From 2001 to 2023, 17 (14%) of 120 neonates with confirmed herpes simplex virus (HSV) infection tested positive for HSV by polymerase chain reaction (PCR) from only mucosal sites without a clinical mucosal lesion. Whether mucosal PCR positivity reflects early infection that may lead to recognizable disease, transient colonization, or a false-positive PCR result remains a clinical conundrum and warrants further study.

Objectives To test our hypothesis that routine year-round testing of specimens from multiple body sites and genotyping of detected virus would describe seasonal changes, increase diagnostic yield, and provide a better definition of clinical manifestations of human parechovirus (PeV-A) infections in young febrile infants. Study design PeV-A reverse-transcriptase polymerase chain reaction (RT-PCR) analysis was incorporated in routine evaluation of infants aged 60 days hospitalized at Nationwide Children's Hospital for fever and/or suspected sepsis-like syndrome beginning in July 2013. We reviewed electronic medical records of infants who tested positive for PeV-A between July 2013 and September 2016. Genotyping was performed with specific type 3 RT-PCR and sequencing. Results Of 1475 infants evaluated, 130 (9%) tested positive for PeV-A in 1 or more sites: 100 (77%) in blood, 84 (65%) in a nonsterile site, and 53 (41 %) in cerebrospinal fluid (CSF). Five infants (4%) were CSF-only positive, 31 (24%) were blood-only positive, and 20 (15%) were nonsterile site-only positive. PeV-A3 was the most common type (85%) and the only type detected in CSF. Although the majority (79%) of infections were diagnosed between July and December, PeV-A was detected year-round. The median age at detection was 29 days. Fever (96%), fussiness (75%), and lymphopenia (56%) were common. Among infants with PeV-A-positive CSF, 77% had no CSF pleocytosis. The median duration of hospitalization was 41 hours. Four infants had bacterial coinfections diagnosed within 24 hours of admission; 40 infants had viral coinfections. Conclusions Although most frequent in summer and fall, PeV-A infections were encountered in every calendar month within the 3-year period of study. More than one-half of patients had PeV-A detected at more than 1 body site. Coinfections were common. PeV-A3 was the most common type identified and the only type detected in the CSF.

OBJECTIVE:To determine when infants in the neonatal intensive care unit (NICU) have the first hearing screen performed, and thus inform targeted testing for cytomegalovirus (CMV)-related hearing loss. STUDY DESIGN:Retrospective review of electronic health records of infants admitted to a Level 4 outborn NICU and had a first hearing screen performed from 8/2016-8/2018. RESULT:Among 1498 infants, 546 (36%) had a first hearing screen performed at age >21 days when a positive CMV PCR test cannot distinguish congenital from postnatal CMV acquisition. While most infants tested at >21 days of age were <34 weeks' gestational age (71%), 18% (n = 100) and 11% (n = 59) were ≥34 and ≥37 weeks' gestation, respectively. CONCLUSION:Targeted CMV testing for failed hearing screen in the NICU is problematic as 36% of infants did not have a hearing screen performed before 21 days of age, supporting the need for CMV screening at NICU admission.

Objective: To determine the frequency of detection of cytomegalovirus (CMV) among infants evaluated for late-onset sepsis in the neonatal intensive care unit (NICU). Methods: This study was a prospective cohort study. Results: During the 13-month study, 84 infants underwent 116 sepsis evaluations, and CMV DNA was detected in saliva in three (4%) infants (median: gestational age 28 weeks, birth weight 950 g), representing 5% (n = 6) of all sepsis evaluations. One infant had CMV DNA detected in saliva in all four sepsis evaluations. Two infants had acquired CMV infection, while the timing of CMV acquisition could not be determined in one infant. Two of the three infants had concomitant Gram-negative bacteremia and urinary tract infections (UTIs), two developed severe bronchopulmonary dysplasia (BPD) and none died. Conclusion: Detection of CMV DNA in saliva occurred in 4% of infants and 5% of sepsis evaluations. Persistence of CMV DNA shedding in saliva made attribution of clinical illness difficult to ascertain.

Human adenovirus has more than 50 serotypes that cause infection in immunocompetent and immunocompromised patients. In patients with aberrant or absent adaptive immunity, adenovirus can cause life-threatening disseminated infection.1Lion T. Adenovirus infections in immunocompetent and immunocompromised patients.Clin Microbiol Rev. 2014; 27: 441-462Crossref PubMed Scopus (474) Google Scholar For example, neonates with disseminated adenovirus infection have mortality rates exceeding 85%.2Ronchi A. Doern C. Brock E. Pugni L. Sanchez P.J. Neonatal adenoviral infection: a seventeen year experience and review of the literature.J Pediatr. 2014; 164 (e1-4): 529-535Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Virus-specific T lymphocytes (VSTs) have been used to treat infections in allogeneic hematopoietic cell transplant recipients3Houghtelin A. Bollard C.M. Virus-specific T cells for the immunocompromised patient.Front Immunol. 2017; 8: 1272Crossref PubMed Scopus (46) Google Scholar and, most recently, in infants with primary immunodeficiencies.4Naik S. Nicholas S.K. Martinez C.A. Leen A.M. Hanley P.J. Gottschalk S.M. et al.Adoptive immunotherapy for primary immunodeficiency disorders with virus-specific T lymphocytes.J Allergy Clin Immunol. 2016; 137: 1498-1505.e1Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar We report the successful isolation and adoptive transfer of maternal adenovirus-specific T lymphocytes enriched by means of cytokine capture to treat disseminated adenovirus infection in a premature infant. Details of the methods and analyses are located in the Methods section and Table E1 in this article's Online Repository at www.jacionline.org. Briefly, maternal haploidentical VSTs were isolated by using the cytokine capture CliniMACS Prodigy Cytokine Capture System (IFN-γ; Miltenyi Biotec, Auburn, Calif) through an Emergency Investigational New Drug (EIND) application, according to the manufacturer's instructions. After VST infusion, the infant had at least weekly chemistries and peripheral blood counts, as well as graft-versus-host disease (GvHD) assessments, to monitor for toxicity. Adenovirus response was monitored by using qualitative and quantitative real-time PCR from plasma, urine, stool, and endotracheal aspirate/nasopharyngeal swabs. Differentiation of maternal and infant's monocytes and lymphocytes in the infant's blood was determined based on HLA immunophenotype. A female infant at 2967 weeks' gestation was born by means of emergency caesarian delivery for breech presentation to a 28-year-old gravida 4, para 1 mother with negative screening results for HIV I/II, hepatitis B and C viruses, syphilis, gonorrhea, chlamydia, and group B Streptococcus species. Pregnancy was complicated by preterm premature rupture of the membranes for 6 days, preterm labor, cigarette smoking, and recreational drug use. Apgar scores at birth were 8 and 9 at 1 and 5 minutes, respectively, and the infant was admitted to the neonatal intensive care unit, receiving nasal continuous positive airway pressure (CPAP) therapy. Birth weight (1480 g, 75%), length (39 cm, 68%), and head circumference (27 cm, 55%) were appropriate for gestational age, and results of physical examination were normal. Ampicillin and gentamicin were administered for 48 hours until blood cultures were sterile. Two days postpartum (day of life [DOL] 2), the mother had bilateral eye drainage and crusting that persisted through DOL 5 and did not respond to topical antimicrobial therapy. She visited the infant and provided skin-to-skin care while providing some maternal milk. The infant also had bilateral yellow eye discharge with mild injected conjunctivae and periorbital edema on DOL 2. Bacterial and chlamydial cultures were negative, and ocular symptoms resolved without antimicrobial therapy. The infant did well without respiratory support until DOL 7, when she experienced marked lethargy with labored breathing, apnea, bradycardia, and desaturation episodes. Nasal CPAP was restarted, a sepsis work-up was performed, and ampicillin and gentamicin were started. Cerebral spinal fluid (CSF) indices were normal, and blood, CSF, and urine bacterial cultures were sterile (see Table E2 in this article's Online Repository at www.jacionline.org). Results of head ultrasonography were normal. Because of persistent lethargy, hypotonia, desaturation episodes, and hypothermia, another sepsis evaluation was performed on DOL 11 to evaluate for viral caused. Antimicrobial therapy was changed to nafcillin, gentamicin, and acyclovir. Blood, CSF, and urine bacterial cultures remained sterile, and results of PCR tests for herpes simplex virus were negative (see Table E2). However, a nasopharyngeal respiratory viral PCR multiplex panel was positive for adenovirus species C. On DOL 13, the infant was transferred to Nationwide Children's Hospital. The infant remained lethargic on CPAP 7 (30% oxygen) therapy, with chest radiography revealing hyperinflated lungs, diffuse bronchial wall thickening, and patchy perihilar and bibasilar opacities. On DOL 14, quantitative plasma adenovirus PCR was more than 2 million DNA copies/mL, and adenovirus DNA was detected by means of qualitative adenovirus PCR in nasal secretions and stool (Table I). The infant also had increased hepatic enzyme levels and thrombocytopenia. She received 1 dose of intravenous immunoglobulin (1 g/kg) and started thrice-weekly intravenous cidofovir (1 mg/kg) with probenecid. High-level adenovirus DNAemia continued, and she had progressive respiratory failure, first requiring mechanical ventilation (DOL 17) and then high-frequency oscillatory ventilation and inhaled nitric oxide (DOL 19). Chest radiography showed worsening bilateral perihilar and lower lung opacities and small bilateral pleural effusions.Table IClinical course, laboratory evaluations, and administered supportive care and antiviral therapiesDOLInterventionsAdenovirus PCR∗Quantitative (plasma) and qualitative (NP, ETT, stool, and urine) PCR results are shown.Laboratory studiesAntiviral therapySupportive carePlasma copies/mL (log10/mL)NP/ETTStoolUrineCr (mg/dL)ALT (U/L)AST (U/L)WBC (× 103/μL)ALCHGB (g/dL)PLT (× 103/μL)LDH (U/L)14IVIG, cidofovir†Intravenous cidofovir (1 mg/kg) was infused over 1 hour thrice weekly starting on DOL 14. Normal saline bolus (10 mL/kg) was administered intravenously over 30 minutes before cidofovir infusion. Starting on DOL 18, enteral probenecid (37.5 mg/kg) was administered through an oral gastric tube 3 hours before cidofovir and at 3 and 9 hours (each 20 mg/kg) after cidofovir.Nasal CPAP>2M (>6.3)PositivePositive—0.549232610.43,12011202—16Cidofovir17Intubated,MV started>2M (>6.3)Positive——0.427742410.93,48812103—18Cidofovir19MV→HFOV, iNO started21Cidofovir>2M (>6.3)Positive—Positive0.504114116.14,6998.9‡Received packed red blood cell transfusion.1093,43522VST infusion23CidofoviriNO stopped—Positive—24545,050 (5.7)Positive—Positive0.41254523.18,5479.6‡Received packed red blood cell transfusion.1741,58925CidofovirHFOV→MV26Extubated→ BiPAP28Cidofovir2916,155 (4.2)PositiveNegative—0.4916193610,4409.649696830Cidofovir———Negative———23.79,7179.0‡Received packed red blood cell transfusion.571—31BiPAP→CPAP1,951 (3.3)———32Cidofovir0.2L O2 NC341,214 (3.1)———0.40212317.99,48711.357080635CidofovirNegative——36424 (2.6)———37Cidofovir43Negative———0.34242612.87,6809.1420—57———0.3229238.35,0637.539962364Negative———0.2736318.86,9527.247280669————0.2836228.26,3146.9‡Received packed red blood cell transfusion.48361276————0.30353610.87,94911.9353697—, No sample acquisition at the indicated time; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BiPAP, bilevel positive airway pressure; Cr, creatinine; Ct, cycle time; ETT, endotracheal tube aspirate; HFOV, high-frequency oscillatory ventilation; HGB, hemoglobin; iNO, inhaled nitric oxide; IVIG, intravenous immunoglobulin; LDH, lactate dehydrogenase; M, million; MV, mechanical ventilation; NC, nasal cannula; NP, nasopharyngeal swab; PLT, platelets; UA, uric acid.∗ Quantitative (plasma) and qualitative (NP, ETT, stool, and urine) PCR results are shown.† Intravenous cidofovir (1 mg/kg) was infused over 1 hour thrice weekly starting on DOL 14. Normal saline bolus (10 mL/kg) was administered intravenously over 30 minutes before cidofovir infusion. Starting on DOL 18, enteral probenecid (37.5 mg/kg) was administered through an oral gastric tube 3 hours before cidofovir and at 3 and 9 hours (each 20 mg/kg) after cidofovir.‡ Received packed red blood cell transfusion. Open table in a new tab —, No sample acquisition at the indicated time; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BiPAP, bilevel positive airway pressure; Cr, creatinine; Ct, cycle time; ETT, endotracheal tube aspirate; HFOV, high-frequency oscillatory ventilation; HGB, hemoglobin; iNO, inhaled nitric oxide; IVIG, intravenous immunoglobulin; LDH, lactate dehydrogenase; M, million; MV, mechanical ventilation; NC, nasal cannula; NP, nasopharyngeal swab; PLT, platelets; UA, uric acid. Given the high mortality associated with neonatal disseminated adenovirus infection and the mother's resolved conjunctivitis presumed to be caused by adenovirus, an EIND application was filed on DOL 17, seeking to infuse maternal haploidentical VSTs enriched through the CliniMACS Cytokine Capture System (IFN-γ). After EIND approval, the infant's mother underwent HLA typing and donor infectious diseases and medical screenings. On DOL 21, the mother underwent 2 blood volume exchange leukapheresis, and her PBMCs were processed for enrichment of haploidentical VSTs by means of cytokine capture (see Table E3 in this article's Online Repository at www.jacionline.org). To this end, the cytokine capture technique yielded 310- and 131-fold enrichment of maternal CD3+CD4+IFN-γ+ and CD3+CD8+IFN-γ+ T cells, respectively. On DOL 22, the infant (1.9 kg) received 2 mL of maternal VSTs containing 1.29 × 105 total CD3+ and 1.00 × 105 CD3+IFN-γ+ cells through slow-push intravenous infusion. Thrice-weekly cidofovir and probenecid were continued. Within 24 hours after VST infusion (DOL 23), inhaled nitric oxide was discontinued. On DOL 24, plasma viral load decreased to 545,050 copies of adenoviral DNA/mL, and hepatic transaminitis and thrombocytopenia resolved (Table I). The infant's absolute lymphocyte counts (ALCs) doubled from a maximum pre-VST infusion ALC of 4,699 (DOL 21) to a maximum post-VST infusion ALC of 10,440 (DOL 29), coinciding with decreases in viral load and detection of maternal HLA-A9+ T cells in the infant's blood (Fig 1, A). T cells of maternal origin peaked 1 week after VST infusion, representing 0.6% and 1.9% of total CD4+ and CD8+ T cells, respectively (Fig 1, B and C). Throughout the follow-up period, CD4/CD8 ratios for infant and maternal T cells in the infant's peripheral blood were age appropriate and differed significantly (Fig 1, D). On DOL 25, the infant was transitioned from high-frequency oscillatory ventilation to conventional mechanical ventilation and ultimately extubated to bilevel positive airway pressure on DOL 26. On DOL 29, plasma adenoviral load decreased to 16,155 DNA copies/mL, and adenovirus was no longer detected in stool and urine. On DOL 31, the infant was transitioned to nasal CPAP at which time plasma viral load was 1,951 adenovirus DNA copies/mL. On DOL 35, results of adenovirus PCR of nasopharyngeal secretions were negative. On DOL 36, 14 days after maternal haploidentical VSTs and 22 days after initiating cidofovir, plasma adenoviral load was less than 500 DNA copies/mL (Table I). Cidofovir was discontinued on DOL 37. The infant did not experience any recurrence in adenovirus detection or infection nor did she manifest any clinical manifestations of GvHD after adoptive VST therapy. On DOL 78, the infant was discharged home on nasal cannula oxygen for bronchopulmonary dysplasia. The child is now 10 months of age and is healthy and developing normally. Her newborn screen, which included SCID screening through T-cell receptor excision circle assessment, revealed the presence of T-cell receptor excision circles and absence of any metabolic disorders. She has not been hospitalized since having recovered from disseminated adenoviral infection, and she has not had any additional immune work-up performed. Viruses can cause disseminated disease in neonates, associating with high infant mortality.2Ronchi A. Doern C. Brock E. Pugni L. Sanchez P.J. Neonatal adenoviral infection: a seventeen year experience and review of the literature.J Pediatr. 2014; 164 (e1-4): 529-535Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Although largely undefined, neonatal susceptibility to viruses likely reflects naive skewing in cellular immune responses to infectious challenge. Specifically, not only do infants lack immune memory, but their lymphocytes also exhibit recent thymic emigrant immunophenotype.5van den Broek T. Borghans J.A.M. van Wijk F. The full spectrum of human naive T cells.Nat Rev Immunol. 2018; 18: 363-373Crossref PubMed Scopus (122) Google Scholar Their naive CD4+ T cells also polarize toward TH2 cytokine responses with decreased production of IFN-γ and TNF-α,6Basha S. Surendran N. Pichichero M. Immune responses in neonates.Expert Rev Clin Immunol. 2014; 10: 1171-1184Crossref PubMed Scopus (261) Google Scholar signature cytokines mediating antiviral immunity. Finally, neonatal tissues possess high proportions of regulatory T cells that can suppress endogenous T-cell activation,7Thome J.J. Bickham K.L. Ohmura Y. Kubota M. Matsuoka N. Gordon C. et al.Early-life compartmentalization of human T cell differentiation and regulatory function in mucosal and lymphoid tissues.Nat Med. 2016; 22: 72-77Crossref PubMed Scopus (202) Google Scholar potentially increasing susceptibility to disseminated viral infection. Immune response to infectious challenge in the premature infant is less well characterized but generally accepted to be less effective than that of full-term infants. Notwithstanding, adjuvant adoptive transfer of maternal VSTs along with concomitant cidofovir resulted in viral clearance and clinical improvement in our premature infant. To our knowledge, this case illustrates the first successful use of haploidentical VSTs to treat maternally transmitted disseminated adenoviral infection in a premature infant and supports investigation into the broader applicability of such rapidly generated antiviral cell therapies beyond the hematopoietic cell transplant setting. We thank the infant's mother, who entrusted us with the care of her infant and acted courageously in making difficult medical decisions on behalf of herself and her child. An EIND application was filed through the US Food and Drug Administration to collect maternal VSTs for infusion into her premature infant with disseminated adenovirus infection. The haploidentical (5/10 HLA-matched) mother underwent 2 blood volume exchange leukapheresis using a Spectra Optia (Terumo BCT, Lakewood, Colo) at Nationwide Children's Hospital, Columbus, Ohio. The unmobilized leukapheresis product was processed at the Cell Therapy Laboratory at the Ohio State University Comprehensive Cancer Center. Informed consent for maternal PBMC collection and VST infusion into the premature infant was obtained after institutional review board approval. Donor PBMCs (1 × 109 total nucleated cells) were processed through the CliniMACS Prodigy Cytokine Capture System (IFN-γ; Miltenyi Biotec). Specifically, the total nucleated cell aliquot was activated by using the Miltenyi Biotec PepTivator-AdV5 Hexon for 4 hours to induce IFN-γ secretion in CD4+ and CD8+ T cells reactive to adenovirus. After incubation, cells were labeled with the CliniMACS IFN-γ Catchmatrix Reagent (consisting of a bispecific antibody conjugate specific for CD45 and IFN-γ and an IFN-γ–specific antibody conjugated to superparamagnetic particles), followed by magnetic enrichment of target cells (IFN-γ+ cells). The resultant cell product was tested for viability, sterility, and cell composition and then resuspended in Plasma-Lyte A at the appropriate dose and volume before transport and intravenous infusion into the infant. Total process time from the end of leukapheresis to infusion of VSTs into the infant was 27 hours. The T-cell threshold to avoid GvHD in the haploidentical donor hematopoietic cell transplant setting is less than 105 CD3+ cells/kg.E1Aversa F. Tabilio A. Velardi A. Cunningham I. Terenzi A. Falzetti F. et al.Treatment of high-risk acute leukemia with T-cell-depleted stem cells from related donors with one fully mismatched HLA haplotype.N Engl J Med. 1998; 339: 1186-1193Crossref PubMed Scopus (1018) Google Scholar Furthermore, haploidentical cytokine capture–enriched VSTs have been administered at cell doses of up to 2.4 × 104 cells/kg in pediatric transplant recipients.E2Feucht J. Opherk K. Lang P. Kayser S. Hartl L. Bethge W. et al.Adoptive T-cell therapy with hexon-specific Th1 cells as a treatment of refractory adenovirus infection after HSCT.Blood. 2015; 125: 1986-1994Crossref PubMed Scopus (105) Google Scholar Therefore the maximum target dose for a single VST infusion was a total CD3+ cell dose (105 cells/kg) and a total IFN-γ+CD3+ cell dose (2.4 × 104/kg). After VST infusion, complete blood counts and renal and hepatic chemistry profiles were sent at least weekly. Quantitative (plasma) and qualitative (nasopharyngeal aspirates, endotracheal tube aspirates, urine, and stool) adenovirus real-time PCR testing (Molecular Laboratory, Nationwide Children's Hospital) and standardized GvHD assessments were also performed at least weekly for 50 days after VST infusion. Low-resolution HLA typing was performed by using standard PCR-based methodology at the Ohio State University Clinical Histocompatibility Laboratory. Peripheral blood was obtained from the infant at weekly intervals for 5 weeks starting 1 week after VST infusion, and each sample was cryopreserved for batch testing. Mouse anti-human HLA-A9 was used to discriminate infant (homozygous HLA-A11) and maternal (HLA-A11 and A23 [A9 split]) cells by using flow cytometry in combination with anti-CD3, CD4, and CD8 antibodies and viability dye (Table E1). Specifically, maternal monocytes were identified as live HLA-A9+ cells within the granular cell gate, as determined by using forward scatter (FSC). Maternal T cells were identified as either live CD3+CD4+/HLA-9A+ or CD3+CD8+/HLA-9A+ cells within the lymphocyte gate, as determined by using FSC. At least 50,000 events were collected per antibody panel and analyzed by using FlowJo single-cell flow cytometry (FlowJo, Ashland, Ore). Maternal (HLA-9A+) cells were quantified, multiplying the infant's ALC measured at each indicated time point by gated percentages of live monocytes and T cells determined initially by means of FSC through flow cytometry, as previously described.Table E1Conjugated mAbs used in flow cytometric analysesAntibody/probeFluorochromeCloneVendorAnti-human CD3Allophycocyanin (APC)-Vio 770REA613Miltenyi BiotechAnti-human CD4Allophycocyanin (APC)REA623Miltenyi BiotechMouse anti-human CD8BB515RPA-T8BD Biosciences (Franklin Lakes, NJ)Anti-human HLA-A9Phycoerythrin (PE)REA127Miltenyi BiotechViabilityGhost Dye Violet 510—Tonbo Bioscience (San Diego, Calif) Open table in a new tab Table E2Diagnostic evaluations performed and antimicrobial therapies administered at an outside hospitalDOL 1DOL 2DOL 8DOL 9DOL 11DOL 12Complete blood count WBC (× 103/μL)16.820.622.217 Segmented neutrophils (%)57797968 Band neutrophils (%)2033Absolute neutrophil count9,91216,27418,20412,070 Lymphocytes (%)24161124ALC4,0323,2962,4424,080 Hemoglobin (g/dL)15.413.612.213 Platelet (× 103/μL)305194288309Inflammatory marker C-reactive protein (mg/L)3.816.8CSF Glucose (mg/dL)6850 Protein (mg/dL)165107 RBCs (cells/μL)2,0952,420 WBCs (cells/μL)414Neutrophils (%)6747Lymphocytes (%)1433Monocytes/macrophages (%)1713Eosinophils (%)11Basophils (%)01Bacterial cultures Eye including Chlamydia speciesNo growth BloodNo growthNo growthNo growth CSFNo growthNo growth UrineNo growthNo growthPCR testing Viral nasopharyngeal panelADV C NP influenza A/BNegative NP respiratory syncytial virusNegative Herpes simplex virusBloodNegativeCSFNegativePooled surface swabsNegativeMeningitis/encephalitis array∗Meningitis/encephalitis array tests for the following pathogens: Cryptococcus gatti/neoformans, Escherichia coli K1, cytomegalovirus, enterovirus, Haemophilus influenzae, human herpesvirus 6, herpes simplex virus 1 and 2, human parechovirus, Listeria monocytogenes, Neisseria meningitides, Streptococcus agalactiae, and Streptococcus pneumoniae. CSFNegativeAntimicrobial therapy Ampicillin, 100 mg/kg every 12 hXXXX Gentamicin, 4 mg/kg dailyXXXXXX Nafcillin, 50 mg/kg every 12 hXX Acyclovir, 20 mg/kg every 8 hXXADV, Adenovirus; NP, nasopharyngeal.∗ Meningitis/encephalitis array tests for the following pathogens: Cryptococcus gatti/neoformans, Escherichia coli K1, cytomegalovirus, enterovirus, Haemophilus influenzae, human herpesvirus 6, herpes simplex virus 1 and 2, human parechovirus, Listeria monocytogenes, Neisseria meningitides, Streptococcus agalactiae, and Streptococcus pneumoniae. Open table in a new tab Table E3Cell processing and product information∗Indicated doses based on patient weight (1.9 kg).PBMC leukapheresisAdenovirus-specific T cellsCell counts Total volume196 mL5.13 mL†A dose of 0.37 mL of final VST product was diluted to 2 mL in Plasma-Lyte A for infusion into the patient. The remaining 5.13 mL was cryopreserved in 4 aliquots. Total nucleated cell count93.79 × 108 cells/kg1.15 × 106 cells/kg Fresh viability (≥90% expected)97%— Volume processed on CliniMACS Prodigy11 mL— Total nucleated cells processed on CliniMACS Prodigy1 × 109 cells—Postprocessing resultsAcceptability criteriaResult IFN-γ+ T-cell purity (%)—≥50% CD3+CD4+ cells90.0% IFN-γ+ T-cell purity (%)—≥50% CD3+CD8+ cells86.4% Fold enrichment, CD3+CD4+IFN-γ+ T cells—≥20-fold310-fold Fold enrichment, CD3+CD8+IFN-γ+ T cells—≥20-fold131-fold Percentage recovery, CD3+CD4+IFN-γ+ T cells—Report only153% Percentage recovery, CD3+CD8+IFN-γ+ T cells—Report only55%Release testing resultsTest methodTarget specificationResult WBC viability (%)—Flow cytometry, 7-AAD≥45%47.2% CD3+IFN-γ+ T-cell viability (%)—Flow cytometry, 7-AAD≥70%91.7% Microbial contamination through Gram staining—Gram stainNo organisms seenNo organisms seen Endotoxin (EU/kg)—Limulus amebocyte lysate≤5.0 EU/kg/h<0.04 EU/kg CD3+IFN-γ+ T-cell dose—Flow cytometry≤2.4 × 104 cells/kg1.00 × 105 CD3+ T-cell dose—Flow cytometry≤1 × 105 cells/kg1.29 × 105Microbial contamination (after release)Test methodTarget specificationResult Aerobic bacteria cultureNo growthAerobic bacteria cultureNo growthNo growth Anaerobic bacteria cultureNo growthAnaerobic bacteria cultureNo growthNo growth7-AAD, 7-Aminoactinomycin D; EU, endotoxin unit.∗ Indicated doses based on patient weight (1.9 kg).† A dose of 0.37 mL of final VST product was diluted to 2 mL in Plasma-Lyte A for infusion into the patient. The remaining 5.13 mL was cryopreserved in 4 aliquots. Open table in a new tab ADV, Adenovirus; NP, nasopharyngeal. 7-AAD, 7-Aminoactinomycin D; EU, endotoxin unit.

Staphylococcus aureus (SA) is a major human pathogen, causing a variety of nosocomial and community-acquired infections. Nasal carriers of SA are at increased risk for healthcare associated infections with this organism. Timely detection of SA and Methicillin-resistant Staphylococcus aureus (MRSA) and decolonization of pre-surgical patients carrying SA are of importance in infection prevention. We sought to evaluate the clinical performance of the Xpert SA Nasal Complete assay (Xpert SA) for detection of SA and MRSA in the nasal specimens from pediatric patients. A total of 504 nasal specimens were collected in the Copan dual swab systems from patients with ages between 0 to 61 years with 91.9% patients ≤21 year-old (n = 463). For each sample, one swab was tested with Xpert SA. The second swab was plated onto Blood agar, Mannitol salt agar and ChromID™ MRSA plate and incubated at 35°C in non-CO2 incubator. The identification of SA and MRSA was compared between Xpert SA and the culture results. Methicillin resistance was determined using conventional methods (susceptibility testing or detection of altered penicillin binding protein). When compared with culture for the identification of SA (n = 481), there was an agreement of 95.0% with sensitivity and specificity being 95.6% and 94.8%, respectively. Among those culture-confirmed and Xpert SA positive samples (n = 131), concordance between Xpert SA and conventional methods for detection of methicillin resistance was 97.0% with sensitivity and specificity being 100% and 96.3%, respectively. Four culture-confirmed methicillin-susceptible SA (MSSA) were identified as MRSA by Xpert SA. Among 504 nasal specimens, 23 (4.6%) samples had invalid or instrument failure results. Nasal swabs collected from pediatric patients (≤21-year-old) had a higher invalid/instrument failure rate (5.0%) than those from adults (0%) (P < 0.001). Xpert SA Nasal Complete assay provides a rapid and sensitive method to detect and differentiate between MSSA and MRSA colonization. The higher invalid rate in pediatric patients and misidentification of MSSA as MRSA by Xpert SA warrant the confirmation by bacterial culture and conventional susceptibility test. A. Leber, Nationwide Children’s Hospital: Research Contractor, Research support.

Detection of CMV by PCR is the preferred method for both diagnosing infection and monitoring therapy. The design of CMV PCR depends on analysis of all available nucleic acid sequences to maximize performance. We describe two patients in whom our in-house CMV PCR was falsely negative (FN) due to two recently emerged mutations in the DNA polymerase gene. In-house CMV PCR targeting a specific 61 bp fragment of the polymerase gene (UL54) has been in use in our lab since 2003. Confirmatory CMV PCR was sent to a reference lab which uses PCR targeting US9 gene. Case 1: 4 months F with familial hemophagocytic lymphohistiocytosis (homozygous PRF1) underwent 10/10 MUD BMT (CMVD+/R−). Plasma CMV was not detected on admission and monitoring was performed weekly. She developed respiratory failure, intubated on D+13 with hemorrhagic respiratory secretions. Repeat PCR of tracheal secretions and plasma detected CMV on D+33, prompting ganciclovir and cytogam. She developed refractory hypoxemia and asystolic cardiac arrest on D+51 (Figure 1a). Case 2: Thirty-two-week F born via C-section for fetal distress noted to have SGA, microcephaly, thrombocytopenia and hyperbilirubinemia at birth, concerning for congenital CMV; urine CMV + (Ct 43.18). Repeat urine and blood PCRs on Day 5 of life were indeterminate. Given initial CMV detection and clinical stigmata, ganciclovir was started. Close analysis in Case 1 of the amplification curve (Figure 1b1) on the 21st sample submitted lead us to sequence the amplicon region and to discover two mutations (C-T) in the probe binding site affecting the sensitivity of UL54 PCR(Figure 1b2). These previous FNs delayed CMV diagnosis and the start of antivirals. For Case 2, the distinct curve was noted on the first sample and was sent for confirmation, resulting in no adverse clinical implications. We subsequently developed a CMV PCR targeting US9 that can detect these mutations. Periodic assessment of all available CMV sequences and close review of amplification curves are essential to prevent FN PCR. With conflicting laboratory and clinical data, clinicians with a high suspicion for CMV should question negatives and if appropriate, ask for PCR using an alternate target. A. Leber, Nationwide Children’s Hospital: Research Contractor, Research support.

Abstract Background Previous studies suggest that RSV increases NP bacterial colonization and may facilitate infection. However, the role of NP colonization with potentially pathogenic bacteria (PPB) in the pathogenesis of RSV bronchiolitis is not well understood. We sought to determine the frequency, type, and density of NP PPB detection in infants with RSV infection compared with healthy controls (HC), and its association with clinical outcomes. Methods Single-center, prospective study of previously healthy infants with RSV infection and age-matched HC. Inpatients (IP) were enrolled within 24 hours of hospitalization, outpatients (OP) at the ED or primary clinics and HC at well-child visits. RSV infection and the following PPB: [S. pneumoniae, M. catarrhalis, H. influenzae, and S. aureus] were detected and quantified by PCR. We compared demographic, clinical characteristics, and outcomes of care according to NP PPB detection. Results From 2010 to 2018, we enrolled 815 infants: 664 with RSV infection [IP, 560; OP, 104] and 151 HC. RSV+ OP (6.1 [3.7–10.7] months) and HC (6.9 [3.8–10.8] months) were older than IP (2.5 [1.4–5.4] months; P < 0.001). Identification of ≥1 PPB was 89% in RSV+ infants [IP, 88%; OP, 90%] versus 63% of HC (P < 0.0001). While H. influenzae or >1 PPB detection was higher in RSV infection (P < 0.001), S. aureus detection predominated in HC (P < 0.05; Figure 1). Frequency of S. pneumoniae detection was comparable between groups; however, S. pneumoniae loads were one log higher in RSV+ infants versus HC (P = 0.001) adjusted for antibiotic use. Differences in colonization rates remained different in RSV+ infants versus HC across age ranges (<3, 3–6, >6–12, and >12–24 months; Figure 2). Last, RSV patients (both IP and OP) with S. pneumoniae or H. influenzae detection had fever more frequently (70%–74% vs. 25%–47%; P < 0.0001), higher clinical disease severity scores (P = 0.01), and higher blood neutrophil counts (34%–36% vs. 16%–19%; P < 0.001), versus those with M. catarrhalis, S. aureus detection or PCR negative. In addition, NP detection of H. influenzae in RSV children was associated with higher frequency of atelectasis/consolidation by chest X-ray (P < 0.005). Conclusion These data suggest that NP colonization with PPB is high in infants with RSV infection independent of age, and that specific bacteria, namely S. pneumoniae and H. influenzae, are associated with enhanced clinical disease severity. Disclosures A. Leber, Nationwide Children’s Hospital: Research Contractor, Research support. O. Ramilo, Janssen Scientific Affairs, LLC: Consultant, Consulting fee. A. Mejias, Janssen: Grant Investigator and Scientific Advisor, Consulting fee and Research grant. Abbvie: CME talks, Speaker honorarium.

EV and HPeV are known to cause febrile illness in young infants. The differences in epidemiologic and clinical features between these infections are not well studied. We analyzed the prevalence, clinical manifestations, virologic data and outcomes of EV and HPeV in infants aged 4–60 days evaluated at Nationwide Children’s Hospital. We retrospectively reviewed EMRs of all infants aged 4–60 days undergoing sepsis evaluation with a positive EV and/or HPeV PCR from any site between January 2015 and September 2016. EV and HPeV were detected by in-house developed Real-time PCR. We analyzed cycle threshold values for positive samples. Of 713 patients tested, 151 (21%) were positive for EV and 77 (11%) were positive for HPeV in at least one site. Median age was 24 days (IQR 16–37) in EV+ group and 29 days (IQR 18–41.5) in the HPeV+ group. The proportion of EV infection increased in early summer to early fall while HPeV infection peaked during mid spring to early fall months. HPeV infection was associated with higher and longer duration of fevers (HPeV and EV maximum T median = 102.2°F and 101.8°F, respectively, P < 0.01); HPeV and EV median fever duration = 2 days and 1 day, respectively, P < 0.01). HPeV patients had lower WBC, absolute neutrophil and lymphocyte counts (HPeV WBC median=5.8, IQR 4.1–7.5; EV WBC median=9.5, IQR 6.8–12.4; P < 0.01). Meningitis was common in both infections: 75 patients had a positive CSF for EV (49.7%) and 32 patients for HPeV (41%), P = 0.26. Patients who were EV+ in CSF had more CSF pleocytosis than those HPeV+ in CSF (median CSF WBC=50 vs 4.5, respectively; P < 0.0001). Unlike EV patients, 43% of HPeV patients with positive CSF testing did not have CSF pleocytosis. PICU admitted patients had lower Ct value in CSF HPeV+ patients (P < 0.01). All patients recovered fully at the time of discharge. EV patients had coinfection in 36% vs. 28.6% in HPeV patients (P = 0.3). There was no significant difference between the groups in length of stay or hospital charges. EV and HPeV had overlapping features in this large study as both were common causes of infection in infants undergoing sepsis evaluation with similar seasonality. Although meningitis was common in both infections, EV patients had more pleocytosis while majority HPeV patients had normal CSF cell counts. O. Ramilo, Abbvie: Board Member, Consulting fee; Regeneron: Board Member, Consulting fee; Janssen: Board Member and Investigator, Consulting fee and Research grant; NIH: Grant Investigator, Research grant; A. Leber, BioFIre Diagnostics: Research Contractor and Scientific Advisor, Research support, Speaker honorarium and Travel expenses

HPeV have been associated with severe disease in young infants. Of the 17 genotypes described, HPeV 1 and 3 have been the most frequently reported. The epidemiology and clinical features associated with different genotypes have not been well defined. We analyzed the prevalence, genotypes, and clinical manifestations and outcomes of HPeV in infants ≤ 60 days evaluated at Nationwide Children’s Hospital, Columbus, Ohio. We retrospectively reviewed EMRs of all infants ≤ 60 days undergoing sepsis evaluation with a positive HPeV PCR from any site between July 2013 and September 2016. All available HPeV CSF, blood, and superficial site specimens were typed by PCR or Sanger sequencing (types assigned per GenBank®). Of 1,265 patients tested, 131 (10%) were positive for HPeV in at least one site, of which 100 had available isolates for genotyping. Median age was 30 days (IQR 19–39), 55% were male. HPeV3 was identified in 87 (87%), HPeV4 in 6, HPeV1 in 5, and HPeV5 and 6 were identified in one infant each. For comparisons we grouped types 1, 4, 5 and 6 into HPeV0 (n = 13). The circulation of HPeV peaked in the months of July to October independent of the type. However, while HPeV0 were identified only in second half of the year, HPeV3 was detected year round. HPeV3 patients had higher temperatures (P < 0.05). There were no significant differences between HPeV3 vs. HPeV0 in age, gender, presenting symptoms, length of stay, PICU admission and CBC. ALT values were higher in HPeV0 patients (P < 0.01). CSF indices were also similar in both groups. Of the positive CSF isolates for HPeV, 43% had no pleocytosis; all CSF isolates typed were HPeV3. HPeV4 was found in blood and superficial sites. HPeV1, 5 and 6 were only found in superficial sites and more commonly with coinfections (enterovirus [EV], rhinovirus, group B streptococcus). There were 4 PICU admissions, 3 of them had HPeV3 and 1 had HPeV1 (patient also had Rhinovirus/EV bronchiolitis). All patients recovered at the time of discharge. HPeV was commonly identified in infants ≤ 60 days undergoing sepsis evaluation. HPeV3 was the most common type in this age group. HPeV4 also caused viremia, while other infrequent types were identified with coinfections. O. Ramilo, Abbvie: Board Member, Consulting fee; Regeneron: Board Member, Consulting fee; Janssen: Board Member and Investigator, Consulting fee and Research grant; NIH: Grant Investigator, Research grant; A. Leber, BioFIre Diagnostics: Research Contractor and Scientific Advisor, Research support, Speaker honorarium and Travel expenses

An outbreak of enterovirus D68 (EV-D68) caused severe respiratory illness in 2014. The disease spectrum of EV-D68 infections in children with underlying medical conditions other than asthma, the role of EV-D68 loads on clinical illness, and the variation of EV-D68 strains within the same institution over time have not been described. We sought to define the association between EV-D68 loads and sequence variation, and the clinical characteristic in hospitalized children at our institution from 2011 to 2014.May through November 2014, and August to September 2011 to 2013, a convenience sample of nasopharyngeal specimens from children with rhinovirus (RV)/EV respiratory infections were tested for EV-D68 by RT-PCR. Clinical data were compared between children with RV/EV-non-EV-D68 and EV-D68 infections, and among children with EV-D68 infections categorized as healthy, asthmatics, and chronic medical conditions. EV-D68 loads were analyzed in relation to disease severity parameters and sequence variability characterized over time.In 2014, 44% (192/438) of samples tested positive for EV-D68 vs. 10% (13/130) in 2011-13 (p<0.0001). PICU admissions (p<0.0001) and non-invasive ventilation (p<0.0001) were more common in children with EV-D68 vs. RV/EV-non-EV-D68 infections. Asthmatic EV-D68+ children, required supplemental oxygen administration (p = 0.03) and PICU admissions (p <0.001) more frequently than healthy children or those with chronic medical conditions; however oxygen duration (p<0.0001), and both PICU and total hospital stay (p<0.01) were greater in children with underlying medical conditions, irrespective of viral burden. By phylogenetic analysis, the 2014 EV-D68 strains clustered into a new sublineage within clade B.This is one of the largest pediatric cohorts described from the EV-D68 outbreak. Irrespective of viral loads, EV-D68 was associated with high morbidity in children with asthma and co-morbidities. While EV-D68 circulated before 2014, the outbreak isolates clustered differently than those from prior years.

Infants with Congenital CMV Infection: Is It Worth Doing the Lumbar Puncture? Christopher Ouellette, MD; Andrea Ronchi, MD; Asuncion Mejias, MD, PhD; Susana Chavez-Bueno, MD; Douglas Salamon, MB(ASCP)SV; Lorenza Pugni; Fabio Mosca; Pablo J. Sanchez MD, FIDSA; Pediatrics, Division of Infectious Diseases, Nationwide Children’s Hospital, Columbus, OH; Pediatrics, University of Texas Southwestern, Dallas, TX; Center for Vaccines and Immunity, The Research Institute at Nationwide Children’s Hospital, Columbus, OH; University of Oklahoma Health Sciences Center, Oklahoma City, OK; Department of Laboratory Medicine, Nationwide Children’s Hospital, Columbus, OH; Neonatal Intensive Care Unit, Department of Clinical Sciences and Community Health, Universita degli Studi di Milano, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milan, Italy

Background. During 9 May 2010-7 May 2011, an outbreak of pertussis-like illness (incidence, 80 cases per 100 000 persons) occurred in Franklin County, Ohio. The majority of cases were identified by IS481-directed polymerase chain reaction (PCR), which does not differentiate among Bordetella species. We sought to determine outbreak etiology and epidemiologic characteristics.Methods. We obtained demographic, clinical, and vaccination-related data from the Ohio Disease Reporting System and Impact Statewide Immunization Information System. We tested sera from 14 patients for anti-pertussis toxin (PT) antibodies and used species-specific PCR on 298 nasopharyngeal specimens.Results. Reported cases totaled 918. IS481 results were available for 10 serologically tested patients; 5 of 10 had discordant anti-PT antibody and IS481 results, suggestive of Bordetella holmesii, which lacks PT and harbors IS481. We identified specific Bordetella species in 164 of 298 specimens tested with multitarget PCR; B. holmesii and Bordetella pertussis were exclusively detected among 48 (29%) and 112 (68%), respectively; both were detected in 4 (2%). Among 48 patients with B. holmesii infections, 63% were aged 11-18 years, compared with 35% of 112 patients with B. pertussis infections (P =.001). Symptoms were similar among B. holmesii- and B. pertussis-infected patients. Adolescent pertussis ("Tdap") booster vaccinations were more effective against B. pertussis than B. holmesii (effectiveness: 67% and 36%, respectively; 95% confidence intervals, 38%-82% and -33% to 69%, respectively).Conclusions. We report the first documented mixed outbreak of B. pertussis and B. holmesii infections. Bordetella holmesii particularly affected adolescents. Although laboratory capacity limitations might inhibit routine use of multitarget PCR for clinical diagnosis, focused testing and enhanced surveillance might improve understanding the burden of B. holmesii infection.

The use of herpes simplex virus (HSV) polymerase chain reaction for diagnosis of HSV disease involving the central nervous system has not translated into widespread use for the detection of DNAemia. We report our 6-year experience using blood polymerase chain reaction testing for HSV infection in neonates and older children with HSV disease. (J Pediatr 2012;161:357-61)