Anne Bett

Viral etiology of severe pneumonia among Kenyan infants and children.

CONTEXT Pneumonia is the leading cause of childhood death in sub-Saharan Africa. Comparative estimates of the contribution of causative pathogens to the burden of disease are essential for targeted vaccine development. OBJECTIVE To determine the viral etiology of severe pneumonia among infants and children at a rural Kenyan hospital using comprehensive and sensitive molecular diagnostic techniques. DESIGN, SETTING, AND PARTICIPANTS Prospective observational and case-control study during 2007 in a rural Kenyan district hospital. Participants were children aged 1 day to 12 years, residing in a systematically enumerated catchment area, and who either were admitted to Kilifi District Hospital meeting World Health Organization clinical criteria for severe pneumonia or very severe pneumonia; (2) presented with mild upper respiratory tract infection but were not admitted; or (3) were well infants and children attending for immunization. MAIN OUTCOME MEASURES The presence of respiratory viruses and the odds ratio for admission with severe disease. RESULTS Of 922 eligible admitted patients, 759 were sampled (82% [median age, 9 months]). One or more respiratory viruses were detected in 425 of the 759 sampled (56% [95% confidence interval {CI}, 52%-60%]). Respiratory syncytial virus (RSV) was detected in 260 participants (34% [95% CI, 31%-38%]) and other respiratory viruses were detected in 219 participants (29%; 95% CI, 26%-32%), the most common being Human coronavirus 229E (n = 51 [6.7%]), influenza type A (n = 44 [5.8%]), Parainfluenza type 3 (n = 29 [3.8%]), Human adenovirus (n = 29 [3.8%]), and Human metapneumovirus (n = 23 [3.0%]). Compared with well control participants, detection of RSV was associated with severe disease (5% [corrected] in control participants; adjusted odds ratio, 6.11 [95% CI, 1.65-22.6]) while collectively, other respiratory viruses were not associated with severe disease (23% in control participants; adjusted odds ratio, 1.27 [95% CI, 0.64-2.52]). CONCLUSION In a sample of Kenyan infants and children admitted with severe pneumonia to a rural hospital, RSV was the predominant viral pathogen.

A Preliminary Study of Pneumonia Etiology Among Hospitalized Children in Kenya

Abstract Background. Pneumonia is the leading cause of childhood death in the developing world. Higher-quality etiological data are required to reduce this mortality burden. Methods. We conducted a case-control study of pneumonia etiology among children aged 1–59 months in rural Kenya. Case patients were hospitalized with World Health Organization–defined severe pneumonia (SP) or very severe pneumonia (VSP); controls were outpatient children without pneumonia. We collected blood for culture, induced sputum for culture and multiplex polymerase chain reaction (PCR), and obtained oropharyngeal swab specimens for multiplex PCR from case patients, and serum for serology and nasopharyngeal swab specimens for multiplex PCR from case patients and controls. Results. Of 984 eligible case patients, 810 (84%) were enrolled in the study; 232 (29%) had VSP. Blood cultures were positive in 52 of 749 case patients (7%). A predominant potential pathogen was identified in sputum culture in 70 of 417 case patients (17%). A respiratory virus was detected by PCR from nasopharyngeal swab specimens in 486 of 805 case patients (60%) and 172 of 369 controls (47%). Only respiratory syncytial virus (RSV) showed a statistically significant association between virus detection in the nasopharynx and pneumonia hospitalization (odds ratio, 12.5; 95% confidence interval, 3.1–51.5). Among 257 case patients in whom all specimens (excluding serum specimens) were collected, bacteria were identified in 24 (9%), viruses in 137 (53%), mixed viral and bacterial infection in 39 (15%), and no pathogen in 57 (22%); bacterial causes outnumbered viral causes when the results of the case-control analysis were considered. Conclusions. A potential etiology was detected in >75% of children admitted with SP or VSP. Except for RSV, the case-control analysis did not detect an association between viral detection in the nasopharynx and hospitalization for pneumonia.

Incidence and Severity of Respiratory Syncytial Virus Pneumonia in Rural Kenyan Children Identified through Hospital Surveillance

BACKGROUND Although necessary for developing a rationale for vaccination, the burden of severe respiratory syncytial virus (RSV) disease in children in resource-poor settings remains poorly defined. METHODS We conducted prospective surveillance of severe and very severe pneumonia in children aged <5 years admitted from 2002 through 2007 to Kilifi district hospital in coastal Kenya. Nasal specimens were screened for RSV antigen by immunofluorescence. Incidence rates were estimated for the well-defined population. RESULTS Of 25,149 hospital admissions, 7359 patients (29%) had severe or very severe pneumonia, of whom 6026 (82%) were enrolled. RSV prevalence was 15% (20% among infants) and 27% during epidemics (32% among infants). The proportion of case patients aged 3 months was 65%, and the proportion aged 6 months was 43%. Average annual hospitalization rates were 293 hospitalizations per 100,000 children aged <5 years (95% confidence interval, 271-371 hospitalizations per 100,000 children aged <5 years) and 1107 hospitalizations per 100,000 infants (95% confidence interval, 1012-1211 hospitalizations per 100,000 infants). Hospital admission rates were double in the region close to the hospital. Few patients with RSV infection had life-threatening clinical features or concurrent serious illnesses, and the associated mortality was 2.2%. CONCLUSIONS In this low-income setting, rates of hospital admission with RSV-associated pneumonia are substantial; they are comparable to estimates from the United States but considerably underestimate the burden in the full community. An effective vaccine for children aged >2 months (outside the age group of poor responders) could prevent a large portion of RSV disease. Severity data suggest that the justification for RSV vaccination will be based on the prevention of morbidity, not mortality.

Added Value of an Oropharyngeal Swab in Detection of Viruses in Children Hospitalized with Lower Respiratory Tract Infection

ABSTRACT Paired nasopharyngeal and oropharyngeal swabs collected from 533 children hospitalized with lower respiratory tract infection were assessed by multiplex reverse transcription-PCR. Oropharyngeal swabs increased the number of viral infections detected by 15%, compared to collection of a nasopharyngeal swab alone. This advantage was most pronounced for detection of influenza, parainfluenza, and adenovirus.

Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR

Highlights • Sequence variation at probe target site inhibited detection of a new RSV-B variant.• RSV-B virus diversity was consistent with real-time RT-PCR sensitivity.• Reduced PCR insensitivity could underestimate disease prevalence in clinical settings.• Regular check of primer and probe target sites for rapidly evolving viruses is key.

Transmission patterns and evolution of respiratory syncytial virus in a community outbreak identified by genomic analysis.

Abstract Detailed information on the source, spread and evolution of respiratory syncytial virus (RSV) during seasonal community outbreaks remains sparse. Molecular analyses of attachment (G) gene sequences from hospitalized cases suggest that multiple genotypes and variants co-circulate during epidemics and that RSV persistence over successive seasons is characterized by replacement and multiple new introductions of variants. No studies have defined the patterns of introduction, spread and evolution of RSV at the local community and household level. We present a whole genome sequence analysis of 131 RSV group A viruses collected during 6-month household-based RSV infection surveillance in Coastal Kenya, 2010 within an area of 12 km2. RSV infections were identified by regular symptom-independent screening of all household members twice weekly. Phylogenetic analysis revealed that the RSV A viruses in nine households were closely related to genotype GA2 and fell within a single branch of the global phylogeny. Genomic analysis allowed the detection of household-specific variation in seven households. For comparison, using only G gene analysis, household-specific variation was found only in one of the nine households. Nucleotide changes were observed both intra-host (viruses identified from same individual in follow-up sampling) and inter-host (viruses identified from different household members) and these coupled with sampling dates enabled a partial reconstruction of the within household transmission chains. The genomic evolutionary rate for the household dataset was estimated as 2.307 × 10 − 3 (95% highest posterior density: 0.935–4.165× 10 − 3) substitutions/site/year. We conclude that (i) at the household level, most RSV infections arise from the introduction of a single virus variant followed by accumulation of household specific variation and (ii) analysis of complete virus genomes is crucial to better understand viral transmission in the community. A key question arising is whether prevention of RSV introduction or spread within the household by vaccinating key transmitting household members would lead to a reduced onward community-wide transmission.

A34 Spread and evolution of respiratory syncytial virus A genotype ON1, coastal Kenya, 2010–2015

A33 Respiratory syncytial virus group B evolutionary trends in the attachment (G) glycoprotein in Kilifi, Kenya, 2003–2015 Everlyn Kamau, Clement Lewa, Graham F. Medley, Patricia A. Cane, D. James Nokes, and Charles N. Agoti Wellcome Trust Research Programme, Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI), Kilifi, Kenya, Department of Global Health and Development, London School of Hygiene and Tropical Medicine, London, UK, Public Health England, Salisbury, UK, School of Life Sciences and SBIDER, University of Warwick, Coventry, UK and Department of Biomedical Sciences, Pwani University, Kilifi, Kenya

Human Coronavirus NL63 Molecular Epidemiology and Evolutionary Patterns in Rural Coastal Kenya

Abstract Background Human coronavirus NL63 (HCoV-NL63) is a globally endemic pathogen causing mild and severe respiratory tract infections with reinfections occurring repeatedly throughout a lifetime. Methods Nasal samples were collected in coastal Kenya through community-based and hospital-based surveillance. HCoV-NL63 was detected with multiplex real-time reverse transcription PCR, and positive samples were targeted for nucleotide sequencing of the spike (S) protein. Additionally, paired samples from 25 individuals with evidence of repeat HCoV-NL63 infection were selected for whole-genome virus sequencing. Results HCoV-NL63 was detected in 1.3% (75/5573) of child pneumonia admissions. Two HCoV-NL63 genotypes circulated in Kilifi between 2008 and 2014. Full genome sequences formed a monophyletic clade closely related to contemporary HCoV-NL63 from other global locations. An unexpected pattern of repeat infections was observed with some individuals showing higher viral titers during their second infection. Similar patterns for 2 other endemic coronaviruses, HCoV-229E and HCoV-OC43, were observed. Repeat infections by HCoV-NL63 were not accompanied by detectable genotype switching. Conclusions In this coastal Kenya setting, HCoV-NL63 exhibited low prevalence in hospital pediatric pneumonia admissions. Clade persistence with low genetic diversity suggest limited immune selection, and absence of detectable clade switching in reinfections indicates initial exposure was insufficient to elicit a protective immune response.

Impact of viral upper respiratory tract infection on the concentration of nasopharyngeal pneumococcal carriage among Kenyan children

Viral upper respiratory tract infection (URTI) predisposes to bacterial pneumonia possibly by facilitating growth of bacteria such as Streptococcus pneumoniae colonising the nasopharynx. We investigated whether viral URTI is temporally associated with an increase in nasopharyngeal pneumococcal concentration. Episodes of symptomatic RSV or rhinovirus URTI among children <5 years were identified from a longitudinal household study in rural Kenya. lytA and alu PCR were performed on nasopharyngeal samples collected twice-weekly, to measure the pneumococcal concentration adjusted for the concentration of human DNA present. Pneumococcal concentration increased with a fold-change of 3.80 (95%CI 1.95–7.40), with acquisition of RSV or rhinovirus, during 51 URTI episodes among 42 children. In repeated swabs from the baseline period, in the two weeks before URTI developed, within-episode variation was broad; within +/−112-fold range of the geometric mean. We observed only a small increase in nasopharyngeal pneumococcal concentration during RSV or rhinovirus URTI, relative to natural variation. Other factors, such as host response to viral infection, may be more important than nasopharyngeal pneumococcal concentration in determining risk of invasive disease.

A27 Using whole genome sequence data and minority variant profiles to elucidate transmission patterns during RSV household outbreaks

well as host/virus interaction. In our study, using 454/Illumina sequencing, we have obtained large amount of whole genome sequences. We designed a preliminary bioinformatics analysis pipeline to classify these NGS reads. First we mapped our nucleotide reads to GenBank reference sequences using BLAST, and classified them by their taxonomic family, such as host, virus and unclassified. Then, for a specific type of virus (e.g. influenza virus, MERS coronavirus), we conducted de novo and reference based assembly of the reads to obtain the full genome sequences for further phylogenetic study. In the future, through advanced bioinformatics tools, we hope to get more detailed information from our large amount of NGS sequences of field/clinical samples, experimental data, especially in the following areas: (i) finding novel pathogens in unclassified sequences; (ii) virus/virus interactions; (iii) pathogen/host interaction.