ARTICLE. Cerebrospinal Fluid Enterovirus Testing in Infants 56 Days or Younger

ARTICLE Cerebrospinal Fluid Enterovirus Testing in Infants 56 Days or Younger Maya Dewan, MD, MPH; Joseph J. Zorc, MD, MSCE; Richard L. Hodinka, PhD; Samir S. Shah, MD, MSCE Objective: To determine whether cerebrospinal fluid (CSF) enterovirus polymerase chain reaction (PCR) testing of febrile neonates is associated with a shorter hospital length of stay (LOS). Design: Retrospective cohort study. Setting: Urban tertiary care children s hospital emergency department. Participants: Febrile infants 56 days or younger evaluated by means of lumbar puncture. Main Exposure: Performance of CSF enterovirus PCR testing. Main Outcome Measure: Hospital LOS. Results: A CSF enterovirus PCR test was performed in 361 of 1231 eligible infants (29.3%); 89 of those tested (24.7%) were positive. The median LOS was 2 days. In multivariable analysis, CSF enterovirus PCR testing was associated with a 26.0% shorter LOS for infants with a positive test result (adjusted coefficient, 0.305; 95% confidence interval, 0.457 to 0.153; P.001) and an 8.0% longer LOS for those with a negative test result (0.075; 0.021 to 0.171; P=.12) compared with untested infants. In stratified analysis, LOS was shorter for all infants 28 days or younger who tested positive regardless of receipt of antibiotics before lumbar puncture. For infants 29 to 56 days old, a positive test result was associated with a shorter LOS only in those not previously receiving antibiotics. The median (interquartile range) turnaround time for CSF enterovirus PCR testing was 22.2 (15.1-27.4) hours, with no effect of turnaround time on LOS. Conclusions: Among infants 56 days or younger, a positive CSF enterovirus PCR test result was associated with a shorter LOS compared with untested infants. The CSF enterovirus PCR test may improve the care of infants with fever. Arch Pediatr Adolesc Med. 2010;164(9):824-830 Author Affiliations: Divisions of Infectious Diseases (Drs Dewan and Shah), Emergency Medicine (Dr Zorc), and General Pediatrics (Drs Hodinka and Shah), Children s Hospital of Philadelphia, Pennsylvania, and the Departments of Pediatrics (Drs Hodinka and Shah), Pathology and Laboratory Medicine (Dr Hodinka), and Epidemiology and Biostatistics (Dr Shah) and the Center for Clinical Epidemiology and Biostatistics (Dr Shah), University of Pennsylvania School of Medicine, Philadelphia. NEONATES AND YOUNG INfants routinely undergo lumbar puncture during the emergency department evaluation of fever. 1,2 Although the prevalence of bacterial meningitis is low, 3 these infants do not typically manifest the classic signs of meningitis found in older children and adults. 4-6 Furthermore, clinical prediction rules incorporating the results of lumbar puncture do not accurately distinguish young infants with aseptic meningitis from those with bacterial meningitis. 7-12 This differentiation is important because aseptic meningitis is usually self-limited, whereas bacterial meningitis requires prompt antibiotic therapy. 4,11 Owing to uncertainty in diagnosis, these infants are often hospitalized to receive empirical antibiotic therapy while awaiting culture results. 4,5,13-16 Cerebrospinal fluid (CSF) enterovirus polymerase chain reaction (PCR) testing can help guide physicians in the management of young febrile infants because it provides for rapid and accurate diagnosis of infants with enterovirus meningitis, 1,17-21 the most common cause of aseptic meningitis. 8,10,12 In older children, CSF enterovirus PCR testing is associated with decreased hospital length of stay (LOS) for those ultimately diagnosed as having enterovirus meningitis. 11,12,20,22 However, these studies did not adjust for additional factors that may affect LOS and did not address the use of CSF enterovirus PCR testing in young infants, where the approach to treatment is often more conservative than that in older children. 11,12,20 King et al 23 found that a positive CSF enterovirus PCR test result during the enterovirus season was associated with a decrease in LOS in infants younger than 3 months. However, that study included only infants tested for enteroviral meningitis and, thus, could not compare LOS between tested and untested patients. Routine enterovirus PCR testing provides ben- 824

efit only if positive test results decrease the LOS and negative test results do not increase the LOS compared with no testing. The objective of this study was to evaluate the impact of CSF enterovirus PCR testing on hospital LOS for infants 56 days or younger. Specifically, we aimed to determine the effect of CSF enterovirus PCR testing on LOS for tested patients (with positive or negative results) compared with untested patients. METHODS STUDY DESIGN AND SETTING This retrospective cohort study was conducted at the Children s Hospital of Philadelphia, an urban tertiary care children s hospital. The Committees for the Protection of Human Subjects of the Children s Hospital of Philadelphia approved this study, with a waiver of informed consent. STUDY POPULATION Febrile neonates and young infants (birth through 56 days of age) who underwent a lumbar puncture in the emergency department between January 1, 2005, and December 31, 2007, were eligible for inclusion in the study. At the Children s Hospital of Philadelphia, infants 56 days or younger routinely undergo lumbar puncture during evaluation for fever. 1,2,16 We excluded infants who had insufficient CSF for cell count determination because lack of CSF precluded further testing, including CSF enterovirus PCR testing. PRIMARY EXPOSURE AND OUTCOME The main exposure was CSF enterovirus PCR testing, classified into 3 categories: not performed, performed and results negative, and performed and results positive. The primary outcome was hospital LOS. STUDY DEFINITIONS All historical data, vital signs, and clinical findings were obtained from the initial evaluation. Patients not tested for enterovirus at initial presentation were classified as CSF enterovirus PCR test not performed. Fever was defined as a reported tactile temperature or a recorded temperature higher than 38.0 C at or before presentation. Other vital sign abnormalities were defined as follows: hypoxia, percutaneous oxygen saturation level less than 90% or receipt of supplemental oxygen; tachypnea, respiratory rate higher than 70/min 24 ; hypothermia, temperature lower than 36.1 C; and hypotension, systolic blood pressure lower than 63 mm Hg. 25 Prematurity was defined as gestational age younger than 37 weeks. Serious bacterial infection was defined as bacterial meningitis, bacteremia, or urinary tract infection. Bacterial meningitis was defined as either the isolation of a known bacterial pathogen from the CSF or, in patients who received antibiotics before evaluation, the combination of CSF pleocytosis and bacteria reported on CSF Gram stain. Bacteremia was defined as the isolation of a known bacterial pathogen from blood cultures excluding commensal skin flora. Urinary tract infection was defined as growth of a single known pathogen meeting 1 of 3 criteria: (1) at least 1000 colony-forming units (CFUs) per milliliter for urine cultures obtained by means of suprapubic aspiration, (2) at least 50 000 CFUs/mL from a catheterized specimen, or (3) at least 10 000 CFUs/mL from a catheterized specimen in association with positive urinalysis results. 26-28 Hyponatremia was defined by serum sodium levels less than 131 meq/l (to convert to millimoles per liter, multiply by 1.0). Thrombocytopenia was defined by platelet counts less than 150 10 3 /µl (to convert to 10 9 per liter, multiply by 1.0). Levels of CSF glucose less than 40 mg/dl (to convert to millimoles per liter, multiply by 0.0555) were considered low. Levels of CSF protein greater than 0.17 g/dl (to convert to grams per liter, multiply by 10.0) for infants 28 days or younger or greater than 0.08 g/dl for infants aged 29 to 56 days were considered elevated. 29 Pleocytosis of CSF was defined by previously published criteria: white blood cell count greater than 22/µL (to convert to 10 9 per liter, multiply by 0.001) for infants 4 weeks or younger and greater than 15/µL for infants older than 4 weeks. 29 Traumatic lumbar puncture was defined by a red blood cell count greater than 500 10 6 /µl 3 (to convert to 10 12 per liter, multiply by 1.0). 30 The enterovirus season was defined as June 1 through October 31 for each year. 31,32 The CSF enterovirus PCR test turnaround time was defined as the difference, in hours, from test ordering to result availability. CSF ENTEROVIRUS PCR TESTING As previously described, 23 enterovirus RNA was extracted from the CSF using an automated MagNAPure LC instrument (Roche Diagnostics Corporation, Indianapolis, Indiana) and total nucleic acid isolation kit (Roche Diagnostics). Amplification and detection were completed using either a sequence detection system (ABI Prism 7000; Applied Biosystems, Foster, California) or a 7500 real-time PCR system (Applied Biosystems). Standard techniques used by the Clinical Virology Laboratory at The Children s Hospital of Philadelphia to minimize the likelihood of false-positive and false-negative results have also been described previously. 23 Because we used automated, magnet bead based extraction technology, blood contamination of the CSF from traumatic lumbar punctures would not be expected to affect the PCR yield. 33 DATA COLLECTION AND STATISTICAL ANALYSIS Data were abstracted from medical records, entered on a standardized data collection form, and analyzed using a statistical software program (STATA 10; Stata Corp, College Station, Texas). Continuous variables were compared using the Wilcoxon rank sum test and are described using mean, median, range, and interquartile range (IQR) values. Categorical variables were compared using the 2 test and are described using counts and percentages. Univariable analyses were conducted to identify factors associated with LOS. Multivariable linear regression was performed to identify factors independently associated with LOS. Because the LOS variable had a skewed distribution (P.001, Shapiro-Wilk test), we used the logarithmically transformed LOS value as the dependent variable. The resulting coefficients were transformed to reflect the percentage difference in LOS. The multivariable model initially incorporated age, CSF white blood cell count, diagnosis of serious bacterial infection, presence of seizures, enterovirus season, and the administration of antibiotics before lumbar puncture based on the a priori hypotheses of their effect on LOS. Additional variables were considered for inclusion in the multivariable model if they were associated with LOS in univariable analyses (P.20). 34 These variables were included in the final multivariable model if they remained statistically significant after adjustment for other factors or if their inclusion in the model resulted in a greater than 10% change in 825

Table 1. Clinical Characteristics of 1231 Febrile Infants 56 Days or Younger Who Underwent Lumbar Puncture in the Emergency Department Characteristic Infants, No. (%) Age category, d 0-14 270 (21.9) 15-28 282 (22.9) 29-42 342 (27.8) 43-56 337 (27.4) Male sex 666 (54.1) Presenting during enterovirus season 535 (43.5) Transported 153 (12.4) Race/ethnicity a Non-Hispanic white 438 (37.6) Non-Hispanic black 565 (48.5) Hispanic 51 (4.4) Other 111 (9.5) Birth history Premature birth 163 (13.2) Cesarean delivery 343 (30.6) b History of present illness Apnea 73 (5.9) Upper respiratory tract infection signs 608 (49.4) Gastrointestinal tract infection signs 425 (34.5) Seizure 90 (7.3) Physical examination Tachypnea 45 (3.7) Hypoxia 31 (2.5) Hypotension 35 (2.8) Lethargy or irritability 222 (18.0) Bleeding or bruising 15 (1.2) Received antibiotics before lumbar puncture 277 (22.5) a Race/ethnicity was unknown for 66 infants. b n=1120. the effect size of the primary association of interest, enterovirus PCR testing and LOS, regardless of statistical significance. 35 Statistical significance was determined a priori as a 2-tailed P.05. The possibility of important subgroup differences (ie, differences in outcome based on enterovirus season, age, antibiotic use before lumbar puncture, or CSF pleocytosis) was explored using interaction terms. The significance of these subgroups in multivariable analysis was evaluated using the likelihood ratio test. Because there was a significant interaction between age and the administration of antibiotics before lumbar puncture (P.001, likelihood ratio test), the analysis was subsequently stratified according to these variables. We examined the association between CSF enterovirus PCR test turnaround time and LOS in the subset of infants for whom CSF enterovirus PCR testing had been performed using linear regression with logarithmically transformed LOS values. The goal of this analysis was to determine whether differences in LOS could be due to test result availability. In univariable analysis, we estimated that 3 years of data collection would provide 80% power ( =.05) to detect a 17% difference in LOS between tested and untested infants based on a mean (SD) LOS of 2.5 (4.6) days. RESULTS STUDY POPULATION Table 2. Puncture in the Emergency Department Infants, No./ Laboratory Result Total No. (%) Hyponatremia 10/1231 (0.8) Abnormal coagulation study results 26/36 (72.2) Peripheral white blood cell count 15 000/µL 186/1231 (15.1) Thrombocytopenia 242/1187 (20.4) Low CSF glucose 109/1135 (9.6) Elevated CSF protein 186/1135 (16.4) Elevated hepatic transaminases a 4/26 (15.4) CSF pleocytosis 271/1231 (22.0) Traumatic lumbar puncture 395/1155 (34.2) Abbreviation: CSF, cerebrospinal fluid. SI conversion factor: To convert white blood cell count to 10 9 per liter, multiply by 0.001. a Hepatic transaminase levels were considered abnormal if the values were 50% greater than the upper limit of normal defined by the Clinical Virology Laboratory at The Children s Hospital of Philadelphia. During the study, 1256 febrile infants 56 days or younger underwent lumbar puncture in the emergency department. Twenty-five infants (2.0%) were excluded owing to insufficient CSF to perform a cell count, and the remaining 1231 infants were included in the subsequent analyses. The median (IQR) patient age was 32 (16-44) days (Table 1). The CSF enterovirus PCR test result was positive in 89 of 361 patients (24.7%) who were tested; most testing (77.6%) occurred during the enterovirus season, although only 535 infants (43.5%) presented during this period. Results of CSF enterovirus PCR testing were positive in 80 of 281 patients (28.5%) during the enterovirus season and in 9 of 81 patients (11.1%) outside the enterovirus season. Results of blood enterovirus PCR testing were positive in 13 of 42 tested infants (31.0%); 11 of these infants (84.6%) also had a positive CSF enterovirus PCR test result, 1 had a negative CSF enterovirus PCR test result, and 1 did not undergo CSF enterovirus PCR testing. Overall, 277 patients (22.5%) received antibiotics before lumbar puncture; 65.3% of infants previously receiving antibiotics were 28 days or younger. Results of initial laboratory studies are given in Table 2. Pleocytosis of the CSF was present in 34.4% of infants undergoing CSF enterovirus PCR testing and in 16.9% of those not tested (P.001, 2 test). HOSPITAL LOS The median (IQR) LOS for all patients was 2 (1-3) days. For patients 28 days or younger, the median LOS was 3 (2-5) days, whereas for patients 29 to 56 days of age, the median LOS was 2 (1-2) days (Figure). In unadjusted analysis, there was no difference in LOS for those tested vs untested (unadjusted coefficient, 0.077; 95% confidence interval [CI], 0.015 to 0.170; P=.10). However, compared with untested patients, the unadjusted LOS was 22.3% shorter in patients with a positive CSF enterovirus PCR test result ( coefficient, 0.252; 95% CI, 0.412 to 0.092; P=.002) and 20.6% longer in those with a negative result (0.187; 0.086 to 0.290; P.001). The median LOS was longer for infants who received antibiotics before lumbar puncture (median [IQR] LOS, 3 [1-3] days) compared with those who did not (2 [1-3] days) (P.001, Wilcoxon rank sum test). 826

Length of Stay, d 10 8 6 4 2 0 Not Tested Negative Positive Age 0-28 d Not Tested Negative Positive Age 29-56 d Figure. Boxplot of the hospital length of stay stratified by age and results of cerebrospinal fluid enterovirus polymerase chain reaction testing. The top and bottom borders of each box mark the 25th and 75th percentiles, respectively, and the whiskers extend to 1.5 times the interquartile range. The black squares denote the median values. Factors associated with a longer LOS in univariable analysis included enterovirus season, younger age, birth by cesarean delivery, and antibiotic use before lumbar puncture (Table 3). Abnormalities in certain laboratory test results, including hyponatremia, an elevated peripheral white blood cell count, thrombocytopenia, a low CSF glucose level, and CSF pleocytosis, were also associated with a longer LOS. In a multivariable analysis, there was no overall difference in LOS between tested and untested infants (adjusted coefficient, 0.004; 95% CI, 0.094 to 0.087; P=.94). However, a positive CSF enterovirus PCR test result was associated with a 26.0% shorter LOS (adjusted coefficient, 0.305; 95% CI, 0.457 to 0.153; P.001), and a negative test result was associated with an 8.0% longer LOS (0.075; 0.021 to 0.171; P=.12) compared with untested infants; the difference in LOS between untested infants and those with a negative enterovirus PCR test result was not statistically significant (P=.12). Stratified analysis demonstrated shorter LOS for all infants 28 days or younger who tested positive for enterovirus compared with untested infants regardless of receipt of antibiotics before lumbar puncture (Table 4). For infants aged 29 to 56 days, a positive test result was associated with a shorter LOS only in patients who did not receive antibiotics before lumbar puncture (Table 4 and etable; http://www.archpediatrics.com). CSF ENTEROVIRUS PCR TEST TURNAROUND TIME AND HOSPITAL LOS The median (IQR) CSF enterovirus PCR test turnaround time was 22.2 (15.1-27.4) hours; the results of CSF enterovirus PCR testing were available before discharge for 317 of the 361 infants tested (87.8%). Most CSF enterovirus PCR test results (90%) were available within 36 hours; 95% of results were available within 48 hours. The difference in median CSF enterovirus PCR test turnaround time between infants with positive (23.1 hours) and negative (22.0 hours) results was not significant (P=.23, Wilcoxon rank sum test). Table 3. Univariable Analysis of Factors Associated With Increased Hospital Length of Stay in Infants 56 Days or Younger Variable Coefficient (95% CI) P Value Age category, d 43-56 Reference 29-42 0.123 (0.009 to 0.237).03 15-28 0.348 (0.235 to 0.462).001 0-14 0.679 (0.564 to 0.793).001 Presenting during enterovirus 0.084 ( 0.002 to 0.171).06 season Race/ethnicity Non-Hispanic white Reference Non-Hispanic black 0.078 ( 0.176 to 0.018).11 Hispanic 0.138 ( 0.082 to 0.357).22 Other 0.110 ( 0.270 to 0.049).18 Birth history Premature birth 0.274 (0.149 to 0.399).001 Cesarean delivery 0.107 (0.011 to 0.202).03 History of present illness Apnea 0.468 (0.297 to 0.638).001 Upper respiratory tract infection 0.230 ( 0.344 to 0.112).001 signs Gastrointestinal tract infection 0.130 ( 0.220 to 0.039).005 signs Seizure 0.452 (0.295 to 0.608).001 Physical examination Tachypnea 0.184 ( 0.428 to 0.411).11 Hypoxia 0.793 (0.538 to 1.049).001 Hypotension 1.110 (0.872 to 1.344).001 Lethargy or irritability 0.051 ( 0.057 to 0.159).35 Interventions Required vasopressors 1.620 (0.914 to 2.329).001 Required intubation 1.510 (1.240 to 1.780).001 Laboratory results Elevated white blood cell count a 0.286 (0.172 to 0.400).001 Low CSF glucose 0.264 (0.119 to 0.409).001 Elevated CSF protein 0.093 ( 0.024 to 0.210).12 Elevated hepatic transaminases 0.448 ( 1.470 to 0.576).38 CSF pleocytosis 0.174 (0.074 to 0.275).001 Traumatic lumbar puncture 0.056 ( 0.351 to 0.148).23 Hyponatremia 1.33 (0.888 to 1.780).001 Thrombocytopenia 0.533 (0.260 to 0.806).001 Serious bacterial infection 0.100 (0.008 to 0.194).03 Antibiotics received before lumbar 0.449 (0.353 to 0.545).001 puncture CSF enterovirus PCR testing Not done Reference Negative results 0.187 (0.086 to 0.290).001 Positive results 0.252 ( 0.412 to 0.092).002 Abbreviations: CI, confidence interval; CSF, cerebrospinal fluid; PCR, polymerase chain reaction. a Defined as a peripheral white blood cell count of 15 000/µL or higher (to convert white blood cell count to 10 9 per liter, multiply by 0.001). There was no difference in turnaround time for the enterovirus PCR testing performed during (22.0 hours) or outside of (22.3 hours) the enterovirus season (P=.50, Wilcoxon rank sum test). Among infants with a positive CSF enterovirus PCR test result, no effect of turnaround time on LOS was observed when stratified by age (age 28 days: coefficient, 0.002; 95% CI, 0.272 to 0.023; P=.86; and age 29-56 days: 0.006; 0.007 to 0.019; P=.33). No effect of turnaround time on LOS was observed when stratified by antibiotic use before lumbar puncture (antibiotic use 827

Table 4. Multivariable Analysis Comparing Changes in LOS for Infants With and Without CSF Enterovirus PCR Testing Stratified by Age and Reception of Antibiotics Before Lumbar Puncture a Age 0-28 d Age 29-56 d Variable Change in LOS% (95% CI) P Value Change in LOS% (95% CI) P Value Antibiotics before lumbar puncture n=181 n=94 Not tested Reference Reference Tested, negative results 4.8 ( 27.1 to 24.6).72 10.6 ( 24.9 to 63.0).61 Tested, positive results 32.5 ( 52.9 to 3.5).03 37.3 ( 61.9 to 3.4).07 No antibiotics before lumbar puncture n=360 n=447 Not tested Reference Reference Tested, negative results 1.5 (14.5 to 20.5).86 12.5 ( 1.9 to 29.0).09 Tested, positive results 26.3 ( 44.4 to 2.1).04 27.1 ( 43.7 to 5.4).02 Abbreviations: CI, confidence interval; CSF, cerebrospinal fluid; LOS, length of stay; PCR, polymerase chain reaction. a Model adjusted for enterovirus season, prematurity, apnea, hypoxia, hypotension, intubation, hyponatremia, elevated white blood cell count, CSF pleocytosis, serious bacterial infection, and seizure. before lumbar puncture: coefficient, 0.001; 95% CI, 0.018 to 0.020; P=.91; and no antibiotic use before lumbar puncture: 0.005; 0.011 to 0.022; P=.53). Finally, there was no effect of turnaround time on LOS ( coefficient, 0.006; 95% CI, 0.002 to 0.014; P=.13) in the subset of patients whose positive enterovirus PCR test result was available before discharge. COMMENT We found that in a large cohort of febrile infants, a positive CSF enterovirus PCR test result was associated with a shorter hospital LOS. The LOS for patients who tested negative was not significantly different compared with the LOS for untested patients. We believe that these findings lend support to routine CSF enterovirus testing for young febrile infants during the enterovirus season. Two important factors affected the association between LOS and CSF enterovirus PCR testing age and the receipt of antibiotics before lumbar puncture. The magnitude of reduction in LOS was similar for patients 28 days or younger and those 29 to 56 days of age; however, infants 28 days or younger had a longer median LOS than did infants 29 to 56 days of age. Febrile infants in this younger age group have a higher prevalence of serious bacterial infection 1,36-41 compared with older infants and children. 42-44 Furthermore, the diagnosis of meningitis is difficult because of the poor sensitivity of clinical examination in identifying young infants with any serious bacterial infection, including bacterial meningitis. 16 Thus, we hypothesize that physicians hospitalize infants 28 days or younger for longer periods than they do infants 29 to 56 days of age because of concern about serious bacterial illness without overt clinical signs to indicate bacterial infection. The magnitude of the decrease in LOS for infants 28 days or younger who were enterovirus PCR positive was similar to that for infants aged 29 to 56 days. The administration of antibiotics before lumbar puncture also affected the association between LOS and CSF enterovirus PCR testing. It is likely that enterovirus testing may disproportionately decrease LOS for patients who received antibiotics before lumbar puncture. The magnitude of reduction in LOS for those with a positive enterovirus PCR test result compared with those not tested was greater in the subsets that received antibiotics before lumbar puncture. Physicians cannot rely on negative culture results to exclude the possibility of bacterial meningitis when antibiotics have been administered before lumbar puncture because CSF bacterial cultures may become sterile within 15 minutes after antibiotic administration. 45 Thus, previous antibiotic therapy may lead to longer hospitalizations as physicians elect to observe pretreated infants longer than infants who were not pretreated. We hypothesize that because of the small risk of bacterial and viral meningitis co-infection, 46 physicians feel confident in attributing fever to enteroviral meningitis in the face of a potentially unreliable culture result, thereby facilitating earlier hospital discharge. We explored the relationship between PCR test turnaround time and LOS and found no significant difference in turnaround time for patients with a positive compared with a negative CSF enterovirus PCR test result. This finding assures us that differences in LOS are not attributable to CSF enterovirus PCR test result availability. A previous study 23 at The Children s Hospital of Philadelphia found that for each 24-hour increase in turnaround time for infants with a positive CSF enterovirus PCR test result, the LOS increased by 13.6%. However, no association was found between LOS and turnaround time in the present study. Over time, the Children s Hospital of Philadelphia has transitioned from limiting enterovirus PCR testing to weekdays to performing enterovirus PCR testing daily. In this study, almost all CSF enterovirus PCR test results were available within 36 hours. Thus, it is possible that we reached a floor effect in which physicians were not comfortable discharging patients sooner than 36 hours despite having a positive CSF enterovirus PCR test result. This study has several limitations. First, as in all observational studies, confounding by indication is possible because infants tested for enterovirus infection may be different from untested infants. We minimized confounding by adjusting for measured confounders, but we could not account for unmeasured confounding. However, because the adjusted LOS was not statistically different for untested infants compared with those who tested 828

negative for organisms, it is likely that such confounding, if present, was accounted for in the multivariable analysis. Second, this study includes data from a single hospital, so generalizability to other institutions may vary according to different standards of treatment for young infants and the use and logistics of enterovirus testing. Third, we believe that the availability of CSF enterovirus PCR test results in a clinically relevant time frame depends on the specimen s timely arrival at the laboratory and on the ability of the laboratory to process the specimen and perform the test quickly. The turnaround time in this study was within a relatively narrow range. Other hospitals with fewer laboratory resources or greater demand for testing may show greater variability in turnaround time and, thus, clinically meaningful associations between test delays and LOS. Specifically, physicians practicing in settings where CSF enterovirus PCR testing is performed by commercial reference laboratories are unlikely to achieve meaningful reductions in LOS by routinely testing for enterovirus. Fourth, although enterovirus infections may occur throughout the year, the benefit of enterovirus testing may be greater during the enterovirus season, when the disease is most prevalent. In this study, too few tests were performed outside of the peak enterovirus season to allow us to comment on the role of enterovirus testing during nonpeak months. Fifth, this study was underpowered to detect differences in certain subgroups. The reduction in LOS was statistically significant for all subgroups except patients aged 29 to 56 days who received antibiotics before lumbar puncture; there were relatively few patients in this subgroup, making this study inadequately powered to detect even modest reductions in LOS in this subgroup. In conclusion, we found that a positive CSF enterovirus PCR test result decreases hospital LOS for febrile infants 56 days or younger. Further studies should assess this practice prospectively in other populations and explore subgroups that may be most likely to benefit from testing. Accepted for Publication: February 15, 2010. Correspondence: Samir S. Shah, MD, MSCE, Division of Infectious Diseases, Children s Hospital of Philadelphia, Room 1526 (North Campus), 34th Street and Civic Center Boulevard, Philadelphia, PA 19104 (shahs@email.chop.edu). Author Contributions: Dr Shah had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Hodinka and Shah. Acquisition of data: Dewan, Zorc, Hodinka, and Shah. Analysis and interpretation of data: Dewan, Hodinka, and Shah. Drafting of the manuscript: Dewan, Zorc, and Hodinka. Critical revision of the manuscript for important intellectual content: Dewan, Zorc, Hodinka, and Shah. Statistical analysis: Dewan and Shah. Obtained funding: Shah. Study supervision: Zorc, Hodinka, and Shah. Financial Disclosure: None reported. Funding/Support: This study was supported by grant K01 AI73729 from the National Institute of Allergy and Infectious Diseases (Dr Shah) and by the Physician Faculty Scholar Program of the Robert Wood Johnson Foundation (Dr Shah). Online-Only Material: The etable is available at http: //www.archpediatrics.com. REFERENCES 1. Baker MD, Bell LM. Unpredictability of serious bacterial illness in febrile infants from birth to 1 month of age. Arch Pediatr Adolesc Med. 1999;153(5):508-511. 2. Baker MD, Bell LM, Avner JR. The efficacy of routine outpatient management without antibiotics of fever in selected infants. Pediatrics. 1999;103(3):627-631. 3. Shah SS, Alpern E. Fever. In: Zauotis L, Change V, eds. Comprehensive Pediatric Hospital Medicine. Philadelphia, PA: Mosby Inc; 2007:314-323. 4. Rorabaugh ML, Berlin LE, Heldrich F, et al. Aseptic meningitis in infants younger than 2 years of age: acute illness and neurologic complications. Pediatrics. 1993; 92(2):206-211. 5. Rotbart HA, McCracken GH Jr, Whitley RJ, et al. Clinical significance of enteroviruses in serious summer febrile illnesses of children. Pediatr Infect Dis J. 1999; 18(10):869-874. 6. Sawyer MH. Enterovirus infections: diagnosis and treatment. Semin Pediatr Infect Dis. 2002;13(1):40-47. 7. Nigrovic LE, Kuppermann N, Macias CG, et al; Pediatric Emergency Medicine Collaborative Research Committee of the American Academy of Pediatrics. Clinical prediction rule for identifying children with cerebrospinal fluid pleocytosis at very low risk of bacterial meningitis. JAMA. 2007;297(1):52-60. 8. Nigrovic LE, Chiang VW. Cost analysis of enteroviral polymerase chain reaction in infants with fever and cerebrospinal fluid pleocytosis. Arch Pediatr Adolesc Med. 2000;154(8):817-821. 9. Mulford WS, Buller RS, Arens MQ, Storch GA. Correlation of cerebrospinal fluid (CSF) cell counts and elevated CSF protein levels with enterovirus reverse transcription-pcr results in pediatric and adult patients. J Clin Microbiol. 2004; 42(9):4199-4203. 10. Lee BE, Chawla R, Langley JM, et al. Paediatric Investigators Collaborative Network on Infections in Canada (PICNIC) study of aseptic meningitis. BMC Infect Dis. 2006;6:68. doi:10.1186/1471-2334-6-68. 11. Ramers C, Billman G, Hartin M, Ho S, Sawyer MH. Impact of a diagnostic cerebrospinal fluid enterovirus polymerase chain reaction test on patient management. JAMA. 2000;283(20):2680-2685. 12. Stellrecht KA, Harding I, Woron AM, Lepow ML, Venezia RA. The impact of an enteroviral RT-PCR assay on the diagnosis of aseptic meningitis and patient management. J Clin Virol. 2002;25(suppl 1):S19-S26. 13. Lee BE, Davies HD. Aseptic meningitis. Curr Opin Infect Dis. 2007;20(3):272-277. 14. Hsiao AL, Baker MD. Fever in the new millennium: a review of recent studies of markers of serious bacterial infection in febrile children. Curr Opin Pediatr. 2005; 17(1):56-61. 15. Baker MD, Avner JR, Bell LM. Failure of infant observation scales in detecting serious illness in febrile, 4- to 8-week-old infants. Pediatrics. 1990;85(6):1040-1043. 16. Baker MD, Bell LM, Avner JR. Outpatient management without antibiotics of fever in selected infants. N Engl J Med. 1993;329(20):1437-1441. 17. Ahmed A, Brito F, Goto C, et al. Clinical utility of the polymerase chain reaction for diagnosis of enteroviral meningitis in infancy. J Pediatr. 1997;131(3):393-397. 18. Byington CL, Taggart EW, Carroll KC, Hillyard DR. A polymerase chain reactionbased epidemiologic investigation of the incidence of nonpolio enteroviral infections in febrile and afebrile infants 90 days and younger. Pediatrics. 1999; 103(3):E27. 19. Stellrecht KA, Harding I, Hussain FM, et al. A one-step RT-PCR assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis. J Clin Virol. 2000;17(3):143-149. 20. Hamilton MS, Jackson MA, Abel D. Clinical utility of polymerase chain reaction testing for enteroviral meningitis. Pediatr Infect Dis J. 1999;18(6):533-537. 21. Monpoeho S, Coste-Burel M, Costa-Mattioli M, et al. Application of a real-time polymerase chain reaction with internal positive control for detection and quantification of enterovirus in cerebrospinal fluid. Eur J Clin Microbiol Infect Dis. 2002; 21(7):532-536. 22. Robinson CC, Willis M, Meagher A, Gieseker KE, Rotbart H, Glodé MP. Impact of rapid polymerase chain reaction results on management of pediatric patients with enteroviral meningitis. Pediatr Infect Dis J. 2002;21(4):283-286. 23. King RL, Lorch SA, Cohen DM, Hodinka RL, Cohn KA, Shah SS. Routine cerebrospinal fluid enterovirus polymerase chain reaction testing reduces hospitalization and antibiotic use for infants 90 days of age or younger. Pediatrics. 2007; 120(3):489-496. 24. Rusconi F, Castagneto M, Gagliardi L, et al. Reference values for respiratory rate in the first 3 years of life. Pediatrics. 1994;94(3):350-355. 829

25. Zubrow AB, Hulman S, Kushner H, Falkner B; Philadelphia Neonatal Blood Pressure Study Group. Determinants of blood pressure in infants admitted to neonatal intensive care units: a prospective multicenter study. J Perinatol. 1995; 15(6):470-479. 26. Zorc JJ, Levine DA, Platt SL, et al; Multicenter RSV-SBI Study Group of the Pediatric Emergency Medicine Collaborative Research Committee of the American Academy of Pediatrics. Clinical and demographic factors associated with urinary tract infection in young febrile infants. Pediatrics. 2005;116(3):644-648. 27. Shaw KN, Gorelick M, McGowan KL, Yakscoe NM, Schwartz JS. Prevalence of urinary tract infection in febrile young children in the emergency department. Pediatrics. 1998;102(2):e16. 28. Hoberman A, Chao HP, Keller DM, Hickey R, Davis HW, Ellis D. Prevalence of urinary tract infection in febrile infants. J Pediatr. 1993;123(1):17-23. 29. Shah SN, Ahmed A, Newland J, Armstrong S, Bell L, Shah SS. Infectious diseases. In: Frank G, Shah SS, Catallozzi M, Zaoutis L, eds. The Philadelphia Guide: Inpatient Pediatrics. Philadelphia, PA: Lippincott Williams & Wilkins; 2005:162-207. 30. Mazor SS, McNulty JE, Roosevelt GE. Interpretation of traumatic lumbar punctures: who can go home? Pediatrics. 2003;111(3):525-528. 31. Khetsuriani N, Lamonte-Fowlkes A, Oberst S, Pallansch MA; Centers for Disease Control and Prevention. Enterovirus surveillance United States, 1970-2005. MMWR Surveill Summ. 2006;55(8):1-20. 32. Strikas RA, Anderson LJ, Parker RA. Temporal and geographic patterns of isolates of nonpolio enterovirus in the United States, 1970-1983. J Infect Dis. 1986; 153(2):346-351. 33. Boom R, Sol CJ, Salimans MM, Jansen CL, Wertheim-van Dillen PM, van der Noordaa J. Rapid and simple method for purification of nucleic acids. J Clin Microbiol. 1990;28(3):495-503. 34. Mickey RM, Greenland S. The impact of confounder selection criteria on effect estimation. Am J Epidemiol. 1989;129(1):125-137. 35. Cohen DM, Lorch SA, King RL, Hodinka RL, Cohn KA, Shah SS. Factors influencing the decision to test young infants for herpes simplex virus infection. Pediatr Infect Dis J. 2007;26(12):1156-1158. 36. Chiu CH, Lin TY, Bullard MJ. Application of criteria identifying febrile outpatient neonates at low risk for bacterial infections. Pediatr Infect Dis J. 1994;13(11):946-949. 37. Chiu CH, Lin TY, Bullard MJ. Identification of febrile neonates unlikely to have bacterial infections. Pediatr Infect Dis J. 1997;16(1):59-63. 38. Kadish HA, Loveridge B, Tobey J, Bolte RG, Corneli HM. Applying outpatient protocols in febrile infants 1-28 days of age: can the threshold be lowered? Clin Pediatr (Phila). 2000;39(2):81-88. 39. Dagan R, Powell KR, Hall CB, Menegus MA. Identification of infants unlikely to have serious bacterial infection although hospitalized for suspected sepsis. J Pediatr. 1985;107(6):855-860. 40. Bachur RG, Harper MB. Predictive model for serious bacterial infections among infants younger than 3 months of age. Pediatrics. 2001;108(2):311-316. 41. Bonadio WA, McElroy K, Jacoby PL, Smith D. Relationship of fever magnitude to rate of serious bacterial infections in infants aged 4-8 weeks. Clin Pediatr (Phila). 1991;30(8):478-480. 42. Caspe WB, Chamudes O, Louie B. The evaluation and treatment of the febrile infant. Pediatr Infect Dis. 1983;2(2):131-135. 43. King JC Jr, Berman ED, Wright PF. Evaluation of fever in infants less than 8 weeks old. South Med J. 1987;80(8):948-952. 44. Roberts KB, Borzy MS. Fever in the first eight weeks of life. Johns Hopkins Med J. 1977;141(1):9-13. 45. Kanegaye JT, Soliemanzadeh P, Bradley JS. Lumbar puncture in pediatric bacterial meningitis: defining the time interval for recovery of cerebrospinal fluid pathogens after parenteral antibiotic pretreatment [published correction appears in Pediatrics. 2002;110(3):651]. Pediatrics. 2001;108(5):1169-1174. 46. Rotbart HA, ed. Human Enterovirus Infections. Washington, DC: ASM Press; 1995. Call for Photographs Archives of Pediatrics and Adolescent Medicine is always seeking interesting cover photographs. Since many of our readers are excellent amateur photographers, we would appreciate submissions of high-quality photographs. Please e-mail them as attachments in.jpg or.tif format to archpediatrics@jama-archives.org or, if necessary, mail them to Archives of Pediatrics and Adolescent Medicine, University of Washington, Child Health Institute, 6200 NE 74th St, Ste 120B, Seattle, WA 98115-8160. Contact Suzanne Shuey, editorial manager, with any questions at (206) 685-3573. Announcement Trial Registration Required. In concert with the International Committee of Medical Journal Editors (ICMJE), Archives of Pediatrics and Adolescent Medicine will require, as a condition of consideration for publication, registration of all trials in a public trials registry (such as http://clinicaltrials.gov). Trials must be registered at or before the onset of patient enrollment. This policy applies to any clinical trial starting enrollment after July 1, 2005. The trial registration number should be supplied at the time of submission. For details about this new policy, and for information on how the ICMJE defines a clinical trial, see the editorials by DeAngelis et al in the September 8, 2004 (2004;292:1363-1364) and June 15, 2005 (2005;293: 2927-2929) issues of JAMA. Also see the Instructions to Authors on our Web site: www.archpediatrics.com. 830