear to be novel and with functional consequences. Overall, this study provides an important baseline characterisation of M. tuberculosis genetic diversity for the Philippines, and will fill a gap in global datasets and aid the development of a nation-wide database for epidemiological studies and clinical decision making. Further, by establishing a molecular barcode for detecting Manila strains it will assist with the design of diagnostic tools for disease control activities.

Complete Genome Sequences of 13 Human Respiratory Syncytial Virus Subgroup A Strains of Genotypes NA1 and ON1 Isolated in the Philippines.

Complete genome sequences of 13 human respiratory syncytial virus strains were determined from samples obtained from children hospitalized in the Philippines between 2012 and 2013 because of acute respiratory infection. We identified amino acid polymorphisms between the NA1 and ON1 genotypes in the P, G, F, and L proteins.

Strain Level Streptococcus Colonization Patterns during the First Year of Life.

Pneumococcal pneumonia has decreased significantly since the implementation of the pneumococcal conjugate vaccine (PCV), nevertheless, in many developing countries pneumonia mortality in infants remains high. We have undertaken a study of the nasopharyngeal (NP) microbiome during the first year of life in infants from The Philippines and South Africa. The study entailed the determination of the Streptococcus sp. carriage using a lytA qPCR assay, whole metagenomic sequencing, and in silico serotyping of Streptococcus pneumoniae, as well as 16S rRNA amplicon based community profiling. The lytA carriage in both populations increased with infant age and lytA+ samples ranged from 24 to 85% of the samples at each sampling time point. We next developed informatic tools for determining Streptococcus community composition and pneumococcal serotype from metagenomic sequences derived from a subset of longitudinal lytA-positive Streptococcus enrichment cultures from The Philippines (n = 26 infants, 50% vaccinated) and South African (n = 7 infants, 100% vaccinated). NP samples from infants were passaged in enrichment media, and metagenomic DNA was purified and sequenced. In silico capsular serotyping of these 51 metagenomic assemblies assigned known serotypes in 28 samples, and the co-occurrence of serotypes in 5 samples. Eighteen samples were not typeable using known serotypes but did encode for capsule biosynthetic cluster genes similar to non-encapsulated reference sequences. In addition, we performed metagenomic assembly and 16S rRNA amplicon profiling to understand co-colonization dynamics of Streptococcus sp. and other NP genera, revealing the presence of multiple Streptococcus species as well as potential respiratory pathogens in healthy infants. A range of virulence and drug resistant elements were identified as circulating in the NP microbiomes of these infants. This study revealed the frequent co-occurrence of multiple S. pneumoniae strains along with Streptococcus sp. and other potential pathogens such as S. aureus in the NP microbiome of these infants. In addition, the in silico serotype analysis proved powerful in determining the serotypes in S. pneumoniae carriage, and may lead to developing better targeted vaccines to prevent invasive pneumococcal disease (IPD) in these countries. These findings suggest that NP colonization by S. pneumoniae during the first years of life is a dynamic process involving multiple serotypes and species.

Laboratory Diagnosis for Outbreak-Prone Infectious Diseases after Typhoon Yolanda (Haiyan), Philippines.

INTRODUCTION: Typhoon Yolanda (Haiyan) hit the central part of the Philippines on November 8, 2013. To identify possible outbreaks of communicable diseases after the typhoon, nasopharyngeal swabs, stool and blood samples were collected from patients who visited the Eastern Visayas Regional Medical Center due to acute respiratory infection (ARI), acute gastroenteritis (AGE) or other febrile illness (OFI) including suspected dengue fever, between November 28, 2013 and February 5, 2014.   Methods: Samples were tested on-site for selected pathogens using rapid diagnostic tests. Confirmation and further analysis were conducted at the Research Institute for Tropical Medicine (RITM) in Manila using polymerase chain reaction (PCR) and sequencing. Residues of the rapid diagnostic tests and samples collected in the filter papers (FTATM card) were transported to Manila under suboptimal conditions. PCR results were compared between the kit residues and the filter papers.   Results: A total of 185 samples were collected. Of these, 128 cases were ARI, 17 cases were AGE and 40 cases were OFI. For nasopharyngeal swab samples, detection rates for enterovirus and rhinovirus residues were higher than the filter papers. For stool samples, rotavirus positive rate for the filter paper was higher than the kit residues. We also managed to obtain the sequence data from some of the kit residues and filter papers.   Discussion: Our results confirmed the importance of PCR for the laboratory diagnosis of infectious diseases in post-disaster situations when  diagnostic options are limited.

Molecular detection and characterization of sapovirus in hospitalized children with acute gastroenteritis 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.

Molecular Characterization of Human Respiratory Syncytial Virus in the Philippines, 2012-2013.

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.

Low adiposity during early infancy is associated with a low risk for developing dengue hemorrhagic fever: a preliminary model.

Dengue virus (DENV) infections range from asymptomatic or mild illness to a severe and potentially life threatening disease, dengue hemorrhagic fever (DHF). DHF occurs in primary DENV infections during early infancy. A prospective clinical study of DENV infections during infancy was conducted in San Pablo, Philippines. We found that infants who developed DHF with a primary DENV infection had higher WHO weight-for-age z scores before and at the time of infection compared to infants with primary DENV infections who did not develop DHF. In addition, TLR 7/8-stimulated tumor necrosis factor-α (TNF-α) production from myeloid-derived cells was higher among well-nourished infants. Leptin augmented TLR 7/8-mediated TNF-α production in monocytes and decreased intracellular cAMP levels. Circulating leptin levels were elevated during early infancy and correlated with WHO weight-for-age z scores. Our data support a plausible hypothesis as to why well-nourished infants are at risk for developing DHF with their first DENV infection.