key: cord-0829232-8lojw5cz
authors: Palmu, Arto A.; Ware, Robert S.; Lambert, Stephen B.; Sarna, Mohinder; Bialasiewicz, Seweryn; Seib, Kate L.; Atack, John M.; Nissen, Michael D.; Grimwood, Keith
title: Nasal swab bacteriology by PCR during the first 24‐months of life: A prospective birth cohort study
date: 2019-01-04
journal: Pediatr Pulmonol
DOI: 10.1002/ppul.24231
sha: f30b60d6d87ae04dfd5fe469f3f496080c924cbd
doc_id: 829232
cord_uid: 8lojw5cz

BACKGROUND: Most respiratory bacterial carriage studies in children are based on cross‐sectional samples or longitudinal studies with infrequent sampling points. The prospective Observational Research in Childhood Infectious Diseases birth cohort study intensively evaluated the community‐based epidemiology of respiratory viruses and bacteria during the first 2‐years of life. Here we report the bacteriologic findings. METHODS: Pregnant women in Brisbane, Australia were recruited between September 2010 and October 2012, and their healthy newborn children were followed for the first 2‐years of life. Parents kept a daily symptom diary for the study child, collected a weekly anterior nose swab and completed an illness burden diary when their child met pre‐defined illness criteria. Specimens were tested for respiratory bacteria by real‐time polymerase chain reaction (PCR) assays and those containing human genomic DNA, deemed as high‐quality, were analyzed. RESULTS: Altogether 8100 high‐quality nasal swab specimens from 158 enrolled children were analyzed. Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae were detected in 42.4%, 38.9%, and 14.8% of these samples, respectively. Concomitant detection of bacteria was common. In contrast, Bordetella pertussis, B. parapertussis, Mycoplasma pneumoniae, Chlamydia pneumoniae, and Simkania negevensis were rarely identified. The prevalence of the three major bacteria was higher with increasing age and in the winter and spring months. Siblings and childcare attendance were the other risk factors identified. CONCLUSIONS: We confirmed the feasibility of frequent nasal swabbing by parents for studying bacterial colonization. PCR detected the major respiratory tract bacteria with expected high frequencies, but atypical bacteria were found rarely in this cohort.

Acute respiratory infections (ARI) are responsible for substantial global morbidity and are important causes of mortality in children from lowincome countries. 1 Carriage of respiratory bacterial pathogens, such as Streptococcus pneumoniae, Moraxella catarrhalis, and Haemophilus influenzae, is a prerequisite for two important childhood ARIs; acute otitis media, which is the most frequently reported pediatric bacterial infection, 2 and bacterial pneumonia, a leading cause of childhood deaths. 1, 3 These bacteria are also carried in the upper airways of healthy young children who are considered the principal reservoirs for maintaining these organisms within populations. 4 (clinicaltrials.gov: NCT01304914). 6 In this prospective, communitybased birth cohort study, children were enrolled at their mothers' antenatal visits and followed from birth until their second birthday.

Recruitment was progressive over 2-years at two metropolitan hospitals (one private and one government-funded). To be eligible to participate, children needed to be healthy, without congenital abnormalities or underlying chronic disorders, and to be born at term (36-42 weeks gestation). Participants were able to leave the study temporarily (such as during family holidays) and re-join at a later date.

The primary aim of the ORChID study was to describe the nature and timing of viral respiratory and gastrointestinal infections during the first 2-years of life. 6 

After enrolment, data on demographic, social, and health history, pregnancy, and birth details were collected by parental interview.

Subsequently, telephone interviews were conducted every 3-months to update feeding, vaccination, and childcare attendance details.

Childcare was categorized as formal (regulated care outside the child's home) or informal (non-regulated care provided by family or friends).

Parents received training to recognize respiratory symptoms. Parents used diary cards to record daily, pre-defined respiratory symptoms, or diagnoses in a designed tick-box format. Parents also recorded in a separate illness impact diary any health visits and antibiotic prescriptions for their child's ARI episodes. 9 Both diaries were returned by mail each month to the research team.

Research personnel collected anterior nasal swabs soon after birth and, following training and receiving written instructions, parents obtained swabs weekly thereafter. All swabs were collected using a plastic-shaft, rayon-budded swab in a transport tube with a foam pad reservoir soaked with viral transport medium (Virocult MW950, Medical Wire & Equipment, Wiltshire, England) as one of the primary aims of the ORChID study was to detect respiratory viruses. Swabs were sent by standard surface mail to the laboratory where they were stored at −80°C, at a median of 3 (interquartile range 2-4) days after swabbing.

Since the swab's viral transport medium contained chloramphenicol and amphotericin B, polymerase chain reaction (PCR) assays rather than culture were used to detect the bacterial pathogens of interest.

For nucleic acid extraction, each swab was resuspended in 2 mL of phosphate buffered saline from which 200 μL was used for extraction.

Samples were extracted on the QIAxtractor automated highthroughput extraction platform using DX reagents following the manufacturer's instructions (Qiagen, Australia). Total DNA and RNA were eluted into 150 μL of elution buffer. Each sample was spiked with whole equine herpesvirus (EHV) to assess nucleic acid extraction quality and presence of potential PCR inhibitors. Any extract having a >3 cycle threshold (Ct) difference to that of the expected value by EHV real-time PCR assay was considered to have failed quality control and the sample was re-extracted. Nasal swab specimen quality was determined by screening for human DNA using an endogenous retrovirus-3 (ERV3) assay as described previously. 10 Swabs with ERV3 Ct values >38 were deemed lower-quality and they, and associated person-time, were excluded from analyses. Table) . Batch testing methods and results for 17 respiratory viruses from the ORChID cohort were available and have been described previously. 6, 7 Ct values <40 for individual bacteria were considered positive. 11 

The characteristics of the 158 enrolled cohort children, of whom 154 provided daily symptom data, are shown in Table 1 Overall, the prevalence of the major respiratory bacterial pathogens in the 8100 high-quality nasal swabs were: S. pneumoniae Table) .

Bacterial prevalence was higher in symptomatic children (Table 2) .

Higher prevalence of bacteria was detected in swabs taken during respiratory symptoms, especially when acute otitis media was diagnosed. This was observed also in swabs negative for any virus (Supplemental Digital Content 4, Table) . Conversely, the prevalence of respiratory symptoms was more common in bacteria-positive Figure) . A similar pattern with even higher differences between the aORs was seen for the other bacterial species (data not shown). In concordance with earlier studies, we also observed the association of bacterial detection with respiratory symptoms. 25 Furthermore, although symptoms were more prevalent in virus- This study confirms previous findings that the main risk factors for carrying respiratory bacterial pathogens are age, having The strengths of the ORChID study include its prospective design, the comprehensive surveillance provided by daily symptom diaries, and weekly sample collections from birth until age 24-months.

Enrolment over two calendar years and follow-up of 4-years allowed for different seasonal and year-to-year epidemiologic patterns.

Considering its intensive nature, cohort retention was good with >70% followed for at least 18-months, and 68% of the maximum expected swabs were collected with a median interval of 1-week as stated in the protocol. 6 Routine laboratory quality assurance included assaying samples for the human DNA marker, ERV3, to ensure human cells were present. While important for viral detection, 10 it seems also relevant for bacteria as we observed considerably higher bacterial detection rates in samples containing ERV-3. Consequently, only highquality nasal swabs were used to avoid compromising sensitivity in the analysis.

There are, however, important limitations to consider. We did not validate upper airway bacterial detection by nasal swab and PCR against the gold standard of NPS culture. However, our current analyses using previously validated PCR targets resulted in expected bacterial prevalence rates. The specificity of the PCR assays are high, yet the ply-PCR may cross-react with oral streptococci. 36 We In conclusion, we have used a novel method to evaluate bacterial carriage in children using nasal swabs optimized initially for viral detection. This method, however, is suitable also for bacterial detection and is especially feasible when frequent sampling is required.

Future analyses will explore interactions between nasal viruses, bacteria, and ARI symptoms, while subtyping will allow carriage dynamics to be explored in greater detail. 

Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory tract infections in 195 countries: a systematic analysis for the Global Burden of Disease Study

Otitis media

Epidemiology of community-acquired pneumonia and implications for vaccination of children living in developing and newly industrialized countries: a systematic literature review

The fundamental link between pneumococcal carriage and disease

Carriage of Mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study

Observational research in childhood infectious diseases (ORChID): a dynamic birth cohort study

Viruses causing lower respiratory symptoms in young children: findings from the ORChID birth cohort

Detection of viruses in weekly stool specimens collected during the first 2 years of life: a pilot study of five healthy Australian infants in the rotavirus vaccine era

The burden of community-managed acute respiratory infections in the first 2-years of life

Nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control

Mailed versus frozen transport of nasal swabs for surveillance of respiratory bacteria in remote Indigenous communities in Australia

Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: updated recommendations from the World Health Organization Pneumococcal Carriage Working Group

Comparison of four different sampling methods for detecting pharyngeal carriage of Streptococcus pneumoniae and Haemophilus influenzae in children

Alternative sampling methods for detecting bacterial pathogens in children with upper respiratory tract infections

Evaluation of swabbing methods for estimating the prevalence of bacterial carriage in the upper respiratory tract: a cross sectional study

The association between anterior nares and nasopharyngeal microbiota in infants hospitalized for bronchiolitis. Microbiome

Culture and PCR detection of Haemophilus influenzae and Haemophilus haemolyticus in Australian Indigenous children with bronchiectasis

Density interactions among Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus in the nasopharynx of young Peruvian children

Molecular surveillance of nasopharyngeal carriage of Streptococcus pneumoniae in children vaccinated with conjugated polysaccharide pneumococcal vaccines

Nasopharyngeal carriage of Streptococcus pneumoniae in Finnish children younger than 2 years old

Otitis-prone children have immunologic deficiencies in naturally acquired nasopharyngeal mucosal antibody response after Streptococcus pneumoniae colonization

A longitudinal study of Streptococcus pneumoniae carriage in a cohort of infants and their mothers on the Thailand-Myanmar border

Epidemiologic studies of Streptococcus pneumoniae in infants: acquisition, carriage, and infection during the first 24 months of life

Epidemiologic studies of Streptococcus pneumoniae in infants. The effects of season and age on pneumococcal acquisition and carriage in the first 24 months of life

Relationship between rhinitis symptoms, respiratory viral infections and nasopharyngeal colonization with Streptococcus pneumoniae, Haemophilus influenzae and Staphylococcus aureus in children attending daycare

Modelling the co-occurrence of Streptococcus pneumoniae with other bacterial and viral pathogens in the upper respiratory tract

Epidemiological markers for interactions among Streptococcus pneumoniae, Haemophilus influenzae, and Staphylococcus aureus in upper respiratory tract carriage

Density, serotype diversity, and fitness of Streptococcus pneumoniae in upper respiratory tract cocolonization with nontypeable Haemophilus influenzae

Prevalence of Bordetella pertussis and Bordetella parapertussis in infants presenting to the emergency department with bronchiolitis

A controlled trial of two acellular vaccines and one whole-cell vaccine against pertussis. Progetto Pertosse Working Group

Bordetella parapertussis infection in children: epidemiology, clinical symptoms, and molecular characteristics of isolates

Ten years' experience with yearround active surveillance of up to 19 respiratory pathogens in children

Simkania negevensis may be a true cause of community acquired pneumonia in children

Crowding and other strong predictors of upper respiratory tract carriage of otitis media-related bacteria in Australian Aboriginal and non-Aboriginal children

Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA

Molecular surveillance of true nontypeable Haemophilus influenzae: an evaluation of PCR screening assays

Nasal swab bacteriology by PCR during the first 24-months of life: A prospective birth cohort study