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Genetic predisposition, RSV infection and respiratory morbidity in preterm infants

Simon B Drysdale, Michael Prendergast, Mireia Alcazar, Theresa Wilson, Melvyn Smith, Mark Zuckerman, Simon Broughton, Gerrard F Rafferty, Sebastian L Johnston, Hennie M Hodemaekers, Riny Janssen, Louis Bont, Anne Greenough


Follow-up

Following discharge from the neonatal or maternity unit, infants were followed prospectively until one year corrected age. The parents were asked to contact the research team when their infant was symptomatic with signs consistent with a LRTI, that is cough, wheeze, and/or shortness of breath. In addition, parents were telephoned every two weeks by researchers to ascertain whether their infant had been or was symptomatic. A researcher visited the home on every occasion an infant had a LRTI and a nasopharyngeal aspirate (NPA) was obtained if the LRTI was confirmed. NPAs were also obtained from all infants hospitalised with an LRTI. Real time reverse transcriptase polymerase chain reaction (PCR) was performed on the NPAs for nine viruses (RSV A and B, rhinovirus, human metapneumovirus, influenza A and B and parainfluenza 1-3) in three multiplexes with an RNA internal control [1]. Adenovirus (DNA virus) was tested by a duplex real time PCR in monoplex assay incorporating a DNA internal control [1]. In addition another multiplex, including an MS2 phage internal control, was developed using previously published primers and probes which tested for enterovirus, parechovirus and human bocavirus.

Genetic analysis

As part of the cohort study, blood or buccal swabs were obtained from the infants prior to maternity unit discharge. Either one mL of blood was collected into an EDTA tube or three buccal swabs were obtained and stored at -20oC until testing. The samples were then sent on dry ice to the National Institute for Public Health and the Environment (RIVM) in Bilthoven, the Netherlands for testing. DNA was isolated from the blood samples or buccal swabs as previously described [6]. Extracted DNA samples were diluted with TE Buffer to 7 ng/µL and sent to KBioscience (Herts, UK) for genotyping to test nine SNPs (ADAM33 rs2787094, IL10 rs1800872, IL19 rs2243191, KLRG1 rs1805723, MMP16 rs2664349, MMP16 rs2664352, NFκB1A rs2233409, SFTPC rs1124 and TGFbR1 rs334353). Two other SNPs (VDR rs10735810 and NOS2A rs1060826) were tested in the Netherlands. The SNPs were chosen as they had previously been associated with an increased risk of severe RSV infection in infants born at term[5, 8, 12] or prematurely [10], an increased risk of developing RDS-[8], a decreased risk of developing BPD [3], an increased risk of recurrent wheeze at one year of age after RSV LRTI [2] or reduced preschool lung function [11]. Genotyping of VDR rs10735810 was performed by a custom TaqMan SNP genotyping assay (Applied Biosystems, Carlsbad, USA) with forward PCR primer GGGTCAGGCAGGGAAGTG, reverse PCR primer TGGCCTGCTTGCTGTTCTT, probes VIC- ATTGCCTCCATCCCTGT and FAM- TGCCTCCGTCCCTGT. NOS2A rs1060826 was performed by using TaqMan SNP genotyping assay C_9458082_10. For each sample 2.5 µL TaqMan Genotyping Master Mix (Applied Biosystems), 0.25 µL TaqMan SNP genotyping assay and 20 ng genomic DNA were used in a total volume of 5 µL. The reaction was run according to the following protocol: 10 min at 95°C, 40 cycles (15 sec, 92°C; 1min, 60°C) and was performed on a 7500 Fast Real Time PCR system (Applied Biosystems).

Lung function measurements at 36 weeks post menstrual age

The infants underwent lung function measurements at 36 weeks PMA whilst still inpatients on the neonatal or maternity unit. Functional residual capacity by helium gas dilution (FRCHe) and compliance (Crs) and resistance (Rrs) of the respiratory system as previously described [7] were assessed. The infants were studied while supine and asleep, they were not sedated and none were ventilated at the time of study. The initial and equilibration helium concentrations were used to calculate FRCHe, which was corrected for oxygen consumption (7 ml/kg/min) and converted to body temperature and water vapour saturated conditions. FRCHe results which were within 10% of each other were averaged. The mean intra-subject coefficient of variability of the measurement of FRCHe was 5%. Compliance (Crs) and resistance (Rrs) of the respiratory system were measured using the single breath occlusion technique. The mean Crs and Rrs results were calculated from at least five technically acceptable occlusions. The mean intra-subject coefficients of variability of the Crs and Rrs measurements were 12% and 11% respectively.

Lung function measurements at one year corrected age

Infants underwent lung function measurements at one year corrected age. FRCHe, Crs and Rrs were assessed. FRC (FRCpleth) and airway resistance (Raw) by plethysmography, as previously described [6] and FRC by the multiple breath wash-in/out technique (FRCMBW) and lung clearance index (LCI) were also assessed. Infants were assessed if they had not been symptomatic with a respiratory tract infection during the previous three weeks. Infants were sedated with chloral hydrate (80 mg/kg). Throughout pulmonary function testing and until the infants had woken, they were monitored by pulse oximetry (Datex-Ohmeda 3800, Hatfield, UK). Once asleep, the infant was placed supine inside a plethysmograph (Department of Medical Engineering, Hammersmith Hospital, London, UK). The infant breathed through an appropriately sized Rendell-Baker face mask, sealed around the nose and mouth with silicone putty. Lung volume (FRCpleth) was calculated from a minimum of three end inspiratory occlusions and related to body weight. Airway resistance (Raw) was calculated electronically during initial inspiration and between 0% and 50% of maximal inspiratory flow [5]. On completion of the plethysmographic measurements, lung volume was assessed by the measurement of FRCHe. FRC (FRCMBW) and lung clearance index (LCI) were assessed by the multiple breath wash-in/out technique. The co-efficient of variation for FRCMBW was 7.9% and for LCI was 6.4%. Finally, Crs and Rrs were measured.

Assessment of respiratory morbidity

Parents completed a respiratory diary for one month when their infant was 11 months corrected age and filled in a respiratory health related questionnaire about their infant when the infant was one year of corrected age. In the respiratory diary, parents documented on a daily basis whether their infant coughed, wheezed, used any respiratory related medications (e.g. inhalers, oral steroids, antibiotics) or they had attended their general practitioner (GP) or had a hospital admission. The respiratory health related questionnaire included questions about the frequency of cough and wheeze in the first year, the use of respiratory related medications (e.g. inhalers, oral steroids, antibiotics) in the first year and whether the infant had ever been diagnosed with asthma by a doctor. The hospital notes were reviewed and all hospitalisations in the first year after birth documented.


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Figure legend: Consort diagram

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