Laboratory diagnosis of CNS infections in children due to emerging and re-emerging neurotropic viruses.

Recent decades have witnessed the emergence and re-emergence of numerous medically important viruses that cause central nervous system (CNS) infections in children, e.g., Zika, West Nile, and enterovirus/parechovirus. Children with immature immune defenses and blood-brain barrier are more vulnerable to viral CNS infections and meningitis than adults. Viral invasion into the CNS causes meningitis, encephalitis, brain imaging abnormalities, and long-term neurodevelopmental sequelae. Rapid and accurate detection of neurotropic viral infections is essential for diagnosing CNS diseases and setting up an appropriate patient management plan. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis/encephalitis. However, the expansion of test menu has led to new challenges in selecting appropriate tests and making accurate interpretation of test results. There are unmet gaps in development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses. Herein we will discuss the advances and challenges in the laboratory diagnosis of viral CNS infections in children. This review not only sheds light on selection and interpretation of a suitable diagnostic test for emerging/re-emerging neurotropic viruses, but also calls for more research on development and clinical utility study of novel molecular assays. IMPACT: Children with immature immune defenses and blood-brain barrier, especially neonates and infants, are more vulnerable to viral central nervous system infections and meningitis than adults. The addition of new molecular assays and next-generation sequencing has broadened diagnostic capabilities for identifying infectious meningitis and encephalitis. There are unmet gaps in the development of rapid, sensitive and specific molecular assays for a growing list of emerging and re-emerging neurotropic viruses.

Multisystem Inflammatory Syndrome of Children: Subphenotypes, Risk Factors, Biomarkers, Cytokine Profiles, and Viral Sequencing.

OBJECTIVE: To assess demographic, clinical, and biomarker features distinguishing patients with multisystem inflammatory syndrome in children (MIS-C); compare MIS-C sub-phenotypes; identify cytokine biosignatures; and characterize viral genome sequences. STUDY DESIGN: We performed a prospective observational cohort study of 124 children hospitalized and treated under the institutional MIS-C Task Force protocol from March to September 2020 at Children's National, a quaternary freestanding children's hospital in Washington, DC. Of this cohort, 63 of the patients had the diagnosis of MIS-C (39 confirmed, 24 probable) and 61 were from the same cohort of admitted patients who subsequently had an alternative diagnosis (controls). RESULTS: Median age and sex were similar between MIS-C and controls. Black (46%) and Latino (35%) children were over-represented in the MIS-C cohort, with Black children at greatest risk (OR 4.62, 95% CI 1.151-14.10; P = .007). Cardiac complications were more frequent in critically ill patients with MIS-C (55% vs 28%; P = .04) including systolic myocardial dysfunction (39% vs 3%; P = .001) and valvular regurgitation (33% vs 7%; P = .01). Median cycle threshold was 31.8 (27.95-35.1 IQR) in MIS-C cases, significantly greater (indicating lower viral load) than in primary severe acute respiratory syndrome coronavirus 2 infection. Cytokines soluble interleukin 2 receptor, interleukin [IL]-10, and IL-6 were greater in patients with MIS-C compared with controls. Cytokine analysis revealed subphenotype differences between critically ill vs noncritically ill (IL-2, soluble interleukin 2 receptor, IL-10, IL-6); polymerase chain reaction positive vs negative (tumor necrosis factor-α, IL-10, IL-6); and presence vs absence of cardiac abnormalities (IL-17). Phylogenetic analysis of viral genome sequences revealed predominance of GH clade originating in Europe, with no differences comparing patients with MIS-C with patients with primary coronavirus disease 19. Treatment was well tolerated, and no children died. CONCLUSIONS: This study establishes a well-characterized large cohort of MIS-C evaluated and treated following a standardized protocol and identifies key clinical, biomarker, cytokine, viral load, and sequencing features. Long-term follow-up will provide opportunity for future insights into MIS-C and its sequelae.

Kinetics of Viral Clearance and Antibody Production Across Age Groups in Children with Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

OBJECTIVES: To improve understanding of transition from viral infection to viral clearance, and antibody response in pediatric patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. STUDY DESIGN: This retrospective analysis of children tested for SARS-CoV-2 by reverse transcription (RT) polymerase chain reaction (PCR) and immunoglobulin G antibody at a quaternary-care, free-standing pediatric hospital between March 13, 2020, and June 21, 2020, included 6369 patients who underwent PCR testing and 215 patients who underwent antibody testing. During the initial study period, testing focused primarily on symptomatic children; the later study period included asymptomatic patients who underwent testing as preadmission or preprocedural screening. We report the proportion of positive and negative tests, time to viral clearance, and time to seropositivity. RESULTS: The rate of positivity varied over time due to viral circulation in the community and transition from targeted testing of symptomatic patients to more universal screening of hospitalized patients. Median duration of viral shedding (RT-PCR positivity) was 19.5 days and time from RT-PCR positivity to negativity was 25 days. Of note, patients aged 6 through 15 years demonstrated a longer time of RT-PCR positivity to negativity, compared with patients aged 16 through 22 years (median 32 vs 18 days, P = .015). Median time to seropositivity, by chemiluminescent testing, from RT-PCR positivity was 18 days, whereas median time to reach adequate levels of neutralizing antibodies (defined as comparable with 160 titer by plaque reduction neutralization testing) was 36 days. CONCLUSIONS: The majority of patients demonstrated a prolonged period of viral shedding after infection with SARS CoV-2. It is unknown whether this correlates with persistent infectivity. Only 17 of 33 patients demonstrated adequate neutralizing antibodies during the time frame of specimen collection. It remains unknown whether immunoglobulin G antibody against spike structured proteins correlates with immunity, and how long antibodies and potential protection persist.

Clinical Utility of Anaerobic and Fungal Blood Cultures in the Pediatric Oncologic Population.

BACKGROUND: If there is a concern for sepsis in a pediatric patient an aerobic blood culture is obtained. A febrile or ill oncology patient will often be evaluated with aerobic, anaerobic, and fungal blood cultures. There is to our knowledge little evidence to confirm the added benefit of broadly obtaining these cultures. METHODS: A retrospective analysis of blood cultures from patients in the oncology and hematopoietic stem cell transplant wards at Children's National Medical Center between January 2010 and April 2017. Positive anaerobic and fungal cultures were evaluated for corollary positive aerobic cultures from the same time frame (+/-24 h). Unique isolates were designated as those positive anaerobic and fungal cultures without positive aerobic cultures and evaluated with detailed chart review. RESULTS: A total of 10,950 cultures were evaluated during the study period: 6579 aerobic, 2391 anaerobic cultures, 1980 fungal. In total, 419 positive aerobic, 140 positive anaerobic, and 43 positive fungal cultures were reviewed. Among these, 22 unique anaerobic cultures and 21 unique fungal cultures met case criteria. Only 7 cultures of obligate anaerobes were observed: 4 Clostridial spp., 2 Propionobacterium acnes, and 1 Peptostreptococcus spp. Thirteen of 21 fungal isolates consisted of a single colony and were regarded as probable contaminants. Penicillium, Cladosporium, and dematiaceous molds were in greatest frequency. Potential risk factors for patients with breakthrough isolates were evaluated and described. CONCLUSIONS: There is little increased yield from the use fungal and anaerobic blood cultures in addition to aerobic blood cultures in the routine evaluation of pediatric oncology and bone marrow transplant patients. Fungal and anaerobic blood cultures and should be reserved for cases with high clinical suspicion.

Why does infant stepping disappear and can it be stimulated by optic flow?

Two independent experiments (n = 22 and n = 22) showed that 2-month-old infants displayed significantly more stepping movements when supported upright in the air than when supported with their feet contacting a surface. Air- and surface-stepping kinematics were quite similar (Experiment 2). In addition, when data were collapsed across both experiments, more air steps and more donkey kicks were seen when infants were exposed to optic flows that specified backward compared to forward translation. The findings challenge the currently accepted heavy legs explanation for the disappearance of stepping at 2 months of age and raise new questions about the visual control of stepping.

Air stepping in response to optic flows that move Toward and Away from the neonate.

To shed further light on the perceptual regulation of newborn stepping, we compared neonatal air stepping in response to optic flows simulating forward or backward displacement with stepping forward on a surface. Twenty-two 3-day-olds performed four 60 s trials in which they stepped forward on a table (Tactile) or in the air in response to a pattern that moved toward (Toward) or away (Away) from them or was static (Static). Significantly more steps were taken in the Tactile and Toward conditions than the Static condition. The Away condition was intermediate to the other conditions. The knee joint activity across the entire trial was significantly greater in the Toward than the Away condition. Within-limb kinematics and between-limb coordination were very similar for steps taken in the air and on the table, particularly in the Toward and Tactile conditions. These findings highlight that visual and tactile stimulation can equally elicit neonatal stepping.

Pediatric urinary tract infections caused by poultry-associated Escherichia coli.

Escherichia coli is the leading cause of urinary tract infections (UTIs) in children and adults. The gastrointestinal tract is the primary reservoir of uropathogenic E. coli, which can be acquired from a variety of environmental exposures, including retail meat. In the current study, we used a novel statistical-genomic approach to estimate the proportion of pediatric UTIs caused by foodborne zoonotic E. coli strains. E. coli urine isolates were collected from DC residents aged 2 months to 17 years from the Children's National Medical Center Laboratory, 2013-2014. During the same period, E. coli isolates were collected from retail poultry products purchased from 15 sites throughout DC. A total of 52 urine and 56 poultry isolates underwent whole-genome sequencing, core genome phylogenetic analysis, and host-origin prediction by a Bayesian latent class model that incorporated data on the presence of mobile genetic elements (MGEs) among E. coli isolates from multiple vertebrate hosts. A total of 56 multilocus sequence types were identified among the isolates. Five sequence types-ST10, ST38, ST69, ST117, and ST131-were observed among both urine and poultry isolates. Using the Bayesian latent class model, we estimated that 19% (10/52) of the clinical E. coli isolates in our population were foodborne zoonotic strains. These data suggest that a substantial portion of pediatric UTIs in the Washington DC region may be caused by E. coli strains originating in food animals and likely transmitted via contaminated poultry meat.IMPORTANCEEscherichia coli UTIs are a heavy public health burden and can have long-term negative health consequences for pediatric patients. E. coli has an extremely broad host range, including humans, chickens, turkeys, pigs, and cattle. E. coli derived from food animals is a frequent contaminant of retail meat products, but little is known about the risk these strains pose to pediatric populations. Quantifying the proportion of pediatric UTIs caused by food-animal-derived E. coli, characterizing the highest-risk strains, and identifying their primary reservoir species could inform novel intervention strategies to reduce UTI burden in this vulnerable population. Our results suggest that retail poultry meat may be an important vehicle for pediatric exposure to zoonotic E. coli strains capable of causing UTIs. Vaccinating poultry against the highest-risk strains could potentially reduce poultry colonization, poultry meat contamination, and downstream pediatric infections.

The epigenesis of wariness of heights.

Human infants with little or no crawling experience surprisingly show no wariness of heights, but such wariness becomes exceptionally strong over the life span. Neither depth perception nor falling experiences explain this extraordinary developmental shift; however, something about locomotor experience does. The crucial component of locomotor experience in this emotional change is developments in visual proprioception-the optically based perception of self-movement. Precrawling infants randomly assigned to drive a powered mobility device showed significantly greater visual proprioception, and significantly greater wariness of heights, than did controls. More important, visual proprioception mediated the relation between wariness of heights and locomotor experience. In a separate study, crawling infants' visual proprioception predicted whether they would descend onto the deep side of a visual cliff, a finding that confirms the importance of visual proprioception in the development of wariness of heights.

Domain differences in early social interactions.

Different social experiences help children develop distinctions between domains of norms. This study investigated whether mothers respond differently to moral, prudential, and pragmatic norms during the 2nd year, a period that precedes the time when children are able to make explicit distinctions between these norms. Sixty mothers of infants between 11 and 23 months were interviewed. Mothers' reports of their initial interventions, changes in intervention following noncompliance, and emotional reactions depended on normative domain. Initial interventions were less differentiated by domain for mothers of older than for mothers of younger children. These findings suggest that children have social experiences in the 2nd year that are associated with distinctions among normative domains.

An 11-Year Review of Lactobacillus Bacteremia at a Pediatric Tertiary Care Center.

OBJECTIVES: To inform clinical decisions on the use of probiotics in a pediatric inpatient setting, we sought to determine the number of cases of Lactobacillus bacteremia as well as associated patient characteristics in a tertiary-care pediatric hospital over an 11-year period. METHODS: Cases of Lactobacillus bacteremia among admitted patients were identified through positive blood culture reports. The clinical chart for each case was reviewed for presenting symptoms and risk factors such as probiotic use, presence of a central venous catheter, immunocompromised state, impaired intestinal function, and age below 3 months. Concurrent total inpatient probiotic administration was assessed. RESULTS: Over an 11-year period, 8 cases of Lactobacillus bacteremia were identified among 127 845 hospital admissions. All cases were associated with systemic signs of infection. Lactobacillus bacteremia patients most frequently had underlying impaired intestinal function and a central venous catheter. Three cases had a history of probiotic use. The peak number of annual cases did not coincide with the peak number of inpatients who received probiotics. CONCLUSIONS: Lactobacillus bacteremia is uncommon and did not correlate with doses of probiotics-administered in the hospital. However, certain populations may be at higher risk and require extra consideration in clinical decision-making regarding use of probiotics.

Risk factors for molecular detection of adenovirus in pediatric hematopoietic stem cell transplantation recipients.

Adenovirus (AdV) infections are a major cause of morbidity and mortality in patients undergoing hematopoietic stem cell transplantation (HSCT). To evaluate the use of molecular AdV testing in HSCT at our institution and identify risk factors for AdV viremia and disease, we performed a retrospective cohort study of all HSCT recipients who had undergone AdV polymerase chain reaction testing over a 2-year period. Two cohorts were identified: cohort 1, comprising patients testing positive for AdV (n = 7) and cohort 2, comprising patients testing negative (n = 36). Overall patient characteristics were not statistically significantly different between the 2 cohorts. A comparison of cohort 1 and cohort 2 identified the following medication exposures as risk factors influencing AdV status: preparatory regimens using fludarabine (relative risk [RR], 8.73; 95% confidence interval [CI], 1.18-64.27; P = .006), melphalan (RR, 3.47; 95% CI, 0.76-15.94: P = .08), and/or cyclophosphamide (RR, 0.18; 95% CI, 0.02-1.4; P = .05), and GVHD prophylaxis with methylprednisone (RR, 3.73; 95% CI, 1.01-13.9; P = .04). AdV-positive patients had higher grades of GVHD and higher rates of GVHD of the gastrointestinal tract (RR, 4; 95% CI, 1.18-13.5; P = .03) compared with AdV-negative patients. Four of the 7 AdV-positive patients had concomitant clinical manifestations of disease, including pneumonia, diarrhea, and/or disseminated disease. Clinical outcomes in symptomatic patients included resolution of disease in 2 patients and death in 2 patients. All 7 AdV-positive patients received antiviral therapy, including 1 patient with severe disseminated disease that resolved after administration of liposomal cidofovir. Our study at a large pediatric HSCT center provides important preliminary data for the development of a prospective trial destined to identify specific HCST patient subpopulations that might benefit most from molecular screening and early preemptive therapy.

Impact of CSF Meningitis and Encephalitis Panel on Resource Use for Febrile Well-Appearing Infants.

OBJECTIVES: To determine whether the BioFire FilmArray Meningitis/Encephalitis (ME) panel is associated with decreased resource use for febrile infants. The ME panel has a rapid turnaround time (1-2 hours) and may shorten length of stay (LOS) and antimicrobial use for febrile well-appearing infants. METHODS: Retrospective cohort study of febrile well-appearing infants ≤60 days with cerebrospinal fluid culture sent in the emergency department from July 2017 to April 2019. We examined the frequency of ME panel use and its relationship with hospital LOS and initiation and duration of antibiotics and acyclovir. We used nonparametric tests to compare median durations. RESULTS: The ME panel was performed for 85 (36%) of 237 infants. There was no difference in median hospital LOS for infants with versus without ME panel testing (42 hours, interquartile range [IQR] 36-52 vs 40 hours, IQR: 35-47, P = .09). More than 97% of infants with and without ME panel testing were initiated on antibiotics. Patients with ME panel were more likely to receive acyclovir (33% vs 18%; odds ratio: 2.2, 95%: confidence interval 1.2-4.0). There was no difference in median acyclovir duration with or without ME panel testing (1 hour, IQR: 1-7 vs 4.2 hours, IQR: 1-21, P = .10). When adjusting for potential covariates, these findings persisted. CONCLUSIONS: ME panel use was not associated with differences in hospital LOS, antibiotic initiation, or acyclovir duration in febrile well-appearing infants. ME panel testing was associated with acyclovir initiation.

Common seasonal respiratory viral infections in children before and during the coronavirus disease 2019 (COVID-19) pandemic.

OBJECTIVES: To describe the incidence of seasonal respiratory viral infections (s-RVIs) before and during the coronavirus disease 2019 (COVID-19) pandemic and to compare virus-specific patient outcomes in pediatric patients. DESIGN: A retrospective cross-sectional study including patient admissions to the Children's National Hospital between October 1, 2015, and December 31, 2020. RESULTS: Among 12,451 patient admissions between March 15 and December 31, 2020 (cohort 1), 8,162 (66%) were tested for severe acute respiratory coronavirus virus 2 (SARS-CoV-2), and 249 (2.0%) were positive. Among 10,986 patient admissions between April 1 and December 31, 2020 (cohort 2), 844 (8%) were tested for s-RV upon admission and 160 were positive. Thus, 1.5% of patient admissions were associated with laboratory-confirmed s-RVIs. Among the 49,901 patient admissions during a viral season between October 1, 2015, and March 31, 2020 (cohort 3), 7,539 (15%) were tested for s-RV upon admission and 4,531 were positive; thus, 9.0% of patient admissions were associated with laboratory-confirmed s-RVIs. hHRV/rENT was the most detected virus, but the detection rate decreased substantially (31% vs 18%; P < .001) during the COVID-19 pandemic. No patients had RSV, influenza, hMPV, hPIV, or hCoV detected upon admission after April 21, 2020. The 3 patient cohorts had no statistically significant difference in the percentage of ICU admissions (10.8% vs 15.0% vs 14.2%; P > .05) or death at discharge (0.8% vs 0.6% vs 0.5%; P > .05). CONCLUSIONS: Compared to COVID-19, s-RVI cases were associated with a higher proportion of inpatient admissions but were similar in ICU admission and death rates in hospitalized pediatric patients. Public health interventions for preventing COVID-19 were highly effective in preventing pediatrics s-RVIs.

ß-Lactamase-Producing, Ciprofloxacin-Resistant Neisseria meningitidis Isolated From a 5-Month-Old Boy in the United States.

Worldwide, there have been few reports of ß-lactamases causing penicillin resistance in Neisseria meningitidis. The first known case of disease in the United States due to a ß-lactamase-producing, ciprofloxacin-resistant N. meningitidis was recently identified. This has potential implications on standard laboratory testing and empiric management of meningococcal disease.

Comparison of Clinical Features of COVID-19 vs Seasonal Influenza A and B in US Children.

Importance: Compared with seasonal influenza, the clinical features and epidemiologic characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and coronavirus 2019 (COVID-19) in US children remain largely unknown. Objective: To describe the similarities and differences in clinical features between COVID-19 and seasonal influenza in US children. Design, Setting, and Participants: This retrospective cohort study included children who were diagnosed with laboratory-confirmed COVID-19 between March 25 and May 15, 2020, and children diagnosed with seasonal influenza between October 1, 2019, and June 6, 2020, at Children's National Hospital in the District of Columbia. Exposures: COVID-19 or influenza A or B. Main Outcomes and Measures: Rates of hospitalization, admission to the intensive care unit, and mechanical ventilator use and the association between underlying medical conditions, clinical symptoms, and COVID-19 vs seasonal influenza. Results: The study included 315 patients diagnosed with COVID-19 (164 [52%] male; median age, 8.3 years [range, 0.03-35.6 years]) and 1402 patients diagnosed with seasonal influenza (743 [53%] male; median age, 3.9 years [range, 0.04-40.4 years]). Patients with COVID-19 and those with seasonal influenza had a similar hospitalization rate (54 [17%] vs 291 [21%], P = .15), intensive care unit admission rate (18 [6%] vs 98 [7%], P = .42), and use of mechanical ventilators (10 [3%] vs 27 [2%], P = .17). More patients hospitalized with COVID-19 than with seasonal influenza reported fever (41 [76%] vs 159 [55%], P = .005), diarrhea or vomiting (14 [26%] vs 36 [12%], P = .01), headache (6 [11%] vs 9 [3%], P = .01), body ache or myalgia (12 [22%] vs 20 [7%], P = .001), and chest pain (6 [11%] vs 9 [3%], P = .01). Differences between patients hospitalized with COVID-19 vs influenza who reported cough (24 [48%] vs 90 [31%], P = .05) and shortness of breath (16 [30%] vs 59 [20%], P = .13) were not statistically significant. Conclusions and Relevance: In this cohort study of US children with COVID-19 or seasonal influenza, there was no difference in hospitalization rates, intensive care unit admission rates, and mechanical ventilator use between the 2 groups. More patients hospitalized with COVID-19 than with seasonal influenza reported clinical symptoms at the time of diagnosis.

Human parechovirus-3 infection: emerging pathogen in neonatal sepsis.

Human parechovirus-3 (HPeV-3) is an emerging pathogen that has been described as a cause of neonatal sepsis. Human parechoviruses are a family of viruses closely related to enteroviruses; however, enteroviral PCR will not detect HPeVs. We present clinical details of neonatal meningoencephalitis and hepatitis-coagulopathy syndrome caused by HPeV-3 infection.

Neonatal stepping in relation to terrestrial optic flow.

This experiment examined whether newborn stepping, a primitive form of bipedal locomotion, could be modulated by optical flow. Forty-eight 3-day-old infants were exposed to optical flows that were projected onto a horizontal surface above which the infants were suspended. Significantly more air steps were elicited by exposure to a terrestrial optical flow specifying forward translation than by a rotating optical flow or a static optical pattern. Thus, a rudimentary coupling between optical flow and stepping is present at birth, suggesting a precocious capacity in the newborn to perceive and utilize visual information specifying self-motion. The findings may help the early diagnosis of infants with visual or visual-motor deficits and the development of visually based interventions for disabled infants.

Do Balance Demands Induce Shifts in Visual Proprioception in Crawling Infants?

The onset of hands-and-knees crawling during the latter half of the first year of life heralds pervasive changes in a range of psychological functions. Chief among these changes is a clear shift in visual proprioception, evident in the way infants use patterns of optic flow in the peripheral field of view to regulate their postural sway. This shift is thought to result from consistent exposure in the newly crawling infant to different patterns of optic flow in the central field of view and the periphery and the need to concurrently process information about self-movement, particularly postural sway, and the environmental layout during crawling. Researchers have hypothesized that the demands on the infant's visual system to concurrently process information about self-movement and the environment press the infant to differentiate and functionalize peripheral optic flow for the control of balance during locomotion so that the central field of view is freed to engage in steering and monitoring the surface and potentially other tasks. In the current experiment, we tested whether belly crawling, a mode of locomotion that places negligible demands on the control of balance, leads to the same changes in the functional utilization of peripheral optic flow for the control of postural sway as hands-and-knees crawling. We hypothesized that hands-and-knees crawlers (n = 15) would show significantly higher postural responsiveness to movements of the side walls and ceiling of a moving room than same-aged pre-crawlers (n = 19) and belly crawlers (n = 15) with an equivalent amount of crawling experience. Planned comparisons confirmed the hypothesis. Visual-postural coupling in the hands-and-knees crawlers was significantly higher than in the belly crawlers and pre-crawlers. These findings suggest that the balance demands associated with hands-and-knees crawling may be an important contributor to the changes in visual proprioception that have been demonstrated in several experiments to follow hands-and-knees crawling experience. However, we also consider that belly crawling may have less potent effects on visual proprioception because it is an effortful and attention-demanding mode of locomotion, thus leaving less attentional capacity available to notice changing relations between the self and the environment.

Molecular and descriptive epidemiologyof multidrug-resistant Enterobacteriaceae in hospitalized infants.

OBJECTIVE: To investigate the epidemiology of multidrug-resistant Enterobacteriaceae (MDRE) in hospitalized infants. METHODS: From 2000 through 2005, active surveillance cultures for MDRE were performed for patients admitted to a 40-bed neonatal intensive care unit (NICU) that provides care for critically ill infants 6 months of age or younger. MDRE epidemiology and the genetic relatedness of MDRE strains determined by repetitive-sequence polymerase chain reaction were analyzed. RESULTS: Active surveillance cultures revealed that 759 (23%) of 3,370 NICU infants (or approximately 1 in 5) developed MDRE colonization or infection and that 613 (72%) of the 853 isolates with epidemiologic data available were healthcare acquired. MDRE colonization occurred more frequently (in 653 infants [86%]) than did MDRE infection (in 106 [14%]). Of the 653 infants with MDRE colonization, 119 (18%) eventually became infected, with 29 (4%) acquiring sterile site infections. The most commonly isolated organisms were the Enterobacter species, accounting for 612 (71%) of the 862 isolates. Molecular epidemiologic analysis revealed that genetic-relatedness clustering (related clusters defined as having a genetic similarity coefficient greater than 95%) varied depending on microbial species. Clustering was detected for 36 (78%) of the 46 Enterobacter aerogenes isolates, 22 (45%) of the 49 Enterobacter cloacae isolates, and 13 (59%) of the 22 Klebsiella pneumoniae isolates. CONCLUSION: Hospitalized infants are at significant risk of acquiring MDRE, specifically Enterobacter species, at the study institution. Active surveillance cultures identified colonized patients who likely contributed to the institutional reservoir of MDRE. Molecular epidemiologic studies suggest that both patient-to-patient transmission and de novo acquisition of resistance play a role in the acquisition of these organisms, and that the clinical significance of such acquisition varies by species. The high percentage of E. aerogenes isolates that demonstrated genetic clustering suggests that monitoring the prevalence of this organism could serve as a useful measure of compliance with infection control procedures.