Human enterovirus 68 (EV-D68) is a historically rarely reported virus linked with respiratory disease. In the past 3 years, a large increase in respiratory disease associated with EV-D68 has been reported, with documented outbreaks in North America, Europe and Asia. In several outbreaks, genetic differences were identified among the circulating strains, indicating the presence of multiple clades. In this report, we analyse archived and novel EV-D68 strains from Africa and the USA, obtained from patients with respiratory illness. Phylogenetic analysis of all EV-D68 sequences indicates that, over the past two decades, multiple clades of the virus have emerged and spread rapidly worldwide. All clades appear to be currently circulating and contributing to respiratory disease.

Coxsackieviruses (CV) A1, CV-A19 and CV-A22 have historically comprised a distinct phylogenetic clade within Enterovirus (EV) C. Several novel serotypes that are genetically similar to these three viruses have been recently discovered and characterized. Here, we report the coding sequence analysis of two genotypes of a previously uncharacterized serotype EV-C113 from Bangladesh and demonstrate that it is most similar to CV-A22 and EV-C116 within the capsid region. We sequenced novel genotypes of CV-A1, CV-A19 and CV-A22 from Bangladesh and observed a high rate of recombination within this group. We also report genomic analysis of the rarely reported EV-C104 circulating in the Gambia in 2009. All available EV-C104 sequences displayed a high degree of similarity within the structural genes but formed two clusters within the non-structural genes. One cluster included the recently reported EV-C117, suggesting an ancestral recombination between these two serotypes. Phylogenetic analysis of all available complete genome sequences indicated the existence of two subgroups within this distinct Enterovirus C clade: one has been exclusively recovered from gastrointestinal samples, while the other cluster has been implicated in respiratory disease.

We report the discovery of two enteroviruses detected in nasopharyngeal samples obtained from subjects with respiratory disease in Peru. Phylogenetic analysis indicated that both viruses belong to a clade within the species Human enterovirus C, which includes the recently characterized human enteroviruses 109 and 104. Members of this clade have undergone significant genomic rearrangement, as indicated by deletions in the hypervariable region of the 59 UTR and the VP1 protein, as well as recombination within the non-structural genes. Our findings and review of published sequences suggests that several novel human enterovirus C serotypes are currently circulating worldwide.

Streptococcus pneumoniae strains comprise >90 serotypes. Here we describe establishment of a MassTag PCR assay designed to serotype S. pneumoniae and demonstrate its utility in tests using 31 paired lung aspirate and nasopharyngeal aspirate samples from children with pneumonia in the Gambia. Serotypes 1, 5, and 14 in were implicated in 90% of lung infections. With 5 exceptions, serotypes found in lung aspirates were also found in nasopharyngeal aspirates. Streptococcus pneumoniae is a major cause of pediatric morbidity and mortality worldwide, particularly in developing countries. A frequent colonizer of the nasopharynx, S. pneumoniae can cause pneumonia, meningitis, and sepsis and annually results in approximately 800,000 deaths in children under 5 years of age (1). A polysaccharide capsule is the major virulence factor in invasive pneumococcal disease (IPD). The capsule is immunogenic and the chemical composition of capsular polysaccharides varies among strains, resulting in the generation of multiple pneumococcal serotypes. Currently, Ͼ90 serotypes are recognized that show various potentials for invasiveness and geographical distributions (2-4). Based on the most common serotypes associated with IPD, a heptavalent pneumococcal conjugate vaccine (PCV7) was licensed in 2000 that included serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. PCV10, licensed in 2008, added 1, 5, and 7F to the existing vaccine serotypes, and PCV13 added serotypes 3, 6A, and 19A in 2009. The PCV7 vaccination program greatly reduced the incidence of IPD caused by vaccine-targeted serotypes (5, 6). However, IPD remains a substantial problem in the developing world, presumably due to limited vaccine access (6, 7).Continued surveillance is essential to determine the geographical prevalence of serotypes involved with IPD and to monitor the replacement of vaccine-targeted serotypes with other serotypes. Historically, serological assays such as the Quellung reaction were used for serotype determination. However, such assays require bacterial culture and substantial investments in time, money, supplies, and operator expertise.MassTag PCR is a multiplex molecular tool that enables rapid, inexpensive detection of bacteria, viruses, fungi, and parasites associated with respiratory, tick, and bloodborne diseases (8-10). It uses a library of distinct low-molecular-mass tags to code microbial gene targets by conjugating each primer species in a multiplex reaction with a distinct mass tag via a photocleavable linkage. After PCR amplification, the identity of the microbial gene target is determined by the presence of its 2 cognate tags, 1 from each primer, detected by mass spectrometry. Here, we adapted this method to develop molecular assays for serotyping S. pneumoniae and evaluated the assay on a set of pediatric clinical samples.MassTag PCR serotyping panels were assembled to detect Ͼ90 recognized serotypes. Primers were designed using Primer3 software. The wzy gene was chosen as a target, except for serotype 3, where the sequence of cap3A gene was us...

Background Native American children have higher rates of morbidity associated with acute respiratory infection than children in the general United States population, yet detailed information is lacking regarding their principal clinical presentations and infectious etiologies. Methods We pursued a comprehensive molecular survey of bacteria and viruses in nasal wash specimens from children with acute respiratory disease collected prospectively over one year (January 1 through December 31, 2009) from 915 Navajo and White Mountain Apache children in their second or third year of life who had been enrolled in an efficacy study of an RSV monoclonal antibody in the first year of life. Results During the surveillance period, 1476 episodes of disease were detected in 669 children. Rates of outpatient and inpatient lower respiratory tract illness were 391 and 79 per 1000 child-years, respectively, and were most commonly diagnosed as pneumonia. Potential pathogens were detected in 88% of specimens. Viruses most commonly detected were respiratory syncytial virus (RSV) and human rhinovirus (HRV); 2009 pandemic influenza A (H1N1) illnesses primarily occurred in the fall. Streptococcus pneumoniae was detected in 60% of subjects; only HRV was significantly associated with S. pneumoniae carriage. The presence of influenza virus, HRV, or S. pneumoniae was not associated with increased risk for lower respiratory tract involvement or hospitalization. Conclusions Acute lower respiratory illnesses occur at disproportionately high rates among young American Indian children, and are associated with a range of common pathogens. This study provides critical evidence to support reducing the disproportionate burden of acute respiratory disease among young Native Americans.