Sepsis is associated with mortality rates as high as 25%

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1 Virl DNAemi nd Immune Suppression in Peditric Sepsis Sm Dvil, MD 1 ; E. Scott Hlsted, MD, PhD 2 ; Mrk W. Hll, MD 3 ; Alln Doctor, MD 1 ; Russell Telford, PhD 4 ; Richrd Holubkov, PhD 4 ; Joseph A. Crcillo, MD 2 ; Gregory A. Storch, MD 1 ; on behlf of the Eunice Kennedy Shriver Collbortive Peditric Criticl Cre Reserch Network Investigtors Objectives: Demonstrte tht DNA viremi is common in peditric sepsis nd quntitte its ssocitions with host immune function nd secondry infection risk. Design: Retrospective nlysis of prospective cohort study. Ptients: Seventy-three children dmitted with sepsis-induced orgn filure. Interventions: None. Mesurements nd Min results: This study ws performed s n ncillry investigtion to single-center prospective 1 Wshington University in St. Louis, St. Louis, MO. 2 University of Pittsburgh, Pittsburgh, PA. 3 Ntionwide Children s Hospitl, Columbus, OH. 4 University of Uth, Slt Lke City, UT. Supported, in prt, by the Wshington University Institute of Clinicl nd Trnsltionl Science grnt UL1TR from the Ntionl Center for Advncing Trnsltionl Sciences; by R01GM (to Dr. Crcillo) from the Ntionl Institute of Generl Medicl Sciences; nd by the following coopertive greements from the Eunice Kennedy Shriver Ntionl Institute of Child Helth nd Humn Development, Ntionl Institutes of Helth, Deprtment of Helth nd Humn Services: U10HD049983, U10HD050096, U10HD049981, U10HD063108, U10HD063106, U10HD063114, U10HD050012, nd U01HD The content is solely the responsibility of the uthors nd does not necessrily represent the officil views of the Ntionl Institutes of Helth. Dr. Dvil received support for rticle reserch from the Ntionl Institutes of Helth (NIH). Dr. Doctor received support for rticle reserch from the NIH, nd his institution received funding from the NIH, Deprtment of Defense, nd Children s Discovery Institute. Dr. Telford received support for rticle reserch from the NIH, nd his institution received funding from the NIH. Dr. Holubkov received support for rticle reserch from the NIH, nd disclosed tht he is Dt Sfety nd Monitoring Bord (DSMB) member for Fibrocell nd Armron Bio; his institution received funding from the NIH/Ntionl Institute of Child Helth nd Humn Development (NICHD) nd he received funding from Pfizer (DSMB Member), St. Jude Medicl (pst; biosttisticl consultnt), nd Medimmune (DSMB). Dr. Crcillo received support for rticle reserch from the NIH, nd his institution received funding from NICHD nd Ntionl Institute of Generl Medicl Services. The remining uthors hve disclosed tht they do not hve ny potentil conflicts of interest. For informtion regrding this rticle, E-mil: smuel.dvil@utsouthwestern.edu Copyright 2017 by the Society of Criticl Cre Medicine nd the World Federtion of Peditric Intensive nd Criticl Cre Societies DOI: /PCC study of children with severe sepsis. Longitudinlly collected, btched, frozen plsm ws exmined using rel time polymerse chin rection for the presence of cytomeglovirus, Epstein-Brr virus, herpes simplex virus, humn herpes virus- 6, torque teno virus, nd denovirus DNA. Innte immune function ws lso mesured longitudinlly vi quntifiction of ex vivo lipopolyscchride -induced tumor necrosis fctor-α production cpcity. Virl DNAemi with virus other thn torque teno virus ws detected in 28 of 73 subjects (38%) nd included cytomeglovirus 5%, Epstein-Brr virus 11%, herpes simplex virus 4%, humn herpes virus-6 8%, nd denovirus 26%. In ddition, torque teno virus ws detected in 89%. Epstein-Brr virus DNAemi ws ssocited with preexisting immune suppression (p = 0.007) Virl DNAemi ws ssocited with preexisting immune suppression nd high risk for the subsequent development of secondry infection (p < 0.05 for both). Subjects with virl DNAemi hd lower innte immune function over time compred with those who were virus negtive (p < 0.05). Conclusions: DNAemi from multiple viruses cn be detected in septic children nd is strongly ssocited with preexisting immune suppression nd secondry infection risk. The role of DNA viruses in the perpetution of impired host defense in this setting should be the subject of prospective study. (Peditr Crit Cre Med 2018; 19:e14 e22) Key Words: immunology; immunosuppression; inflmmtion; peditrics; sepsis; viruses Sepsis is ssocited with mortlity rtes s high s 25% in peditric ptients (1). For decdes, evidence-bsed tretment of sepsis hs been limited to ntimicrobils, supportive cre, nd source control. Filure of novel therpeutics my reflect limited understnding of the host response to sepsis. Improved understnding of this response is necessry in order to develop new pproches to peditric sepsis mngement in hopes of improving outcomes. e14 Jnury 2018 Volume 19 Number 1

2 Online Clinicl Investigtions Recent dult dt suggest tht rectivtion of ltent viruses such s cytomeglovirus occurs frequently in the setting of severe sepsis nd tht the degree of rectivtion is ssocited with the risk for dverse outcomes (2 5). This phenomenon hs not been demonstrted in septic children. In ddition, filure of the host immune system is incresingly recognized s n importnt feture of severe sepsis in dults nd children (6 9). This secondry immune suppression cn ffect both the dptive (lymphocyte) nd innte (monocyte, neutrophil) rms of the immune system. Severe lymphopeni in the context of sepsis hs been ssocited with dverse outcomes in criticlly ill children nd dults (10, 11). In ddition, we hve previously demonstrted tht innte immune suppression, chrcterized by reduced bility of ptients whole blood to produce the proinflmmtory cytokine tumor necrosis fctor (TNF) α upon ex vivo stimultion with lipopolyscchride, is ssocited with incresed risks of secondry infection nd/ or deth cross vriety of criticl dignoses including sepsis (9, 12, 13). The reltionships between sepsis-ssocited immune suppression nd virl rectivtion re unknown in ny ptient popultion. We therefore designed this observtionl study to test the hypotheses tht virl DNA cn be detected in the plsm of criticlly ill children with severe sepsis nd tht this virl DNAemi is ssocited with biomrkers of immune suppression in this popultion. MATERIALS AND METHODS This study represents n ncillry investigtion to single-center prospective study of children with severe sepsis in the PICU t the University of Pittsburgh Medicl Center. The prent study, crried out s pilot study for the Collbortive Peditric Criticl Cre Reserch Network (CPCCRN), included longitudinl mesurement of immune function in enrolled subjects. Stored plsm from these subjects ws used for virl DNA nlysis in this study. The University of Pittsburgh Institutionl Review Bord pproved this study. Ptients meeting the consensus definition of severe sepsis (Systemic Inflmmtory Response Syndrome + suspicion of infection + one or more orgn filures) (14) nd hving n indwelling rteril or centrl venous line for blood drws were eligible for prticiption. Those whose fmilies provided informed consent within 24 hours of the dignosis of sepsis were entered into the study. Non-English speking ptients nd ptients in whom ggressive therpy ws not sought were excluded. Ptients were included in the present study if they hd one or more plsm smples with t lest 100 µl of plsm remining from the prent study. Immune function mesurements nd plsm smples were obtined upon study enrollment nd then twice weekly until ICU dischrge nd stored t 80 C. Demogrphic fetures, results of routine lbortory studies, nd selected clinicl findings were recorded. Vribles of interest included ge, sex, preexisting immune suppression, use of extrcorporel therpies, lymphopeni, neutropeni, nd presence of virl DNAemi. Preexisting immune suppression ws defined s the use of immunosuppressive therpy, history of cncer, or history of trnsplnttion. Lymphopeni ws defined s n bsolute lymphocyte count less thn 1,000 cells/mm 3 for greter thn 48 hours. Neutropeni ws defined s n bsolute neutrophil count less thn 1,000 cells/mm 3 for greter thn 48 hours. Virl DNAemi ws defined s positive polymerse chin rection (PCR) ssy for virus other thn torque teno virus (TTV), s performed in our lbortory, on t lest two of three replictes for ny smple during n episode. TTV DNAemi ws excluded from this definition due to its high prevlence in the helthy popultion. Totl nucleic cid ws extrcted from the thwed plsm using the NuclieSENS esymg utomted nucleic cid extrctor (Biomerieux, Durhm, NC). Lbortory-developed PCR ssys for the following viruses were performed on nucleic cid extrcted from frozen bnked plsm smples: cytomeglovirus (15), Epstein-Brr virus (EBV) (5), herpes simplex virus (5), humn herpes virus-6 (5), TTV (16), nd denovirus (17, 18). Presence of TTV nd level of TTV virl lod were included s seprte vribles. Ptients with preexisting dignoses of virl DNAemi were excluded from nlysis to void including subjects with primry infections. Innte immune function ws quntified t the time of the prent study performnce by mesurement of whole blood ex vivo lipopolyscchride-induced TNFα production cpcity s previously described (12). Briefly, within 60 minutes of smple collection, 50 µl of whole blood ws dded to 500 µl of highly stndrdized stimultion solution contining 500 pg/ml of lipopolyscchride (kits produced by the Immune Surveillnce Lbortory t The Reserch Institute t Ntionwide Children s Hospitl, Columbus, OH) nd incubted for 4 hours t 37 C. The superntnts were then collected nd stored t 80 C for btch nlysis of TNFα. Stimultion ssys were performed in duplicte for ech blood smple, nd TNFα vlues were verged from ech set of duplictes. Stimultion solution, which ws shipped monthly, ws mnufctured nd qulity controlled such tht the intrbtch coefficient of vrition for TNFα production from helthy donor replictes ws determined to be less thn 10% prior to ech shipment. Immune prlysis ws defined, for the purposes of these nlyses, s ex vivo lipopolyscchride-induced TNFα production cpcity less thn 200 pg/ml beyond dy 3 of sepsis s previously described (9). TNFα production cpcity vlues below this threshold hve been ssocited with dverse outcomes from peditric criticl illness in prior single- nd multicenter studies (9, 12, 19). TNFα production cpcity ws lso evluted over time s continuous vrible. Abstrcted clinicl nd lbortory dt were sent to the CPCCRN Dt Coordinting Center for nlysis. In ddition to virl DNAemi, vribles of interest included the development of immune prlysis nd secondry infection. Secondry infection ws defined s cliniclly indicted positive microbiologicl test, culture, or PCR, tht identified new bcteril, fungl, or virl infection fter 48 hours following hospitl dmission. Univrite nlysis of risk fctors ws performed using the Fisher exct test. Those risk fctors with p vlue of less thn 0.1 Peditric Criticl Cre Medicine e15

3 Dvil et l were included in multiple logistic regression models which were fit in order to djust for the effects of covrites ssocited with outcomes of interest. The presence of virl DNAemi ws forced into the models, nd the remining cndidte vribles were entered in stepwise fshion. Adjusted odds rtios (ORs) with 95% CI re shown. A multiple liner regression model ws used to evlute the reltionship between virl DNAemi nd durtion of sty in the PICU. Covrites included ge, gender, nd initil severity of illness t the time of sepsis onset s mesured by Peditric Risk of Mortlity (PRISM) III score (20). Probbility curves were lso generted to exmine time to virl DNAemi for subgroups. Episodes were censored by the dte of lst mesurement. The Wilcoxon rnk-sum test ws used to compre mximum TTV mesurements ginst preexisting immune suppression. TNFα production cpcity (innte immune function) ws lso nlyzed s continuous vrible, with reltionships between TNFα response nd virl DNAemi over time evluted by generlized estimting equtions to ccount for repeting mesures within subjects. Dt re shown s medin nd interqurtile rnge throughout. SAS sttisticl softwre, version 9.4 (Cry, NC) ws used for nlysis. RESULTS One-hundred consecutive ptient episodes meeting the inclusion criteri were enrolled in the originl study, of which 75 hd sufficient plsm vilble to prticipte in the current study. For subjects with multiple septic episodes (n = 2), only the first episode ws used for these nlyses, leving finl smple size of 73 subjects. Of these subjects, 69 (95%) hd TNFα production cpcity dt vilble tht were concurrent with their virl PCR dt. We compred the demogrphics of subjects in the primry study who did (n = 73) nd did not (n = 25) hve smples vilble for this secondry nlysis. We found no significnt differences in ge, gender, cncer or trnsplnt sttus, initil severity of illness (PRISM III score), frequency of immunoprlysis, or lymphopeni between groups (p > 0.05 for ll, dt not shown). Serious underlying diseses were common in the cohort; 21 episodes occurred in subjects who hd received trnsplnt (including three with cncer who hd received hemtopoietic stem cell trnsplnt), nd nine occurred in children who were dignosed with cncer but hd not undergone trnsplnt. A vriety of other underlying dignoses were noted including chronic lung disese, premturity, mitochondril disorders, epilepsy, hydrocephlus, sttic encephlopthy, utoimmune hemolytic nemi, short gut syndrome, nephrotic syndrome, Hirshprung disese, chronic liver filure, nd trisomy 21. Virl PCR testing ws performed on 191 smples (1 9 per subject), nd virl DNAemi ws documented in 28 of the 73 subjects (38%). Demogrphic nd clinicl fetures illustrted in Tble 1 did not differ significntly between subjects with nd without virl DNAemi TABLE 1. The Chrcteristics of Septic Subjects With nd Without Virl DNAemi (Excluding Torque Teno Virus) Chrcteristics Virl DNAemi (n = 28), n (%) No Virl DNAemi (n = 45), n (%) OR (Exct 95% CI) p Age group (yr) (57) 26 (58) 0.97 ( ) (21) 8 (18) 1.26 ( ) 12 6 (21) 11 (24) 0.84 ( ) Femle gender 13 (46) 20 (44) 1.08 ( ) Underlying disese Cncer 4 (14) 5 (11) 1.08 ( ) Trnsplnt 10 (36) 9 (20) 1.94 ( ) No cncer or trnsplnt 14 (50) 31 (69) 0.45 ( ) Preexisting immune suppression b 21 (75) 19 (42) 4.11 ( ) Plsm exchnge 6 (21) 4 (9) 2.80 ( ) Immune prlysis c 11 (39) 10 (22) 2.26 ( ) Lbortory findings Lymphopeni d 10 (36) 17 (38) 0.92 ( ) Neutropeni e 7 (25) 6 (13) 2.17 ( ) OR = odds rtio. Fisher exct test. b Bsed on underlying condition nd/or receipt of immune suppressnt therpy. c Immune prlysis is defined s n ex vivo lipopolyscchride-induced tumor necrosis fctor-α production cpcity < 200 pg/ml beyond 3 d of sepsis. d Lymphopeni is defined s n bsolute lymphocyte count < 1,000 cells/mm 3 for > 48 hr. e Neutropeni is defined s n bsolute lymphocyte count < 1,000 cells/mm 3 for > 48 hr. e16 Jnury 2018 Volume 19 Number 1

4 except for preexisting immune suppression. Preexisting immune suppression ws present in 40 subjects, of whom 21 (53%) hd virl DNAemi. In comprison, of the 33 subjects without preexisting immune suppression, only seven (21%) hd virl DNAemi (p = 0.008). The time to virl DNAemi in septic ptients with preexisting immune suppression ws significntly shorter compred with those without preexisting immune suppression (Fig. 1A) (p = ). Subjects tht left Online Clinicl Investigtions the ICU without virl DNAemi were censored nd no longer considered t risk; the number t risk is shown. In 19 subjects, virl DNAemi ws present in smples collected on study dy 1, wheres nine subjects becme virus positive lter in their sepsis course (medin of 4 d). The detection of individul viruses in subjects with nd without preexisting immune suppression is shown in Tble 2. The presence of preexisting immune suppression ws significntly ssocited with EBV DNAemi (p = 0.007) nd trend towrd being positive for more thn one virus, excluding TTV (p = ). The most commonly detected virus, excluding TTV, ws denovirus which ws present in 13 (33%) of subjects with preexisting immune suppression nd six (18%) of subjects without preexisting immune suppression. There were no significnt differences in ge, gender, rtes of preexisting immune suppression, rtes of lymphopeni, or neutropeni between episodes with nd without denovirus. In only one of the 13 subjects with preexisting immune suppression (nd none of the six without) ws the denovirus dignosed by the cre tem. More thn one virus (excluding TTV) ws identified in eight subjects, including seven with preexisting immune suppression. TTV ws detected in 89% of subjects with no difference in frequency between those with nd without preexisting immune suppression. The medin mximum TTV level, however, ws higher in those with preexisting immune suppression compred with those without preexisting immune suppression (532,331 [ ] vs 5,767 [ ]; p = 0.04). Figure 1. The time to virl DNAemi ws shorter in those with preexisting immune suppression (A). The time to virl DNAemi ws shorter in those who developed secondry infection (B). In these Kpln-Meier plots, subjects re censored t the time of deth or ICU dischrge. The lines represent the probbility of virl DNAemi in the popultion of subjects who remin t risk t given time point. The number of ptients t risk is shown. Sec. = secondry. Fctors ssocited with the development of immune prlysis re Peditric Criticl Cre Medicine e17

5 Dvil et l TABLE 2. Detection of Virl DNAemi in Septic Subjects With nd Without Preexisting Immune Suppression Preexisting Immune Suppression Virus Not Present (n = 33), n (%) Present (n = 40), n (%) p Overll (n = 73), n (%) Cytomeglovirus 0 (0) 4 (10) (5) Epstein-Brr virus 0 (0) 8 (20) (11) Humn herpes virus-6 1 (3) 5 (13) (8) Herpes simplex virus 1 (3) 2 (5) 1 3 (4) Adenovirus 6 (18) 13 (33) (26) TTV positive 30 (91) 35 (88) (89) TTV virl lod upper qurtile 4 (12) 11 (28) (21) Two or more viruses detected (excluding TTV) 1 (3) 7 (18) (11) TTV = torque teno virus. TABLE 3. Univrite nd Multivrite Anlyses of Fctors Associted With Immune Prlysis Immune Prlysis (n = 21), n (%) No Immune Prlysis (n = 52), n (%) OR (Exct 95% CI) b p Adjusted OR (95% CI) χ 2 p Virl DNAemi (excluding TTV) 11 (52) 17 (33) 2.26 ( ) ( ) Age group (yr) (43) 33 (63) 0.43 ( ) (29) 8 (15) 2.20 ( ) 12 6 (29) 11 (21) 1.49 ( ) Femle gender 9 (43) 24 (46) 0.88 ( ) Primry infection Virl 4 (19) 11 (21) 0.88 ( ) Bcteril 14 (67) 32 (62) 1.25 ( ) Fungl 1 (5) 4 (8) 0.60 ( ) Preexisting immune suppression 15 (71) 25 (48) 2.70 ( ) Plsm exchnge 4 (19) 6 (12) 1.80 ( ) TTV positive 18 (86) 47 (90) 0.64 ( ) TTV virl lod upper qurtile 8 (38) 7 (13) 3.96 ( ) Extrcorporel membrne oxygention or dilysis Lbortory findings 6 (29) 9 (17) 1.91 ( ) Lymphopeni 15 (71) 12 (23) 8.33 ( ) < ( ) < Neutropeni 7 (33) 6 (12) 3.83 ( ) OR = odds rtio, TTV = torque teno virus. Immune prlysis is defined s n ex vivo lipopolyscchride-induced tumor necrosis fctor-α production cpcity < 200 pg/ml beyond 3 dy of sepsis. b Fisher exct test. depicted in Tble 3. In univrite nlysis, significnt fctors included upper qurtile TTV virl lod (p = 0.027), lymphopeni (p < 0.001), nd neutropeni (p = 0.042). The finl multivrible logistic regression model for immune prlysis ws significnt only for lymphopeni (OR, 9.8; 95% CI, 2.9 3; p < 0.001) lthough there ws trend towrd n ssocition e18 Jnury 2018 Volume 19 Number 1

6 with virl DNAemi (OR, 3.1; 95% CI, ; p = 0.07). The time to virl DNAemi did not differ significntly in those with nd without immune prlysis (p = 0.27). When TNFα production cpcity ws nlyzed s continuous vrible, innte immune function ws significntly lower over time in children who demonstrted virl DNAemi (excluding TTV) compred with those who were never virus positive (p = 0.026) (Fig. 2). There ws no difference in TNFα production cpcity between these groups on dy 1 of illness (498 [50 1,492] vs 661 [333 1,283] pg/ml; p = 0.37) despite the fct tht 75% of virus-positive subjects with vilble TNFα response dt were lredy virus positive in dy 1 smples. Fctors ssocited with secondry infections re summrized in Tble 4. In univrite nlysis, sttisticlly significnt fctors included virl DNAemi (p = 0.004), femle gender (p = 0.02), nd use of n extrcorporel device (extrcorporel membrne oxygention or renl replcement therpy) (p = 0.043). The finl multivrible logistic regression model for secondry infections included virl DNAemi (odds rtio [OR], 5.5; 95% CI, ; p = 0.003) nd femle gender (OR, 0.24; 95% CI, ; p = 0.012). Thus, while femle gender ws protective, the presence of virl DNAemi ws ssocited with development of secondry infection even when djusting for gender. The time to virl DNAemi, excluding TTV, ws shorter in ptients who developed secondry infection (p = 0.034) (Fig. 1B). Subjects tht left the ICU without virl Figure 2. Children who demonstrted virl DNAemi t ny time (excluding torque teno virus) hd lower innte immune function over time s evidenced by persistently reduced tumor necrosis fctor (TNF) α production cpcity (p = 0.026). Previous studies using this method hve demonstrted TNFα production cpcities of pproximtely 1,000 pg/ml in helthy children (12, 13). Symbols represent medins, error brs represent interqurtile rnges. The numbers of smples vilble t ech time point re shown. Online Clinicl Investigtions DNAemi were censored nd no longer considered t risk; the number t risk is shown. The mortlity for the cohort s whole ws 11% (8/73) with five deths occurring in subjects with virl DNAemi nd three deths occurring in subjects without (p = 0.25). Durtion of ICU sty, however, ws longer in children with virl DNAemi (15 [7 22] vs 8 [4 14] d; p = 0.028). This reltionship remined significnt fter djusting for ge, gender, nd initil PRISM III score (p = for virl DNAemi). DISCUSSION This study demonstrtes tht virl DNAemi is common in criticlly ill children with severe sepsis. Further, the dt suggest n ssocition between virl DNAemi, immune suppression, nd susceptibility to secondry infection in this popultion. Children with preexisting immune suppression were t the gretest risk for virl DNAemi in our cohort, nd children with virl DNAemi demonstrted lower innte immune function over time, s mesured by TNFα production cpcity, compred with children without virl DNAemi. Although lymphopeni ws ssocited with immune prlysis s hs been previously reported (10, 11), lymphopeni ws not ssocited with either virl DNAemi or secondry infection risk in this cohort. Virl DNAemi, however, ws strongly ssocited with secondry infection risk in the multivrite logistic regression model. Our findings rise the possibility tht virl DNAemi, cquired either through new infection or rectivtion of ltent infection, my perpetute host immune suppression nd contribute to secondry infection risk. The phenomenon of sepsisinduced immune suppression is chrcterized by dysfunction of both the innte nd dptive rms of the immune system (21). Apoptosis of peripherl nd splenic lymphocytes (10, 11, 22) often occurs longside decresed monocyte humnleukocyte ntigen-ntigen D relted expression nd reduced whole blood ex vivo lipopolyscchride-induced TNFα production cpcity (9, 21, 23). These fetures, if severe nd persistent, hve been found in multiple dult nd peditric studies of severe sepsis to be ssocited with the subsequent development of secondry infection nd deth (6, 7, 9, 10, 12, 24). Neither immune prlysis, s defined by TNFα production cpcity below threshold of 200 pg/ml beyond dy 3 of sepsis, nor lymphopeni were ssocited with Peditric Criticl Cre Medicine e19

7 Dvil et l TABLE 4. Univrite nd Multivrite Anlyses of Fctors Associted With Secondry Infection Secondry Infection (n = 31), n (%) No Secondry Infection (n = 42), n (%) OR (Exct 95% CI) p Adjusted OR (95% CI) χ 2 p Virl DNAemi (excluding TTV) 18 (58) 10 (24) 4.43 ( ) ( ) Age group (yr) (58) 24 (57) 1.04 ( ) (13) 10 (24) 0.47 ( ) 12 9 (29) 8 (19) 1.74 ( ) Femle gender 9 (29) 24 (57) 0.31 ( ) ( ) Primry infection Virl 9 (29) 6 (14) 2.45 ( ) Bcteril 20 (65) 26 (62) 1.12 ( ) Fungl 3 (10) 2 (5) 2.14 ( ) Preexisting immune suppression 18 (58) 22 (52) 1.26 ( ) Plsm exchnge 7 (23) 3 (7) 3.79 ( ) TTV positive 29 (94) 36 (86) 2.42 ( ) TTV virl lod upper qurtile 9 (29) 6 (14) 2.45 ( ) Extrcorporel membrne oxygention or dilysis Lbortory findings 10 (32) 5 (12) 3.52 ( ) Lymphopeni 11 (35) 16 (38) 0.89 ( ) Neutropeni 6 (19) 7 (17) 1.20 (0.29, 4.74) OR = odds rtio, TTV = torque teno virus. Fisher exct test. virl DNAemi or secondry infection risk in this cohort. When the TNFα response ws evluted s continuous vrible, however, children with virl DNAemi demonstrted filure to recover innte immune function over time compred with children without virl DNAemi. It is possible, therefore, tht virl DNAemi nd/or fctors tht promote its presence my perpetute innte immune suppression in wy tht plces the host t risk for nosocomil infection despite TNFα response greter thn 200 pg/ ml. The concept of virl DNAemi s predictor of immune system filure nd infection risk should be the subject of further study in lrger, multicenter cohort. Wlton recently demonstrted tht rectivtion of ltent viruses ws linked to high rtes of secondry infections nd mortlity in dult septic ptients (5). Our dt re the first to demonstrte this phenomenon in septic children. Of the viruses tht were detected in our study, cytomeglovirus nd EBV were detected only in subjects with preexisting immune suppression; however, the other four viruses tested were detected in ptients with nd without known immune suppression. EBV nd cytomeglovirus DNAemi hve been well described in the immune suppressed popultion nd our findings re consistent with this literture. Interestingly, denovirus ws very commonly detected. Adenovirus is recognized s cuse of severe infection in immunosuppressed children (25) nd ws recently shown to be common cuse of fever of unknown source in nonimmunosuppressed children 2 36 months old (26). It is lso known tht denovirus cn exist s ltent infection with the potentil for subsequent rectivtion, prticulrly in the setting of infection with species C (27). The cre tem only dignosed one episode of denovirus, underlining the potentil need for enhnced surveillnce to dignose the burden of denovirus infection in criticl illness. Becuse there re possible therpies for serious denovirus infections (28), it will be importnt to follow up on this observtion in future studies. TTV is n nellovirus tht is not currently linked to ny humn disese, lthough its genome cn be detected in the blood of s mny s 60% of helthy people (5). Interestingly, while TTV ws present in 89% of our subjects, high levels of the virus were ssocited with preexisting immune suppression. Though not significnt in multivrible nlyses, the strong ssocition of high TTV virl lod with immune prlysis in univrite nlyses is intriguing nd is deserving of future study in lrger cohort, s evidence in dults suggests tht TTV my serve s biomrker of immune suppression fter trnsplnttion (29, 30). e20 Jnury 2018 Volume 19 Number 1

8 Online Clinicl Investigtions There re severl limittions to this study. First, despite being the lrgest study of its kind in children, the smple size is smll nd this cohort my not be representtive of the lrger popultion of criticlly ill children. The subjects in the current study my hve different rtes of virl DNAemi or immune dysfunction compred with broder popultion of criticlly ill children. No sttisticlly significnt conclusions could be drwn s to whether specific type of primry infection (bcteril, virl, or fungl) ws ssocited with virl DNAemi. Further, this study does not define mechnism by which virl DNAemi occurs. The lbortory methods employed in this study do not estblish whether episodes of DNAemi represented new, primry virl infections or rectivtion of ltent virus. Becuse nucleic cids were extrcted from plsm rther thn from whole blood, viruses ltent within WBCs were not evluted. The reltionships between virl DNAemi, nosocomil infection, nd immune function, however, strongly suggest potentilly importnt reltionship between virl DNAemi nd impirment of the host immune response. Finlly, this study does not estblish cuse nd effect reltionships between virl DNAemi, immune function, nd outcomes. Rther, it highlights novel ssocitions tht should be the subject of prospective study. CONCLUSIONS In conclusion, virl DNAemi ws common in children with severe sepsis nd ws most strongly ssocited with preexisting immune suppression. There were lso strong ssocitions of virl DNAemi with secondry infection risk nd lower innte immune function over time. Cusl reltionships between virl DNAemi nd immune dysfunction in the setting of peditric severe sepsis, long with the mesurement of virl DNAemi s potentil mrker of immune suppression, should be investigted in lrger multicenter trils. An understnding of this biology my help inform the identifiction of therpeutic trgets nd determine whether tretment of these DNA viruses cn improve outcomes in septic children. ACKNOWLEDGMENTS We wish to cknowledge the technicl ssistnce of Mri Cnnell, Shiel Mson, Andrew Wlton, Jennifer Jones, RN, nd Luther Springs. Eunice Kennedy Shriver Ntionl Institute of Child Helth nd Humn Development Collbortive Peditric Criticl Cre Reserch Network investigtors: Jeri Burr MS, RN-BC, CCRN, University of Uth nd Deprtment of Peditrics; Alln Doctor, MD, St. Louis Children s Hospitl nd Wshington University Deprtment of Peditrics; Joseph A. Crcillo, MD, Children s Hospitl of Pittsburgh Deprtment of Criticl Cre Medicine; Christopher J. L. Newth, MD, FRCPC, Children s Hospitl Los Angeles nd University of Southern Cliforni Deprtment of Peditrics; Dvid L. Wessel, MD, Children s Ntionl Medicl Center nd George Wshington University Deprtment of Peditrics; Kthleen L. Meert, MD, Children s Hospitl of Michign nd Wyne Stte University Deprtment of Peditrics; J. Michel Den, MD, University of Uth nd Deprtment of Peditrics; Murry M. Pollck, MD, Phoenix Children s Hospitl; Robert A. Berg, MD, Children s Hospitl of Phildelphi nd University of Pennsylvni Deprtment of Anesthesiology; Thoms Shnley, MD, C. S. Mott Children s Hospitl nd University of Michign Deprtment of Peditrics; Rick Hrrison, MD, Mttel Children s Hospitl nd University of Cliforni Los Angeles Deprtment of Peditrics; Richrd Holubkov, PhD, University of Uth nd Deprtment of Peditrics; Mrk W. Hll, MD, Ntionwide Children s Hospitl, The Ohio Stte University College of Medicine Deprtment of Peditrics; Tmmr L. Jenkins, MSN RN, PCNS-BC, Robert F. Tmburro MD, the Eunice Kennedy Shriver Ntionl Institute of Child Helth nd Humn Development. REFERENCES 1. 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