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We sought to determine the frequency and effects of nosocomial respiratory viral infections (RVIs) in premature neonates, including those who may be asymptomatic.
We performed a year-long surveillance for RVIs in infants <33 weeks gestational age admitted to two Syracuse neonatal intensive care units (NICUs). Infants were enrolled within 3 days of NICU admission and were sampled for RVIs until discharge using a multiplex PCR assay capable of detecting 17 different respiratory viruses or subtypes.
26 of 50 prematurely born infants (52%) tested positive for a respiratory virus at least once during their birth hospitalization. Testing positive for a respiratory virus was significantly associated with longer length of stay (70 days vs. 35 days, p = 0.002) and prolonged ventilatory support (51 vs. 13 days, p = 0.002). Infants who tested positive for a respiratory virus during their birth hospitalization had more than twice the rate of developing bronchopulmonary dysplasia (BPD; p < 0.05).
Nosocomial RVIs were frequent in our study population, despite the absence of clinical indicators of illness. Length of hospital stay was significantly longer and a diagnosis of BPD was more common in those premature infants who had respiratory viruses detected.
Recent work has challenged the widely-held assumption that newborns in neonatal intensive care units (NICUs) are protected from infections present in the community. Outbreaks of common respiratory viruses among hospitalized infants have been described, including influenza virus, respiratory syncytial virus (RSV), coronavirus, parainfluenzavirus (PIV), adenovirus, enterovirus and rhinovirus [1–8]. In most of these published studies, virus detection was ascertained only in symptomatic infants. In others, surveillance for respiratory virus pathogens was initiated only after an outbreak was established. Of note, respiratory viral infections (RVIs) were detected in minimally symptomatic and even asymptomatic infants when widespread screening was instituted during outbreaks . Moreover, premature infants often have atypical symptoms of infection, such as feeding difficulty, periodic breathing or apnea [8–10]. Reports of NICU outbreaks generally have focused on a single infectious agent, and studies extending to multiple viruses are hampered by the technical limitations associated with routine assays such as antigen detection and virus culture. Recent developments in multiplex polymerase chain reaction (PCR) testing permit rapid, sensitive testing of multiple viruses from a single specimen, including viruses for which culture is unavailable and/or unreliable or for which rapid antigen assays do not exist. In this study we report on the results of a year-long surveillance study in two NICUs in Syracuse, New York, in which infants’ nasopharyngeal specimens were tested in longitudinal fashion from birth to discharge for 17 different viruses.
A prospective, observational study of premature infants in two NICUs in Syracuse, NY, during one calendar year (2009) was performed. Infants were deemed eligible if they were born at less than 33 weeks 0 days gestational age and were available for enrollment within 3 days of arrival in the NICU, which included transfer from another facility or following hospital birth. Neonates with medical and surgical diagnoses were included. Gestational age was determined by menstrual dates and confirmed by ultrasound. Infants were excluded if they had a known or suspected immune deficiency, or were born to an HIV-positive mother. After obtaining informed consent, nasopharyngeal flocked swabs (Copan Diagnostics, Inc., Corona, CA) were collected from each infant by study personnel trained in the proper procedure for obtaining a nasopharyngeal specimen (including intubated infants to ensure sample consistency) within 3 days of birth and on a regular twice weekly (Monday/Thursday) schedule thereafter until discharge. Sampling was deferred on a particular day if it was deemed to place the infant at risk. Specimens were placed in 1.5 mL universal transport media (Copan) and frozen at −70° C prior to testing with the xTAG® Respiratory Viral Panel (RVP, Luminex Molecular Diagnostics, Inc., Toronto, ON, Canada). RVP is a multiplex PCR assay that detects influenza A H1, H3, and nonspecific; influenza B; respiratory syncytial virus (RSV) A and B; parainfluenza (PIV) 1, 2, 3 and 4; coronavirus 229E, NL63, HKU-1 and OC43; rhinovirus/enterovirus; adenovirus; and human metapneumovirus (hMPV). Clinical and demographic data were collected from the medical records. At each sampling timepoint, the use of mechanical ventilation and percent of inspired oxygen was recorded, along with the results of any microbiologic testing performed for appropriate clinical purposes. Clinical events (episodes of oxygen desaturation, bradycardia or apnea) were recorded daily according to the existing criteria being used routinely by the nursing staff in both NICUs. To qualify as an event, the aforementioned change had to persist for longer than 20 seconds. Oxygen saturation below 88% and a heart rate below 100 beats per minute were the cutoff points used for desaturation and bradycardia events, respectively. Clinical deteriorations were defined as a sustained (lasting three days) increase in daily oxygen desaturation events from the prior baseline rate, an increase in oxygen requirement (as judged by the current inhaled oxygen concentration required for stable oxygen saturations, excluding transient desaturations associated with feeding), or an increased level of respiratory support (moving from nasal cannula to continuous positive airways pressure, to intubation or mechanical ventilation). A diagnosis of bronchopulmonary dysplasia (BPD) was made if an infant required supplemental oxygen at 36 weeks post-conceptional age.
The two NICUs in the study were a level 3 unit with a daily average census of approximately 12 infants, and a level 4 regional center with a daily average census of approximately 55 infants. Throughout the study period, each NICU operated under its usual infection control procedures: all staff employed an extended hand and arm scrub on arrival to the unit, and standard precautions were in place at all times. All direct patient contact by medical staff required gloves. Contact precautions also were in place (gown and glove) for patients known to be colonized or infected with methicillin-resistant Staphylococcus aureus (MRSA). MRSA screening by PCR was performed on all patients at the regional center on a weekly basis. All children (age 17 yrs and younger) were excluded from the units and adult visitors were excluded if ill. Parents who had respiratory symptoms were discouraged from visiting but allowed to visit if they wore gowns, gloves, and a surgical mask. In the regional center NICU, twin and triplet births were separated into different nursing areas. No healthcare personnel were aware of the results of the respiratory virus detection tests during the study.
Statistical comparisons between groups were performed using Fisher exact test or student t test as appropriate. Time to infection was calculated as the interval between birth date and infection date for the first infection and the interval between previous and current infection dates for repeated infections. The marginal effects of several variables, including NICU units, gender, gestational age and birth weight on time to infection were presented by Kaplan-Meier infection-free probability curves and tested by log-rank test. A Cox proportional hazards regression model was further fitted to examine the effect of each covariate, adjusting the effect of other covariates. A random-effect term was included to adjust the dependence among the multiple observations from the same infant. The proportional hazards assumption and possible outliers were visually examined through Schoenfeld and Deviance residual plots. A path model, focusing on the direct and indirect effects of infection on the hospital stay, was proposed and confirmed by using SAS9.2 proc CALIS under which the algorithm was optimized via a Quasi-Newton method. Statistical significance was defined at a level of 0.05.
The research was approved by the Institutional Review Boards of St Joseph’s Hospital Health Center and Crouse Irving Memorial Hospital, both of Syracuse, NY (IRBPHS #2008.147 and #2008.4657, respectively).
Approximately half of the parents approached for the study consented to enrollment. The most common reason given for refusal was a concern for an additional procedure on a very small baby, which may have skewed our population towards enrolling neonates of later gestational age. Fifty infants were enrolled into the study; 27 (54%) were male. The average gestational age was 28 weeks (range 24 to 32). Eighteen infants (36%) had gestational ages <28 weeks. The average duration of birth hospitalization was 54.6 days. Twenty-five infants (50%) were intubated at some time during hospitalization, for a mean of 12.5 days. Twenty-eight infants (56%) required supplemental oxygen at some time during hospitalization, and 17 (34%) met criteria for diagnosis of BPD. Eleven infants were enrolled from the level 3 NICU, and 39 were from the level 4 regional NICU. One infant died.
A total of 708 specimens were obtained and tested from the 50 patients over the 52 week period, for an average of 13.6 specimens per week. No weeks were missed but the level of enrollment varied throughout the year – at any one time the number of active patients from the level 3 NICU varied between zero and four, and at the level 4 NICU between two and eight. 66 specimens tested positive for at least one respiratory virus, giving twenty-six (52%) neonates who tested positive for a respiratory virus on at least one occasion. The mean time to detection of the first positive sample was 24 days (range 0 to 92). For each infected patient an average of 2.5 swabs were positive during their hospital stay (range 1 to 13). Viruses detected in the 26 positive patients were PIV3 (13 patients), hMPV (9 patients), RSV-B (8 patients), RSV-A (7 patients), PIV-2 (7 patients), Entero/Rhinovirus (7 patients) and Influenza B (4 patients). Eighteen samples (28% of the positive swabs) included more than one virus and some infants were positive for different viruses at different times. There was evidence of seasonality of infection with particular viruses, with clusters of detection being observed (Figure 1). Fourteen patients had sequential positive specimens for the same virus, with specimens positive over a range of 3–13 days, however because we obtained specimens only twice weekly an accurate determination of duration cannot be made. The viruses that had episodes of sequential detection were hMPV, RSV B, PIV 3 and EV/RV.
Kaplan-Meier curves were created reflecting location, sex, gestational age and birth weight in order to identify potential predictors of infection. The infection rate from RVIs was similar between the two NICUs (5 of 11 from NICU 1, and 21 of 39 from NICU 2, p = 0.74). The Kaplan-Meier curves comparing each location for sex, birth weights or gestational age were not statistically significantly different (Figure 2 – online only), and the data from each NICU therefore were aggregated for comparison of infected and uninfected infants.
Clinical comparisons between infected and uninfected infants are shown in table 1. Infected infants compared with uninfected infants had a longer average length of stay (70 days versus 35 days, p = 0.002), need for intubation (17 of 26 infected infants versus 7 of 24 uninfected infants, p = 0.01), average duration of intubation (19 days versus 5 days, p = 0.03), and duration of supplemental oxygen requirement (51 days versus 13 days, p = 0.002). Nineteen of the 26 infected infants required supplemental oxygen at some point during their hospitalization, compared with 9 of the 24 uninfected infants (p = 0.006). Seventeen infants required oxygen at an adjusted gestational age of 36 weeks (the criteria for diagnosis of bronchopulmonary dysplasia, BPD); 12 of the 17 were among the virus-infected infants and the remaining 5 were uninfected. The probability of acquiring infection was significantly higher for infants diagnosed with BPD (Figure 3, p < 0.05 – online only).
Supplemental oxygen requirements also were higher in the infected cohort. We identified only a single timepoint when one uninfected infant required an inhaled concentration of oxygen above 50%, an event that occurred at 36 separate timepoints in the infected group.
A Cox proportional hazards model showed modestly significant effects of gestational age after adjusting for the effect of NICU units and sex (Table 2 – online only). At each NICU, an infant with lower gestational age was more likely to be infected. The effect of birth weight in the Cox model was reversed compared with the marginal effect seen in Kaplan-Meier analysis, but one child with massive hydrocephalus had a very high birth weight for gestational age which skewed the model. When this outlier was removed the datapoints tended to cluster with no clear trend (data not shown).
We proposed a path model to determine whether virus infection directly extended length of hospital stay or whether this effect was mediated by the need for intubation and oxygen use. The model was confirmed in SAS using Covariance Analysis of Linear Structural Equations. The effect of RVI on length of stay appeared to be mediated partially through effects on the respiratory system, as there were significant standardized indirect effects of infection on length of stay (p = 0.001). When two outliers were excluded (one who died on day of life 1, another who was discharged to an outlying hospital and lost to follow-up) infection was significantly correlated to length of stay through both direct (p < 0.003) and indirect (p < 0.0005) effects (Figure 4 – online only).
Table 1 shows clinical events in infected and uninfected infants. On average, the infected infants had nearly double the number of desaturation events compared with the uninfected cohort (7.1 episodes / day versus 3.9 episodes / day, p < 0.0001). Episodes of bradycardia were the same between the two groups, while there were significantly fewer apnea events among infected infants. This latter finding was likely due to confounding of infected infants were more likely being mechanically ventilated, which precluded apnea.
Most infants were evaluated for bacterial infection at the time of birth, and again later at the discretion of the clinical team. A total of 118 blood cultures were obtained from the 50 study patients. There were significantly more cultures obtained from the virus-infected infants than from the virus-uninfected infants (70 versus 38, p = 0.008). 11 cultures were positive for bacteria from 6 of the virus-infected infants compared with 6 positive bacterial cultures from 4 of the virus-uninfected infants (23.1% of virus-infected infants had a positive culture, compared to 16.7% of virus-uninfected infants (p = 0.73 by Fisher exact test). Of the 11 positive bacterial cultures in infants infected with a viral pathogen, some were repeatedly positive for the same organism. In addition, one virus-infected infant had two separate blood cultures growing coagulase-negative Staphylococcus and Klebsiella, and another had three separate cultures positive for Klebsiella sp, MRSA and Candida sp. The 6 positive blood cultures from infants who never had a virus detected occurred in 4 infants with 2 sets being repeatly positive for Staphylococcus epidermidis. All clinical deteriorations and clinical events noted in study patients were reviewed, and changes in respiratory support were associated with coincident oxygen desaturation events. Compared with virus-infected infants, uninfected infants displayed few clinical deteriorations beyond the first few days of life; 38 clinical deteriorations occurred in 26 infected infants, versus 8 clinical deteriorations in 24 uninfected infants. During 27 (71%) clinical deteriorations, RVI was detected simultaneously. All 14 episodes of sequential detections of RVIs were associated with clinical deteriorations. Additionally, 7 of the 11 positive blood cultures in the virus-infected group were associated with deteriorations. Specific details of each of the neonates with RVI detections are shown in table 3 (online only).
We documented a remarkable rate of otherwise unsuspected and unrecognized respiratory viral infections among premature neonates during their birth hospitalizations. The detection of viral nucleic acid from the respiratory tract of asymptomatic or minimally symptomatic infants (none of the infants in the study displayed symptoms typical of RVI or were evaluated for RVI by the NICU clinicians) raises the question of whether these events represent true infection. In the absence of data documenting seroconversion, our observation documenting 14 instances of prolonged serial detection suggest ongoing virus replication rather than transient carriage. Moreover, of the viruses included in the detection panel, only adenovirus is known to persist in the nasopharynx, although respiratory viruses have been detected in asymptomatic older children. Detection of viral nucleic acids in nasopharyngeal secretions in our study correlated significantly and directly with several specific negative outcomes, inferring a possible causal relationship. Additionally, our findings suggest the need for enhanced virus surveillance and optimized infection control.
Although outbreaks of RVIs in NICUs have been described, this study confirms the importance of systematic surveillance in detecting infections that otherwise would be unrecognized. Premature neonates with RVIs can display atypical symptoms or can have no overt, recognizable symptoms. However, the detection of RVIs within a NICU can have profound clinical implications. Infants with detectable RVIs, even those without overt symptoms, had greater supplemental oxygen requirements, high likelihood of mechanical ventilation, required longer durations of oxygenation, and were hospitalized longer. Persistent detection of virus was associated with clinical deteriorations. In addition, there was a higher rate of BPD in the virus-infected population, an observation clearly worthy of further study. The expression and release of pro-inflammatory cytokines in the lung have been implicated in the etiology of BPD , in association with chorioamnionitis  and prolonged mechanical ventilation [14, 15]. Nosocomial RVIs may exacerbate the inflammation that already occurs secondary to positive pressure ventilation, prematurity and/or supplemental oxygen therapy and may contribute to the development of chronic lung disease of prematurity. The effect of RVI on length of stay appeared to be mediated through effects on the respiratory system. Path analysis and linear regression analysis of our data showed significant direct and indirect effects of infection on length of stay.
Prolonged hospitalization is itself a risk-factor for nosocomial infection, supported by the finding in our study that the average length of time until first detecting an RVI was 24 days. Pre-existing lung disease may predispose infants to infection, as endotracheal intubation and feeding tubes (both markers of clinically compromised infants) have been previously identified as risk factors for acquisition of RVI .
A limitation of our study is that it only covered one calendar year and a relatively small number of neonates. The potential for cross-contamination of specimens was limited by collection of samples in separate sealed containers using universal precautions. Laboratory contamination was limited by using dedicated trained personnel and strict sample identification identical to that employed for our hospital’s clinical specimens. Sample containers remained sealed from collection until laboratory RNA extraction. To ensure a consistent sample set throughout the year, the collection schedule was adhered to as much as reasonably practical with exceptions made only for times when an infant could not safely be tested, and repeat attempts were made as soon as possible. There were no weeks when specimen collection did not occur. A limitation of our study is the difficulty in proving that detection of an RVI is due to ongoing viral replication. Seroconversion data might support this supposition, but this testing was not performed. In addition, the association between detection of an RVI and prolonged hospitalization, increased requirements for oxygenation and respiratory support, and a higher rate of BPD, does not in itself prove causation.
We documented transmission of respiratory pathogens to premature neonates during their birth hospitalization despite presumed vigilance related to infection control practices in these high-risk settings. These practices included the use of barriers (gowns and gloves), hand washing before and after each patient contact, the use of masks for anyone with respiratory symptoms, and the exclusion of all children. During this study, we did not explore the sources of infection, however most of the infants had contact only with the healthcare personnel and their parents. We noted that several twin infants had identical viruses detected at the same time, despite being in separate nursing areas. Our findings highlight the limitations of current infection control practices and raise questions as to whether more aggressive measures need to be implemented. Specifically, one needs to ask whether the presence or sequelae of these infections justify more intensive monitoring of infections and assessment of strategies of prevention and control guidelines.
During a year-long surveillance for respiratory viral infections in two neonatal intensive care units, we found that more than half of the preterm infants had at least one unrecognized, asymptomatic respiratory virus infection; among this group, hospitalization was prolonged and BPD more prevalent.
Funding support was derived from The Children’s Miracle Network of New York (to JBD and NJB) and the NIAID Division of Intramural Research (Z01-AI000943 to HFR). The study sponsors did not contribute to the study design, implementation, data collection or analysis, or to the decision to publish. No honorarium, grant or other payment was given to anyone to produce this work.
The authors have no relevant conflicts of interest to declare.