Serotype 3 Antibody Response and Antibody Functionality Compared to Serotype 19A Following 13-Valent Pneumococcal Conjugate Immunization in Children.

BACKGROUND: Prevention of infections in children vaccinated with 13-valent pneumococcal conjugate vaccine (PCV13) may be less effective against serotype 3 than 19A. OBJECTIVE: The aim of this study was to to determine differences in IgG and functional antibody for serotype 3 versus 19A following PCV13 immunization, in IgG antibody levels induced by PCV13 compared to naturally-induced immunity, and assess effectiveness of PCV13 against serotype 3 and 19A in prevention of acute otitis media (AOM) and colonization among 6-36-month-old children. METHODS: Samples were from a prospective, longitudinal, observational cohort study conducted in Rochester, NY. Pneumococcal detection was by culture. 713 serum were tested for antibody levels by enzyme-linked immunosorbent assay, 68 for functional antibody by opsonophagocytosis and 47 for antibody avidity by thiocyanate bond disruption. PCV13 effectiveness in preventing AOM and colonization was determined by comparison of pre-PCV13 detection of serotypes 3 and 19A to post-PCV13. RESULTS: The proportion of children who reached the antibody threshold of ≧0.35 µg/mL after PCV13 was higher for serotype 19A than serotype 3. Only serotype 19A showed significant increase in PCV13-induced opsonophagocytosis assay titers and antibody avidity. Serotype 3 naturally-induced immune children showed a positive trend of increase in antibody level as children got older, but not PCV13-immunized children. PCV13 effectiveness was not identified in preventing AOM or colonization for serotype 3 but effectiveness of 19A was confirmed. CONCLUSIONS: PCV13 elicits lower antibody levels and lower effectiveness to serotype 3 versus serotype 19A. Post-PCV13-induced antibody levels for serotype 3 are likely insufficient to prevent AOM and colonization in most young children.

Window of Susceptibility to Acute Otitis Media Infection.

BACKGROUND: Contemporary, quantitative data are needed to inform recommendations and decision-making regarding referral and surgeon endorsement of tympanostomy tube placement in young children with recurrent acute otitis media (AOM). METHODS: A prospective, observational cohort study of 286 children in a primary care pediatric practice setting, who had at least 1 AOM (range 1-8). Children were followed longitudinally from 6 to 36 months old. AOMs were microbiologically confirmed by tympanocentesis for diagnostic accuracy. A window of susceptibility (WOS) was defined as AOMs closely spaced in time with no gap in occurrence >6 months. For prediction of total number of AOMs, we used a quasi-poisson generalized linear model. RESULTS: Eighty percent of AOMs occurred during child age 6 to 21 months old. Seventy two percent of WOS intervals were <5 months and 97% were <10 months. Clinically applicable models were developed to predict which children would benefit most from tympanostomy tubes. Significant predictors were child age at the first AOM (P < .001) and daycare attendance (P = .03). The age of a child when 2, 3, or 4 AOMs had occurred allowed prediction of the number of additional AOMs that might occur. After insertion of tympanostomy tubes, 16 (52%) of 31 children had no additional AOMs. CONCLUSIONS: Recurrent AOM occurs in a narrow WOS and number of AOMs can be predicted at time of AOM based on child age and daycare attendance. Insertion of tympanostomy tubes likely occurs in many children after the WOS to recurrent AOM has passed or only 1 more AOM may be prevented at most.

Pathogenesis of Streptococcus pneumoniae serotype 3 during natural colonization and infections among children and its IgG correlate of protection in a mouse model.

Current licensed pneumococcal conjugate vaccines (PCVs) are effective against pneumococcal diseases caused by the serotypes contained in the PCvs However; several studies evaluating pneumococcal colonization and acute otitis-media (AOM) prevention in young children vaccinated with PCV13, observed less effectiveness against serotype-3. One possible reason for less effectiveness may be release of the capsular polysaccharide (CPS) of serotype-3 (CPS-3) as an immune evasion mechanism. Here we evaluated free CPS-3 levels released from 6 clinical isolates from young children compared to WU2 strain and to serotype-19A CPS (CPS-19A) released in vitro when interacting with nasopharyngeal, middle-ear and lung cell-lines. Clinical serotype-3 strains showed greater release of CPS than WU2 with the interaction to 2 cell-lines and all 6 clinical serotype-19A strains. We next evaluated CPS-3 vs CPS-19A levels in middle-ear fluid (MEF) and the nasopharynx (NP) of young children and found higher levels of CPS-3 compared to CPS-19A in MEF during AOM but not in NP secretions during colonization. With anti-CPS-3 IgG in MEF and NP secretions at time of health and onset of AOM, a significant negative correlation (r = -0.75, p < 0.05) between unbound anti-CPS-3 IgG levels and free- anti-CPS-3 in MEF were found, and a significant lower detection of unbound anti-CPS-3 IgG in NP at the time of health with serotype-3 SPN (p < 0.05) compared to irrelevant SPN serotypes were found. In a mouse model of AOM and pneumonia, we sought a correlate of protection against serotype-3 infection using human serum-derived anti-CPS-3 IgG. We conclude that serotype-3 clinical isolates from children release more capsule than WU2 strains or 19A strains during in vitro testing; release more capsule in the MEF of children during AOM than serotype 19A; unbound anti-CPS-3 IgG levels negatively correlate with free-anti-CPS-3; and a level of 2.8 µg/ml anti-CPS-3 antibody protects mice from AOM and pneumonia but not colonization.

Dynamic changes in otopathogens colonizing the nasopharynx and causing acute otitis media in children after 13-valent (PCV13) pneumococcal conjugate vaccination during 2015-2019.

The otopathogens colonizing the nasopharynx (NP) and causing acute otitis media (AOM) have shown dynamic changes following introduction of pneumococcal conjugate vaccines. Five hundred eighty-nine children were prospectively enrolled, 2015-2019. Two thousand fifty-nine visits (1528 healthy, 393 AOM, and 138 AOM follow-up) were studied. Two thousand forty-two NP and 495 middle ear fluid (MEF) samples by tympanocentesis from 319 AOM cases were cultured for bacterial identification and antibiotic susceptibility. Streptococcus pneumoniae (Spn) isolates were serotyped by Quellung, and multi-locus sequence type (ST) determined by genomic analysis. Haemophilus influenzae (Hi) was the most common otopathogen cultured from MEF during AOM (34% in MEF) followed by Spn (24% in MEF), then Moraxella catarrhalis (Mcat) (15% in MEF). NP isolates during healthy visit were Mcat (39%), Spn (32%), Hi (12%). 48.6% of Hi isolates from MEF were beta-lactamase-producing. Spn non-susceptibility to penicillin and other antibiotics was high. The most common Spn serotypes associated with AOM (and colonizing the NP during healthy visits) were 35B, 23B, and 15B/C. ST558 and ST199 were the most common sequence types. During 2015-2019, Hi was the most common otopathogen cultured from MEF during AOM among young children. Pneumococcal AOM was most commonly caused by non-PCV13 serotypes of Spn, predominantly 35B, 23B, and 15B/C. Resistance to common antibiotics among Spn strains showed an increasing trend.

Transition of Serotype 35B Pneumococci From Commensal to Prevalent Virulent Strain in Children.

In our community-based prospective cohort study in young children, we observed a significant increase in pneumococcal serotype 35B nasopharyngeal (NP) commensal colonization during the 2011-2014 timeframe, but these strains were not associated with disease. Beginning in 2015 and continuing through to the present, the serotype 35B virulence changed, and it became the dominant bacteria isolated and associated with pneumococcal acute otitis-media (AOM) in our cohort. We performed comparative analyses of 250 35B isolates obtained from 140 children collected between 2006 and 2019. Changes in prevalence, clonal-complex composition, and antibiotic resistance were analyzed. Seventy-two (29%) of 35B isolates underwent whole-genome sequencing to investigate genomic changes associated with the shift in virulence that resulted in increased rates of 35B-associated AOM disease. 35B strains that were commensals and AOM disease-causing were mainly associated with sequence type (ST) 558. Antibiotic concentrations of ß-lactams and ofloxacin necessary to inhibit growth of 35B strains rose significantly (2006-2019) (p<0.005). However, only isolates from the 35B/ST558 showed significant increases in MIC50 of penicillin and ofloxacin between the years 2006-2014 and 2015-2019 (p=0.007 and p<0.0001). One hundred thirty-eight SNPs located in 34 different genes were significantly associated with post-2015 strains. SNPs were found in nrdG (metal binding, 10%); metP and metN (ABC transporter, 9%); corA (Mg2+ transporter, 6%); priA (DNA replication, 5%); and on the enzymic gene ldcB (LD-carboxypeptidase, 3%). Pneumococcal serotype 35B strains was a common NP commensal during 2010-2014. In 2015, a shift in increasing number of AOM cases occurred in young children caused by 35B, that was associated with changes in genetic composition and antibiotic susceptibility.

COVID-19 Pandemic Impact on Respiratory Infectious Diseases in Primary Care Practice in Children.

Background: The coronavirus disease 2019 (COVID-19) pandemic led to day care and school closures and children staying home for several months. When they gradually returned, aggressive regulations were implemented in New York State to reduce viral transmission. Method: An ongoing prospective study occurring in the Rochester, NY region, focused on early childhood respiratory infectious diseases, afforded an opportunity to assess the impact of the pandemic on the incidence of these illnesses in a primary care outpatient setting. Physician-diagnosed, medically attended infection visits were assessed in two child cohorts, age 6-36 months old: from March 15 to December 31, 2020 (the pandemic period) compared to the same months in 2019 (prepandemic). Nasopharyngeal colonization by potential otopathogens during healthy/well-child and acute otitis media (AOM) visits was evaluated. Results: One hundred and forty-four children were included in the pandemic cohort and 215 in the prepandemic cohort. The pandemic cohort of children experienced 1.8-fold less frequent infectious disease visits during the pandemic (p < 0.0001). Specifically, visits for AOM were 3.7-fold lower (p < 0.0001), viral upper respiratory infections (URI) 3.8-fold lower (p < 0.0001), croup 27.5-fold lower (p < 0.0001), and bronchiolitis 7.4-fold lower (p = 0.04) than the prepandemic cohort. Streptococcus pneumoniae (p = 0.03), Haemophilus influenzae (p < 0.0001), and Moraxella catarrhalis (p < 0.0001) nasopharyngeal colonization occurred less frequently among children during the pandemic. Conclusion: In primary care pediatric practice, during the first 9 months of the COVID-19 pandemic, significant decreases in the frequency of multiple respiratory infections and nasopharyngeal colonization by potential bacterial respiratory pathogens occurred in children age 6-36 months old.

Haemophilus influenzae Prevalence, Proportion of Capsulated Strains and Antibiotic Susceptibility During Colonization and Acute Otitis Media in Children, 2019-2020.

BACKGROUND: The objective of this study was to determine the prevalence, proportion of encapsulated strains and antibiotic susceptibility of Haemophilus influenzae isolated from young children. METHODS: Children, 6 months to 30 months old, were prospectively enrolled from September 2019 to September 2020 at Rochester, NY, pediatric clinics. H. influenzae isolates from nasopharynx (NP) at healthy visits and disease isolates from NP and middle ear fluid (MEF) at onset of acute otitis media (AOM) were characterized by capsular typing, ß-lactamase production and antibiotic susceptibility. RESULTS: Samples from 565 healthy visits and 130 AOM visits were collected. H. influenzae was detected 5.9% and 27% in the NP from healthy and AOM visits, respectively. In the MEF, H. influenzae was isolated in 43% of samples. Eight percent of H. influenzae isolates were encapsulated, 88% type f. Overall 39.7% of isolates were ß-lactamase producing; 43% for MEF isolates. Ampicillin, trimethoprim/sulfamethoxazole, erythromycin and clarithromycin nonsusceptibility were found in more than 25% of isolates. None of the encapsulated H. influenzae isolates were positive for ß-lactamase production or ampicillin nonsusceptibility. 9.2% of isolates were ß-lactamase negative, ampicillin resistant (ß-lactamase negative, ampicillin resistant + ß-lactamase negative, ampicillin intermediate). CONCLUSIONS: The prevalence of H. influenzae in the NP of young children is very low at times of health, but H. influenzae is highly prevalent in MEF at onset of AOM. Nontypeable H. influenzae accounts for >90% of all H. influenzae isolates. Type f predominated among encapsulated strains. ß-lactamase production and antibiotic nonsusceptibility among H. influenzae strains isolated from the NP and MEF are common.

Comparison of specific in-vitro virulence gene expression and innate host response in locally invasive vs colonizer strains of Streptococcus pneumoniae.

Among Rochester NY children, a dramatic increase in nasopharyngeal (NP) colonization by non-vaccine pneumococcal serotypes 35B and 15A occurred during years 2010-2015, after introduction of 13-valent pneumococcal conjugate vaccine (PCV13). In our population, serotype 35B strains colonized in the nasopharynx (NP) but infrequently caused acute otitis media (AOM) whereas serotype 15A strains displayed virulence, evidenced by causing AOM. To explain the virulence difference, virulence genes expression between 35B and 15A, as well as the host's immune response during asymptomatic colonization were analyzed. We investigated differences in regulation of 19 virulence genes for differences in virulence using RT-PCR in 20 35B and 14 15A strains and measured gene expression of 9 host innate cytokines in the NP to assess the mucosal inflammatory response during asymptomatic colonization. Comparing 35B versus 15A strains, genes for competence ComA and RrgC were upregulated; capsular (Cps2D) and virulence genes (PfbA, PcpA and PhtE) were downregulated among 35B strains. PavB, LytA, LytB, NanA, CiaR, PhtD, LuxS, PspA and pneumolysin (Ply) showed no difference. IL17 and IL23 gene expression were > tenfold higher during 35B compared to 15A strain asymptomatic colonization. Only IL23 showed significant difference. In the first 5 years after introduction of PCV13, serotype 35B strains emerged as asymptomatic colonizers and 15A strains emerged to cause AOM in young children. Various genes (PfbA, PcpA, Cps2D and PhtE) among tested in this analysis were downregulated in 35B whereas ComA and RrgC were significantly upregulated. For the host's cytokine response, IL23 proinflammatory response which is essential for the differentiation of Th17 lymphocytes in the NP of children with 35B strains was significantly higher than the response to 15A during asymptomatic colonization.

A molecular analysis of the GAA gene and clinical spectrum in 38 patients with Pompe disease in Japan.

Pompe disease is an autosomal recessive disorder caused by acid α-glucosidase (GAA) deficiency, which results in the accumulation of glycogen in lysosomes in multiple tissues, including cardiac, skeletal, and smooth muscle cells. Thus far, 558 sequence variants of the GAA gene have been published in the Pompe Disease Mutation Database, and some mutations appear with considerable frequency in particular ethnic groups, such as Caucasians, Taiwanese, Chinese, and Koreans. However, the GAA mutation pattern in Japanese patients remains poorly understood. We analyzed the relationship between the genetic and clinical features of 38 mostly Japanese patients with Pompe disease from 35 unrelated families. We identified 28 different GAA gene mutations, including 7 novel mutations, by a GAA gene analysis. c.546G > T (22.9%) and c.1857C > G (14.3%) were the most common mutations and accounted for 37.1% of the total mutant alleles. In the six patients with infantile-onset Pompe disease (IOPD), c.1857C > G was also the most common mutation. In addition, there were 13 homozygotes (5 with the c.546G > T) among the 35 families, which is the highest frequency reported thus far. Regarding the initial symptoms, cardiomegaly was the most common (3/6 = 50%) in IOPD patients, while muscle weakness was observed the most frequently in patients with late-onset Pompe disease (LOPD) (15/30 = 50%). Notably, all IOPD patients who showed respiratory distress at the time of onset require respiratory assistance at present (4/4 = 100%). Regarding the presenting symptoms, cardiomegaly (6/6 = 100%) and hepatomegaly (4/6 = 66.7%) were more commonly seen in IOPD, and muscle weakness (24/29 = 82.7%) was observed more frequently in LOPD. Respiratory assistance is required at present in 33.3% of IOPD patients and 50% of LOPD patients, and 20% of IOPD patients and 29.6% of LOPD patients are wheelchair users. These individual clinical courses may be influenced by the timing of the diagnosis and treatment; for example, in 2007, an ERT orphan drug for treatment of Pompe disease, Alglucosidase alfa, was made available in Japan, and there were 5 (5/6 = 83.3%) wheelchair users diagnosed from 2008 to 2009 (cases 32-38) and 4 (4/27 = 14.8%) from 2010 to 2015 (cases 1-31). These findings underscore the importance of the early diagnosis and treatment.

Molecular diagnosis of 65 families with mucopolysaccharidosis type II (Hunter syndrome) characterized by 16 novel mutations in the IDS gene: Genetic, pathological, and structural studies on iduronate-2-sulfatase.

Mucopolysaccharidosis type II (MPS II: also called as Hunter syndrome) is an X-linked recessive lysosomal storage disorder characterized by the accumulation of extracellular glycosaminoglycans due to the deficiency of the enzyme iduronate-2-sulfatase (IDS). Previous observations suggested that MPS II can be classified into two distinct disease subtypes: (1) severe type of MPS II involves a decline in the cognitive ability of a patient and (2) attenuated type of MPS II exhibits no such intellectual phenotype. To determine whether such disease subtypes of MPS II could be explained by genetic diagnosis, we analyzed mutations in the IDS gene of 65 patients suffering from MPS II among the Japanese population who were diagnosed with both the accumulation of urinary glycosaminoglycans and a decrease in their IDS enzyme activity between 2004 and 2014. Among the specimens examined, we identified the following mutations: 33 missense, 8 nonsense, 7 frameshift, 4 intronic changes affecting splicing, 8 recombinations involving IDS-IDS2, and 7 other mutations including 4 large deletions. Consistent with the previous data, the results of our study showed that most of the attenuated phenotype was derived from the missense mutations of the IDS gene, whereas mutations associated with a large structural alteration including recombination, splicing, frameshift, and nonsense mutations were linked to the severe phenotype of MPS II. Furthermore, we conducted a homology modeling study of IDS P120R and N534I mutant as representatives of the causative mutation of the severe and attenuated type of MPS II, respectively. We found that the substitution of P120R of the IDS enzyme was predicted to deform the α-helix generated by I119-F123, leading to the major structural alteration of the wild-type IDS enzyme. In sharp contrast, the effect of the structural alteration of N534I was marginal; thus, this mutation was pathogenically predicted to be associated with the attenuated type of MPS II. These results suggest that a combination of the genomic diagnosis of the IDS gene and the structural prediction of the IDS enzyme could enable the prediction of a phenotype more effectively.

Genetic characterization of measles virus in the Philippines, 2008-2011.

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.

Molecular Epidemiology of the Human Rhinovirus Infection in Mongolia during 2008-2013.

Rhinovirus infections are common in all age groups world-wide, and they occur throughout the year. In this study, we examined 2,689 nasopharyngeal swabs collected in Mongolia during 2008-2013. Human rhinoviruses (HRVs) were detected in 295 (11.0%) samples, and 85 (28.8%) patients were co-infected with other respiratory viruses. HRV was co-detected with bocavirus, human coronavirus, and respiratory syncytial virus in 21 (24.7%), 17 (20.0%), and 14 (16.5%), respectively. We tested 170 (57.6%) of the 295 HRV-positive samples: 117 HRV strains were typed by using the VP4/VP2 method and 53 by using 5' UTR method. We found HVR-A, HVR-C, and HVR-B infections in 80 (47.1%), 76 (44.7%), and 14 (8.2%) samples, respectively.

Molecular evolution of enterovirus 68 detected in the Philippines.

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.

Genetic characterization of human respiratory syncytial virus detected in hospitalized children in the Philippines from 2008 to 2012.

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.

Respiratory viruses from hospitalized children with severe pneumonia in the Philippines.

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.

Detection of non-polio enteroviruses from 17 years of virological surveillance of acute flaccid paralysis in the Philippines.

Acute flaccid paralysis (AFP) surveillance has been conducted as part of the World Health Organization (WHO) strategy on poliomyelitis eradication. Aside from poliovirus, which is the target pathogen, isolation, and identification of non-polio enteroviruses (NPEVs) is also done by neutralization test using pools of antisera which can only identify limited number of NPEVs. In the Philippines, despite the significant number of isolated NPEVs, no information is available with regard to its occurrence, diversity, and pattern of circulation. In this study, a total of 790 NPEVs isolated from stool samples submitted to the National Reference Laboratory from 1992 to 2008 were analyzed; neutralization test was able to type 55% (442) of the isolates. Of the remaining 356 isolates, which were untyped by using neutralization test, 348 isolates were analyzed further by RT-PCR targeting the VP1 gene. A total of 47 serotypes of NPEV strains were identified using neutralization test and molecular typing, including 28 serotypes of human enterovirus B (HEV-B), 12 serotypes of HEV-A, and 7 of HEV-C. The HEV-B group (625/790; 79%) constituted the largest proportion of isolates, followed by HEV-C (108/790; 13.7%), HEV-A (57/790; 7.2%), and no HEV-D. Coxsackievirus (CV) B, echovirus (E)6, E11, and E13 were the most frequent isolates. E6, E11, E13, E14, E25, E30, E33, CVA20, and CVA24 were considered as endemic strains, some NPEVs recurred and few serotypes existed only for 1-3 years during the study period. Despite some limitations in this study, plural NPEVs with multiple patterns of circulation in the Philippines for 17 years were identified.

Acute respiratory infections due to enterovirus 68 in Yamagata, Japan between 2005 and 2010.

To clarify the epidemiology of enterovirus 68 (EV68), which is one of the most rarely identified enteroviruses, virus isolation and molecular screening using RT-PCR was performed on 6307 respiratory specimens collected at pediatric clinics in Yamagata, Japan between 2005 and 2010. In the years 2005-2009, 10, 1, 2, 0, and 2 (40) EV68-positive cases, respectively, were identified by RT-PCR. In 2010, 40 cases were identified altogether: 2 by isolation only, 26 by RT-PCR only, and 12 by both isolation and RT-PCR. Phylogenetic analysis indicated that plural genetically distinct clusters co-circulated. These results suggest that that difficulty in EV68 isolation leads to an underestimation of the prevalence of EV68 infections.

Detection of human rhinovirus C viral genome in blood among children with severe respiratory infections in the Philippines.

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.

Enterovirus 68 among children with severe acute respiratory infection, 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.

Interruption of the circulation of an indigenous measles genotype and the introduction of other genotypes after a mass vaccination campaign in the Philippines.

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.