prematurity; Neonatal Intensive Care Unit; viral respiratory infection Almost four million babies are born in the United States each year with approximately 12% of those births occurring prematurely.(1) Pre-term infants suffer significant respiratory morbidity due to lung immaturity at birth, especially those born before 32 weeks gestation. The more severe cases are diagnosed with bronchopulmonary dysplasia (BPD) based on oxygen requirement near term corrected gestational age. However, infants born at less than 32 weeks who do not develop BPD and those born moderate to late pre-term, 32 to< 37 weeks gestation, also have an increased prevalence of respiratory symptoms and rehospitalization due to respiratory problems during their first year of life as well as a greater degree of respiratory symptoms at preschool age. (2, 3) Viral respiratory infections contribute to poor respiratory outcomes and are the most common pathogens identified in children under the age of 18 years hospitalized for community acquired pneumonia. (4) In addition to well documented outbreaks, a prior surveillance study suggested a high burden of on-going respiratory viral infections in preterm infants born at less than 32 weeks gestation while they are still hospitalized in the Neonatal Intensive Care Unit (NICU).(5) NICU infections with human rhinovirus also have been described in both extremely and moderately preterm infants and postulated as a cause of significant respiratory morbidity. (6) A recent report identified respiratory viral infections in a number of clinically significant systemic illnesses in the NICU population and suggested that testing for viral respiratory pathogens may be helpful in the diagnostic evaluation of infants developing signs of sepsis after the first 72 hours of age (late-onset sepsis). (7) We sought to determine the full extent of viral respiratory infections in the extremely to moderately preterm population in the NICU and during the first four months following discharge from the hospital. This study is part of a larger study of infant immune system development and respiratory function (Prematurity, Respiratory outcomes, Immune System and Microbiome [PRISM] study) that is part of the Respiratory Pathogens Research Center at the University of Rochester. We hypothesized that the risk of respiratory viral infections in pre-term babies in the NICU was significantly lower than term infants residing in the community. Secondarily, we hypothesized that the rate of respiratory viral infections in preterm infants would rise to match the term infants' rate of infection once they were discharged from the NICU. read English. In addition, term infants were not eligible if they were admitted to the NICU for any period of time, and preterm infants born at 36 0/7 weeks through 36 6/7 weeks gestation were excluded because they were not routinely admitted to the NICU. Parents were approached within 24-72 hours of birth and all newborns were enrolled by 7 days of life. The Research Subjects Review Board of the University of Rochester approved the study and all parents provided informed consent.
At the initial visit, information was obtained regarding the birth history of the child and the maternal medical history including medication use or medical problems during pregnancy. Parents also self-reported family demographic information. Nose and throat swabs were obtained from each newborn on study day one and then weekly during hospitalization, monthly following discharge until 12 months CGA, and again at three years of age. Results of research testing were not shared with the clinical team. Samples through four months after discharge are included in this report. In addition, families were reminded at each visit to notify the study team if a child developed respiratory symptoms that reached a score of ≥3 on the "COAST" score.(8-10) When a respiratory illness was identified, a study visit was completed as soon as possible. At all visits following hospitalization, parents provided the child's interval medical history.
In addition to our prospective, active surveillance, we reviewed the charts of all enrolled preterm infants who underwent a late onset sepsis evaluation (>72 hours after birth) to determine if the illness episode was associated with a viral respiratory infection.
During the study period, the University of Rochester Medical Center NICU was a regional level IV, 60 bed unit organized into nine 6-8 bed "rooms" opening into a common corridor with four negative pressure isolation rooms. Patients with suspected or proven viral illness were isolated promptly, before a definitive diagnosis was made. Visitor restrictions were in place from mid-December to mid-March limiting visitors to four for each infant with no visitors permitted under the age of 14 years. Influenza vaccination or surgical mask use was required of staff each winter and strongly encouraged for family members. Sibling visits were allowed outside the winter months but required review by a NICU nurse to obtain an updated immunization history and review of symptoms. At all times visitors were asked to refrain from entering the NICU if they had symptoms of a respiratory illness.
Hand hygiene for staff included hand sanitizing and gloves for all patient contact. All patients were assigned a stethoscope and infants < approximately 34 weeks gestation at birth were cared for in incubators until able to maintain temperatures in < 27°C beds. Palivizumab was not administered to hospitalized infants.
Separate flocked swabs (Copan, FLOQSwabs ™ catalog # 525CS01, Copan, Murrieta, CA) were used to obtain samples from the nares and oropharynx/tonsillar region using a tongue depressor. Specimens were immediately combined in 3mL of Universal Transport Media (UTM, Cat # 330CHL, Quidel (formerly Diagnostic Hybrids), Athens, OH), shaken, placed on ice and transported to the laboratory.
Total nucleic acid was extracted using 200μL of UTM with the QIAamp® Viral RNA Mini Kit on a QIAcube (Qiagen, Valencia, CA) with a final volume of 75μL. TaqMan® Array Card (TAC) technology was used on the ViiA7 instrument (Life Technologies, Carlsbad, CA) as previously described, with primer and probe modifications as outlined (Table I; available at www.jpeds.com). (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) Targets included influenza A and B, respiratory syncytial virus (RSV), parainfluenza virus (PIV) 1, 2 and 3, human rhinovirus (hRV), enterovirus (EV), adenovirus (ADV), coronavirus 1 through 4, (229, NL63, OC43, and HKU1 respectively), human metapneumovirus (hMPV), human bocavirus (hBoV), and human parechovirus (hPeV).
Groups were compared by 2-sample t-test for continuous variables and chi-square test for categorical variables. Corresponding non-parametric version of Wilcoxon Rank Sum test and Fisher Exact test were used for confirmation. Survival analysis was applied to study the infection-free curves of pre-term babies during NICU hospitalization versus term babies in the community, and of both cohorts in the community, controlling for other covariates. For the NICU versus community comparison, time to first infection was calculated as the interval between birth date and infection date for the first infection for pre-term babies and discharge date and infection date for term babies. For the comparison of both cohorts in the community, time to first infection was the interval between discharge date and infection date. Time to repeat infection was the interval between previous and current infection dates. Log-rank test and Kaplan-Meier non-parametric estimation of infection-free probability curves were used to compare days to infection between groups, e.g., cohort (pre-term versus term), sex (female versus male), and others. Further, the intensity model (29) using the model-based covariance estimate and coupled with stepwise variable selection was used to explore the effect of demographics and to account for within-subject correlation. All statistical analyses were conducted using Version 9.4 of the SAS System for Windows (SAS Institute Inc., Cary, NC, USA).
From March 2013 through March 2015, we approached 539 full term and 297 pre-term eligible families and enrolled 93 pre-term and 96 term infants. Of the pre-term cohort, the largest numbers of subjects were between 23.1 to 25.6 weeks gestation and 30.1 to 31.9 weeks gestation with the remainder fairly equally divided between the remaining two week blocks (Table II) . The term and pre-term cohorts were generally well matched although, as expected, there were significantly more pre-term infants born by caesarean delivery than term infants and more multiple births among the pre-term cohort (Table II) .
Due to variability in the length of hospitalization the number of specimens from each infant ranged from 1 to 18 with a total of 618 NICU samples that were fairly evenly distributed over all four seasons and represented 119 weeks at risk (Figure 1 ; available at www.jpeds.com). Eighty-9 of 96 term infants contributed one nose/throat swab during the birth hospitalization.
Post discharge, we obtained a total of 489 samples (range of 1-6 per subject) during the first four months following hospitalization with 235 samples contributed by the pre-term group. These samples were also fairly equally divided over all four seasons ( Figure 1 ).
Four infants with viral respiratory infections were identified in the NICU during the 119 weeks at risk. All four were less than 28 weeks gestation at birth and had been in the NICU an average of 11 weeks (Table III) . Two infants were ill with respiratory symptoms within 48 hours of the weekly sampling. Log-rank test and Kaplan-Meier curve estimators suggest that the risk of acquiring a respiratory viral infection in pre-term infants in the NICU was significantly lower than in the term cohort living in the community and the risk was not associated with mode of delivery, multiple birth or sex (Figure 2 , A). These findings were confirmed in the intensity model with only a younger age as measured by the CGA significantly increasing the daily infection rate (HR 0.951, p=0.002) when delivery mode, multiple births, sex, race and ethnicity were included in the model.
Thirty-one pre-term infants (33%) had 71 late-onset sepsis evaluations in the NICU. Six infants had testing for respiratory viruses concomitant with routine bacterial cultures for the evaluation of sepsis and two had viruses identified in the clinical laboratory via molecular methods that also were identified in weekly research samples as noted above, (RSV, coronavirus 4) ( Table III ). The first infant had symptoms including sneezing, progressive congestion, and cough and the clinical team suspected a viral respiratory infection. RSV was identified in the clinical laboratory and by weekly study sampling obtained the following day. The second infant had tachypnea and tachycardia and the following day had an elevated temperature to 38.1°C. Coronavirus 4 was identified by the clinical lab and in the study sample the following day. The third infant underwent a sepsis evaluation for bacterial infection nine days before hRV was identified in a study sample. Worsening respiratory function prompted the sepsis investigation that included a tracheal aspirate for bacterial culture. Mycoplasma culture but no viral diagnostic studies were performed and the symptoms were attributed to evolving BPD when all routine cultures were negative. A weekly study specimen obtained four days before the sepsis evaluation was negative for respiratory pathogens. At the time of hRV identification no specific symptoms were noted in the infant. The fourth infant had influenza B identified in a weekly sample, had not undergone a sepsis evaluation in the prior three months and was clinically asymptomatic. Thus, only 2 of 71, (2.8%), sepsis evaluations identified a viral respiratory infection and both infants had symptoms suggestive of the diagnosis.
Following hospitalization, a majority of infants acquired a viral respiratory infection in the subsequent four months of life (Table IV; available at www.jpeds.com). Seventy-one percent of term babies were infected within four months with 27% acquiring a viral respiratory infection in the first two months of life. Pre-term infants had a slightly higher rate of infection, with 37% acquiring at least one infection in the two months after discharge. However, the difference in the likelihood of acquiring at least one respiratory viral infection in the first four months between the two groups while living in the community was not significant (p=0.39). Further, the Log rank test suggested no difference in the infection-free probability curves between the two groups after hospital discharge (Figure 2, B) . The immediate respiratory viral infection rate after hospital discharge was not associated with mode of delivery, multiple birth or sex by the marginal analyses. Although hRV was the predominant virus detected in both groups, 12 different viral species were identified in infants in the community (Table V ; available at www.jpeds.com).
The number of sick visits for respiratory symptoms was not different between the two groups of infants living in the community. Fourteen percent of pre-term infants had 1-2 sick visits in the first four months following discharge, compared with 17% of term infants (P=0.66), (Table VI; available at www.jpeds.com).
The intensity model was applied to determine the factors associated with the time to acquisition of a viral respiratory infection once discharged from the hospital and included age as measured by CGA, delivery mode, multiple birth, sex, race and ethnicity, with days to infection as the outcome. The model fitting after variable selection procedures, showed that the daily infection rate for all infants following hospital discharge was higher for younger infants as measured by a smaller CGA (range for the pre-term cohort was 38.4 to 64.3; full term cohort range was 40.1 to 59.7). Singleton births, boys and white race were associated with a higher daily infection rate when other covariates were held constant (Table VII) .
We prospectively evaluated a large group of pre-term and term newborns for viral respiratory infections from birth through hospital discharge followed by the first four months in the community and found a very low rate (4%) of viral respiratory infections in our NICU environment. This is in contrast to the findings of Bennett et al (5) who followed 50 pre-term infants with bi-weekly sampling for one year and noted a viral respiratory infection in 52%. Our NICU infection rate was significantly lower than both the rate in term infants living in the community and in pre-term infants once discharged from the hospital. Other variables that were associated with pre-term birth were not associated with the risk of acquiring a viral respiratory infection while still being cared for in the NICU suggesting that the location of care was the key factor responsible for this finding. Our data support the conclusion that it is possible to limit the frequency of respiratory viral infections in premature infants in the NICU.
Our NICU employs standard infection prevention strategies including hand hygiene and gloves for all patient contact with visitor restrictions during the winter months and exclusion of staff and visitors with respiratory symptoms throughout the year. These measures are similar to those reported by Homaira et al in their prospective surveillance study of nosocomial RSV infection where a similar low rate of infection was detected.(30) Although Bennett et al reported that all staff performed an extended hand and arm scrub on arrival to their units, with gloves used for all patient contact there is no information given on hand hygiene before and after patient care or visitation practices so it is difficult to compare practices between the centers.
During the 24 months of this study our unit was arranged in multi-patient rooms and since that time we have moved to a new facility with all single patient rooms.
Although not yet formally evaluated, we speculate that many families visit more frequently and stay for more extended periods when there are single patient rooms such that our low infection rate may have been due to inadvertent limitations on family visitation in the previous physical space.
Our data are consistent with those of Ronchi et al (7) who found that hospitalized infants with respiratory viral infections were likely to have symptoms of congestion and rhinorrhea and be tested based on clinical suspicion. We did not find substantial undetected respiratory viral infections associated with nonspecific concerns for sepsis in our NICU but rather that infants with respiratory infections had suggestive symptoms. Because only 2.8% of sepsis evaluations in the study population were associated with viral detection by surveillance sampling, including viral investigation routinely with sepsis evaluations will have very low yield in this NICU.
The acquisition of a viral respiratory infection in the NICU setting has been linked with a longer length of hospital stay as well as markers of more significant lung disease of prematurity. (5) In this regard it is interesting to note the lower rate of chronic lung disease in our NICU VLBW population from 2006-2014 (17.1%) than comparable units that belong to the Vermont Oxford Network (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) 25 .4%) with a risk adjusted observed to expected average of −12% (data available from authors upon request).
Once discharged from the hospital both preterm and term infants acquired viral respiratory infections at a similar rate and reported an equivalent number of symptomatic illnesses. Male sex, white race, and younger age were associated with an increased daily risk of acquiring an infection. Because our study design focuses on the first six months of life, it is difficult to compare our results with other studies. However, respiratory infection rates have been reported to be higher in younger infants than in children over the age of 12 months, with male sex a risk factor for acquiring hRV infection.(31) White race and young age also have been associated with severity of bronchiolitis suggesting that our findings are consistent with prior research. (32) The strengths of this study include the prospective, longitudinal design with repeated sampling of a large number of pre-term and term infants. Additionally, the study spanned all four seasons of the year and included infants while hospitalized and also while living in the community, both when well and ill with respiratory symptoms. Our study has limitations. First, our center is a regional referral center creating some difficulties for enrollment into long term prospective studies and limiting the percentage of subjects we were able to enroll.
In addition, this study included only one NICU and infection rates appear to vary substantially between different centers based upon limited prior reports.
Another potential limitation is the frequency of sample collection. We obtained nose and throat samples from our population once weekly while in the NICU and this may have led to a decreased detection rate. Previous studies have shown that respiratory samples obtained from the nasal turbinates with a flocked swab have similar sensitivity to nasopharyngeal aspirates and that adding a throat swab to a nasal swab improves the detection of respiratory viruses. (33, 34) In addition, viral identification by PCR is highly sensitive and the TAC platform has been shown to have at least equivalent detection of viral nucleic acid as other commercially available detection systems. (12) Prior studies also have identified extended periods of shedding of respiratory viruses (≥7 days), especially in younger age cohorts, suggesting that our sampling should have been sufficient to identify infections in our NICU population. (5, (35) (36) (37) (38) (39) Our pre-and postdischarge TAC platform and sampling techniques were identical and readily detected viral infections in both term and preterm infants after discharge, supporting the study design. A further limitation is that we did not obtain respiratory samples specifically at the time of suspected sepsis while the pre-term infants were in the NICU and our sampling schedule differed between hospitalized infants and those living in the community. Nevertheless, our data suggest that weekly sampling was sufficient to identify both symptomatic and asymptomatic infections in hospitalized pre-term infants. Once living in the community the monthly sampling schedule likely missed asymptomatic infections in both pre-term and term infants. However, as the schedule was the same between these two groups and we were comparing the infection rates between them, we do not believe this limitation substantially changes our results. Table 1 Primers and Probes used in TAC Detection System Table 4 Total Note: P value = 0.39 for test of same rate of ever infection between two cohorts at home. Full-term cohort, N=76 due to study attrition Pre-term cohort, N=76 due to death (2), transfer to outside hospital (2), continuing hospitalization (13) Caserta et al. Page 21 Table 6 Number of Illness Visits in First 4 Months after Hospital Discharge by Cohort Note: P value = 0.66 for test of same rate of ever sick visit between two cohorts at home. Full-term cohort, N=76 due to study attrition Pre-term cohort, N=76 due to death (2), transfer to outside hospital (2), continuing hospitalization (13) 