We engineered DMAb-ZK190, encoding the mAb ZK190 neutralizing antibody, which targets the ZIKV E protein DIII domain. In vivo-delivered DMAb-ZK190 achieved expression levels persisting >10 weeks in mice and >3 weeks in non-human primate (NHPs), which is protective against ZIKV infectious challenge. This study is the first demonstration of infectious disease control in NHPs following in vivo delivery of a nucleic acid-encoded antibody, supporting the importance of this new platform.Zika virus (ZIKV) infection is endemic to several world regions, and many others are at high risk for seasonal outbreaks. Synthetic DNA-encoded monoclonal antibody (DMAb) is an approach that enables  Esquivel et al. deliver DNA-encoded ZK190 antibody (DMAb-ZK190) by intramuscular administration. DMAb-ZK190 targets the E protein of Zika virus, and it controls Zika virus infection in mice and non-human primates when delivered prior to infection, offering an alternative strategy for rapid protection. Zika virus (ZIKV) is a mosquito-borne infection that has become an important global public health concern, with over 2 billion people at risk. ZIKV infection carries significant risks during pregnancy resulting in severe developmental defects in newborns, including microcephaly and severe cognitive impairment. Guillain-Barré syndrome and other neurological symptoms have also been observed in a subset of infected individuals.In vivo delivery of synthetic nucleic acid expression vectors encoding engineered mAb genes represents a possible alternative novel approach, with great potential to alleviate the critical challenges with recombinant mAb biologics.Individuals who recover from infection develop ZIKV-specific, protective antibodies, and passive transfer of sera from naturally infected or vaccinated individuals protects mice against lethal ZIKV infection.in vivo to mice and rhesus macaques through intramuscular (IM) administration facilitated by adaptive constant current electroporation (CELLECTRA), resulting in in vivo immunoglobulin (Ig) production and secretion of functional mAb in circulation for several months. When animals were challenged, these engineered DMAbs provided rapid protection against ZIKV infection and pathogenesis first in mice, protecting from infection and preventing damage in the immune-privileged testes. Engineered DMAb-ZK190 was also expressed in NHPs and protected rhesus macaques against ZIKV strain PRVABC59 infection, displaying a dramatic protection against viral load. To our knowledge, this is the first demonstration of in vivo expression and prevention of infection with a nucleic acid-encoded antibody in a NHP model. Taken together, the data support further study of DMAb delivery for the prevention of ZIKV and other infectious diseases.We engineered synthetic plasmid DNA-encoding mAb (DMAb) cassettes expressing the potent anti-ZIKV mAb ZK190 (DMAb-ZK190), a clone that binds uniquely to the ZIKV E antigen and is protective in mice,mAb clone ZK190 was isolated from human peripheral blood mononuclear cells (PBMCs) following ZIKV infection.in vitro. A quantitative ELISA was performed on cell supernatants following transfection of HEK293T cells with DMAb-ZK190 or DMAb-ZK190-LALA to confirm plasmid expression and IgG secretion (The ZK190 heavy chains (HCs) and light chains (LCs) were engineered into both a dual-plasmid and single-plasmid DNA DMAb platform. Delivery of the single plasmid or co-delivery of the two plasmids results in expression of full-length human IgG1 DMAb-ZK190 or DMAb-ZK190-LALA. DMAb plasmid expression was first tested ecretion .in vivo stability of mAb expression from a DNA plasmid.C57BL/6 mice were injected with dual-plasmid construct DMAb-ZK190 (200 μg) or dual-plasmid construct DMAb-ZK190-LALA (200 μg) and hyaluronidase by IM injection, followed by electroporation  delivery. Peak expression levels for DMAb-ZK190 and DMAb-ZK190-LALA in mouse sera, as measured by ELISA detecting human IgG, reached a mean of 27.0 μg/mL (±2.6 SEM) and 62.1 μg/mL (±6.4 SD), respectively. Notably, significant human IgG1 expression persisted 10 weeks A and 1B 50) were calculated, demonstrating DMAb-ZK190 and DMAb-ZK190-LALA equivalency in binding capacity to their recombinant mAb counterpart. We compared DMAb-ZK190 binding with recombinant ZK190 as a standard, demonstrating identical binding to ZIKV E protein (Sera collected on day 7 from DMAb-ZK190- and DMAb-ZK190-LALA-administered mice bound to recombinant ZIKV E protein. Binding was first assessed by ELISA on dilutions of DMAb-administered mouse serum (n = 5 mice/group), in comparison with recombinant protein ZK190 C and 1D. protein .Binding activity was further confirmed by western blots loaded with ZIKV E protein and probed with equal concentrations of serum (13 ng/mL) from DMAb-administered mice E and 1F.in vitro microneutralization assays. A flow cytometry-based microneutralization assay was performed utilizing dilutions of sera from DMAb-administered mice and ZIKV strain H/PF/2013 (MOI of 0.35). After 4 days, the cells were fixed and stained with the anti-flavivirus mouse mAb 4G2. The cells were incubated with a goat anti-mouse IgG conjugated to Alexa Fluor488  and analyzed by flow cytometry. We observed similar results, with DMAb-ZK190 and DMAb-ZK190-LALA exhibiting comparable neutralization half-maximum inhibitory concentration (IC50) titers to the corresponding ZK190 IgG control  for 1.5 h and then added to confluent Vero cells. 4 days later, cells were fixed and infected cells were identified by immunostaining with a pan-flavivirus mAb. The percent infected cells for each serum dilution was compared to the percent infected cells in wells that received virus alone.50]). The DMAb-ZK190 day 7 serum neutralized PR209 infection with a MN50 dilution factor of 97, which corresponded to a serum DMAb IgG level of 10.5 ng/mL , DMAb-ZK190-LALA (200 μg) plasmid DNA, or negative control vector pVax1 (200 μg) via IM-EP injection. At 2 days post-DMAb administration, mice were challenged with a lethal dose of ZIKV virus   was delivered intraperitoneally (i.p.) to a parallel group of mice 1 day prior to infection. DMAb expression levels assayed at day +2 following ZIKV infection were 7.9–26.7 μg/mL for DMAb-ZK190 and 8.5–39.1 μg/mL for DMAb-ZK190-LALA (To assess ]/mouse) A. For di190-LALA B. Both D190-LALA C–2E. All190-LALA  compared190-LALA .Figure 25-PFU dose of ZIKV (strain PR209). IFNAR−/− mice (n = 8/group) were administered DMAb-ZK190 (200 μg), DMAb-ZK190-LALA (200 μg), or control pVax1 by IM injection followed by IM-EP. 2 days later, mice were challenged with ZIKV (105 PFU). Recombinant ZK190 mAb was administered i.p. to a separate group (n = 8) 1 day prior to infection , in parallel with a naive control group (n = 5)  A. We det (n = 5) B. ZIKV i (n = 5) D. As exp (n = 5) B, and vi (n = 5) E.Figure We assayed anti-ZIKV E protein endpoint binding titers for all challenged animals from sera obtained on day 35 post-challenge. We observed lower anti-E protein total IgG antibody titers in the DMAb-administered animals , furtherin vivo-delivered anti-ZIKV DMAb (DMAb-ZK190) in a rhesus macaque model of ZIKV infection. To our knowledge, this is the first successful demonstration of protection against ZIKV, or any infectious disease, with a nucleic acid-encoded antibody in a NHP model. ZIKV infection has become endemic in many regions of the world, and strategies for direct in vivo delivery of highly potent mAbs, like DMAbs, would be valuable for conferring rapid, transient preventative protection against ZIKV infection in high-risk populations. DMAb-ZK190 protection in the rhesus macaque model is an important step forward for this technology and for further translation of this approach to the clinic.Our studies describe the first protection with an Sexual transmission of ZIKV is well documented, and approaches to prevent infection in immune-privileged sites are critical to halting human-to-human transmission and vertical transmission from mother to child. In our studies, we demonstrated protection in mice against the highly pathogenic ZIKV strain, PR-209. DMAb-ZK190- and DMAb-ZK190-LALA-administered animals were completely protected against testicular damage and atrophy after either high- or low-dose challenges, with histologically normal testes, characterized by normal spermatogenesis and maturing spermatids. Untreated control animals in the low sublethal dose group displayed severe destruction of the testes and epididymis, including severe organ atrophy with parenchymal loss, edema, necrosis, and loss of maturing sperm. Importantly, analysis of viral loads in multiple tissues demonstrates that DMAb delivery protected against systemic and disseminated infection to multiple organs. DMAb delivery expresses for several months, significantly extending the duration of recombinant mAb in serum. Our study further extends the data on ZK190, demonstrating protection in male mice and, importantly, protection against severe infection in rhesus macaques.in vitro and in mice may be related to experimental design.ZIKV epidemiology overlaps with other flaviviruses, including Dengue virus (DENV), yellow fever virus, and West Nile virus.We observed that protection by DMAb-ZK190 in NHPs was not associated with any enhancement of infection. Even at low serum levels, DMAb-ZK190 had a positive effect on controlling infection in 5/5 animals and significantly lowered viral loads in 4/5 NHPs. We further show that DMAb-ZK190-administered animals have lower anti-ZIKV E protein total IgG titers, further supporting DMAb efficacy. DMAbs delayed the course of infection, and the detected viral load is likely the result of a host anti-DMAb antibody response and not due to lack of efficacy; however, further study of possible ZIKV escape mutants would be highly informative. Additional DMAb cocktail development targeting multiple ZIKV E protein domains may provide more sterilizing protection. NHPs are known to develop anti-human ADA against human mAbs,We evaluated the expression of both single-plasmid and dual-plasmid constructs in mice, demonstrating protective efficacy even at low doses of the DMAb-ZK190 single-plasmid construct . Importain vivo that lasts weeks to months in mice, a potential advantage for spacing more frequent biological infusions of mAb that are associated with recombinant mAb delivery. Delivery of a DMAb could potentially span an entire ZIKV infection cycle and could be paired with a long-term prophylactic vaccine to provide rapid protection against ZIKV in at-risk populations ahead of vaccine-induced amnestic immune responses. This would be highly useful during an outbreak. Unlike other gene-encoded approaches, the lack of antivector serology and expression of the DMAb have advantages for the generation of in vivo immunity, which could be useful in infection control prior to vaccination being an option. This study demonstrates the feasibility of using nucleic acid for mAb delivery in NHP models, and it supports further development for human translation for multiple disease conditions, including to control infectious diseases like ZIKV.Importantly, the DMAb produces transient expression of anti-ZIKV mAbs HEK293T  and Vero CCL-81 (ATCC CCL-81) cells were maintained in DMEM  supplemented with 10% fetal bovine serum  and 1% penicillin and streptomycin (Thermo Fisher Scientific). Cell lines were routinely tested to ensure mycoplasma-free culture conditions.ZIKV strains MR766 (a kind gift from Dr. Susan Weiss) and PR209  were amplified in Vero cells, and stocks were titered by standard plaque assay on Vero cells.−/− (Mutant Mouse Resource and Research Center [MMRRC] repository-The Jackson Laboratory) mice  were housed and treated in a temperature-controlled, light-cycled facility. All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) board (protocol 112761), according to guidelines consistent with the Guide for the Care and Use of Laboratory Animals, 8th edition (the Guide); the Public Health Service Policy on Humane Care and Use of Laboratory Animals (PHS Policy revised 2015); and the Animal Welfare Act and Animal Welfare Regulations (AWRs). All animal research adheres to the standards outlined in OLAW Assurance (A3432-01).The 5- to 6-week-old female C57BL/6  and 4- to 6-week-old IFNARTen rhesus macaques used in this study were of Chinese origin and acquired by BIOQUAL . Animals were housed and cared for at BIOQUAL in accordance with local, state, and federal policies in an Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)-accredited facility. All animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at BIOQUAL. All animals were screened for ZIKV and confirmed seronegative. The animals were divided into two groups based on weight consisting of 2 males and 3 females; the weight of each group averaged 4.98 kg, with a minimum and maximum of 5.30 and 6.00 kg, respectively. The age range of animals was between 4 and 5 years.The ZK190 HC and LC families are VH3-30 and VK3-20, respectively.de novo  and inserted into the DMAb expression vector. In a dual-plasmid system, HCs and LCs were expressed from separate plasmids, resulting in three constructs: DMAb-ZK190-HC, DMAb-ZK190-LC, and DMAb-ZK190-LALA-HC. In a single-plasmid system, HCs and LCs are expressed on the same plasmid, resulting in two constructs: DMAb-ZK190 and DMAb-ZK190-LALA.mAb ZK190 was isolated from human PBMCs, as previously described.6 cells/well in a 12-well tissue culture-treated plate. Cells were transfected with 0.5 μg/DMAb-plasmid using GeneJammer (Agilent Technologies), and cell supernatants were harvested 40 h later. Human IgG DMAb concentration was quantified by ELISA.At 1 day prior to transfection, HEK293T cells were plated at 0.25 × 10For quantification of total human IgG1κ in cell supernatants, and mouse sera in 2SO4. A BioTek Synergy2 plate reader was used to read plates at an OD of 450 nm. Endpoint titers were calculated using the method described in Tebas et al.The 96-well, high-binding immunosorbent plates were coated with 5 μg/mL ZIKV envelope protein (GenScript) and incubated overnight at 4°C. The following day, plates were washed with PBS-T (1% Tween 20) and were incubated in PBS containing 5% non-fat milk and 0.02% Tween 20 for 90 min at 37°C. The plates were washed and incubated with diluted series of samples for 1 h at 37°C. After another wash, the plates were incubated with anti-human IgG (HC + LC [H+L]) conjugated to horseradish peroxidase  in 1:5,000 dilution for 1 h at 37°C. After the final wash, the plates were developed using SigmaFast OPD substrate for 25 min in the dark, then stopped using 2N Hin vivo expression and evaluate binding and neutralization activity. Mice develop an immune response to human antibodies; therefore, to observe long-term kinetics of DMAbs, sera were collected from mice transiently depleted of CD4+ and CD8+ T cells.Mice received IM injections of DMAb DNA (50 μg/leg) in the tibialis anterior or quadriceps muscles that had been treated with hyaluronidase , followed by electroporation (IM-EP) using the CELLECTRA 3P adaptive constant current devicein vivo DMAb pharmacokinetics.Rhesus macaques received 3 sequential administrations of DMAb-DNA (6 mg/administration) on days 0, 3, and 6 by IM injection to the quadriceps muscle, followed by IM-EP using the CELLECTRA 5P adaptive constant current device . Serum was collected longitudinally to monitor Sample lanes on a NuPAGE 4%–12% Bris-Tris gel (Thermo Fisher Scientific) were loaded with 200 ng ZIKV E protein (GenScript) that was reduced with NuPAGE Sample Reducing Agent (10×) (Thermo Fisher Scientific) for 10 min at 70°C. SeeBlue Pre-stained Protein Standard (Thermo Fisher Scientific) was used as a standard marker. After gel electrophoresis, the samples were transferred to polyvinylidene fluoride (PVDF) membrane Immobilon-FL  using an iBlot 2 system (Thermo Fisher Scientific). The membrane was blocked in OBB  for 1 h on a shaker. Sera from individual, DMAb-administered mice were used to probe the membrane (13 ng/mL DMAb-ZK190 or DMAb-ZK190-LALA), diluted in OBB containing 0.1% Tween 20 in 1:2,000 dilution. After 1 h of incubation, the membrane was washed with PBS-T. The membrane was supplied with goat anti-Mouse IRDye 680RD  in OBB containing 0.1% Tween 20 and 0.01% SDS in 1:25,000 dilution and was incubated in the dark for 1 h on a shaker. The membrane was washed three times and was scanned using Odyssey CLx Imager .4 Vero cells/well were washed two times with 1× PBS before sera-virus samples were added to wells, and plates were kept at 37°C for 1.5-h incubation. An equal volume (100 μL) of complete DMEM  was added to wells, and plates were returned to the 37°C incubator. After 4 days of incubation, cells were fixed with 4% formaldehyde solution for 45 min, followed by three washes with 1× PBS containing 0.1% v/v Triton X-100. Cells were incubated with mouse anti-flavivirus group antigen mAb  for 1.5 h, washed three times with 1× PBS containing 0.1% v/v Tween 20, incubated with a cocktail of IRDye 800CW-anti-mouse IgG secondary antibody (LI-COR Biosciences) + CellTag 700 (LI-COR Biosciences) for 1 h, washed three times with 1× PBS containing 0.1% v/v Tween 20, and left to dry overnight in dark.Sera samples were heat inactivated at 56°C for 30 min before starting the assay. Sera samples were initially diluted 10-fold and then serially diluted 2-fold eight times, all in serum-free DMEM. To each sera dilution, 100 PFU ZIKV strain PR209 in equal volume (50 μL) serum-free DMEM was added, and samples were kept at 37°C for 1.5 h. Flat-bottom 96-well plates with 2.00 × 1050 of neutralization by each volunteer sera was calculated by non-linear regression analysis using Prism 7.Plates were scanned on a LI-COR Odyssey CLx scanner, and the ratio of infected cells:total cells in each well was calculated by dividing the value of the 800-nm signal by the value of the 700-nm signal. The neutralization percentage of each sera dilution was calculated by the following equation: 100 × (1 – ((sample 800/700 ratio)/(virus only 800/700 ratio)). The MNNeutralization of ZIKV infection by mAbs was measured using a microneutralization flow cytometry-based assay. Serum samples were heat inactivated before performing the assay. Different dilutions of mAbs were mixed with ZIKV (MOI of 0.35) for 1 h at 37°C and added to 5000 Vero cells/well in 96-well flat-bottom plates. After 4 days, the cells were fixed with 2% formaldehyde, permeabilized in PBS 1% fetal calf serum (FCS) 0.5% saponin, and stained with the mouse mAb 4G2. The cells were incubated with a goat anti-mouse IgG conjugated to Alexa Fluor488  and analyzed by flow cytometry. The neutralization titer is expressed as the percent reduction versus sera dilution. Day 0 (pre-bleed) and day 7 samples from the same mice are included on each graph.−/−  mice received DMAb-ZK190 (200 μg DMAb-DNA), DMAb-ZK190-LALA (200 μg DMAb-DNA), or an irrelevant control DMAb vector (200 μg pVax1) via IM injection followed by IM-EP 2 days prior to infection. At 1 day prior to infection, ZK190 protein IgG mAb (1.0 mg/kg) was administered to a parallel group of mice by i.p. injection. Mice received a bilateral i.p. injection of low-dose (105 PFU) or high-dose (106 PFU) ZIKV(PR-209).The 4- to 6-week-old IFNARAll mice were monitored twice daily for weight loss and survival for 20 days. The percent change in weight was calculated based on the pre-infection weight. Animals were euthanized if they succumbed to hind limb paralysis or lost ≥25% of their total weight. Independent from weight loss, mice were also euthanized upon complete paralysis of their hind limbs. Blood was collected 2 days post-infection to assess the amount of human IgG in the serum.4 PFU ZIKV strain PRVABC59.The 4- to 5-year-old male and female rhesus macaques received DMAb-ZK190 (n = 5/group) via IM injection followed by IM-EP 10, 7, and 4 days prior to infection or were naive controls (n = 5). Macaques were challenged at BIOQUAL with 106 genome copies/μL) were prepared to generate a standard curve. The following primer and probe sets were used: forward primer 5′-CCGCTGCCCAACACAAG-3′, reverse primer 5′-CCACTAACGTTCTTTTGCAGACAT-3′, and probe 5′-FAM-AGCCTACCTTGACAAGCAGTCAGACACTCAA-BHQ1-3′.RNA was extracted from sera and tissues utilizing the QIAGEN RNeasy kit and RNA stocks were prepared at 1 ng/μL. One-step qRT-PCR was performed using the FastPROBE 1-step qRT-PCR Lo-ROX Kit . Each reaction was set up according to the manufacturer’s protocol and run on an Applied Biosystems Fast 7500 Real-Time PCR instrument (Thermo Fisher Scientific). Serial dilutions of ZIKV RNA , and baked at 37°C overnight. The sections were de-paraffinized using two changes of xylene and rehydrated by immersing in 100%, 90%, and then 70% ethanol. The sections were stained for nuclear structures using Harris hematoxylin  for 2 min, followed by differentiation in 1% acid alcohol (Surgipath) and treatment with Scott’s tap water for 2 min. Subsequently, the sections were counterstained for cytoplasmic structures using eosin (Surgipath) for 2 min. The slides were dehydrated with 70%, 90%, and 100% ethanol; cleared in xylene; and mounted using Permount (Thermo Fisher Scientific). Slides were imaged on a Nikon80i Upright microscope using NIS Elements BR software. Testis and ovary tissue histopathology was evaluated by the PennVet Comparative Pathology Core. This evaluation was performed in a blinded fashion.50 and IC50 values were calculated using a non-linear regression of log  versus response. Sample size calculations for two independent proportions were calculated with alpha 0.05 and power 0.90. A minimum of n = 5 mice was calculated to be needed in order to ensure adequate power. Survival data were expressed using Kaplan-Meier survival curves with p values calculated by log rank (Mantel-Cox) test. Viral load was analyzed using one-way ANOVA with multiple comparisons. Data were considered significant when p < 0.05. The lines in all graphs represent the mean value and error bars represent the SD. No samples or animals were excluded from the analysis. Randomization was not performed for the animal studies. Samples, excluding those sent to the histopathology core for analysis, and animals were not blinded before performing each experiment. Data that support the findings of this study are available from the corresponding author upon reasonable request.Standard curves and graphs were prepared using GraphPad Prism 6/7. ECA.P. and R.N.E. designed and performed experiments, analyzed data, and wrote the manuscript. S.B.K. performed experiments, analyzed data, and wrote the manuscript. D.H.P. performed experiments and wrote the manuscript. M.B., S.S., K.A., P.B., K.S., M.B., H.C., and J.W.A. performed experiments. A.C.D., S.R., J.C., J.Y., T.R.F.S., K.B., G.G. participated in data analysis. K.M., D.C., L.H., and D.B.W. provided administrative, technical, or supervisory support.K.M. received grants from DARPA and Inovio, consulting fees from Inovio related to DNA vaccine development, and has a pending patent application (to Inovio) for the delivery of DNA-encoded monoclonal antibodies. J.W.A., J.M., S.R., J.C., J.Y., T.R.F.S., K.B., G.G., and L.H. are employees of Inovio Pharmaceuticals and as such receive salary and benefits, including ownership of stock and stock options, from the company. K.S., M.B., and D.C. are employees of Humabs BioMed: a subsidiary of VirBiotechnology. D.B.W. has received grant funding, participates in industry collaborations, has received speaking honoraria, and has received fees for consulting, including serving on scientific review committees and board services. Remuneration received by D.B.W. includes direct payments or stock or stock options, and, in the interest of disclosure, he notes potential conflicts associated with this work with Inovio and possibly others. In addition, he has a patent DNA vaccine delivery pending to Inovio. All other authors declare no competing interests.


Assuming that part of Methionine (Met) is converted into Cystine (Cys), but ignoring the rates with which such phenomenon occurs may lead to an excessive supply of Met in poultry diets. Such inconvenient could be easily avoided with the knowledge of the ideal Met:Cys/Total sulfur amino acids (TSAA) ratio and the rates of Met conversion into Cys.Met sources did not affect performance. Met:Cys/TSAA ideal ratio was determined using curvilinear-plateau regression model. Both optimum body weight gain and feed conversion ratio were estimated in 1007 g/day and 1.49, respectively, at 52% Met/TSAA ratio. Feed intake was not affected by Met:Cys/TSAA ratios. In the labelled amino acid assay, the rates with which Met was converted into Cys ranged from 27 to 43% in response to changes in Met:Cys/TSAA ratios, being higher at 56:44.Based on performance outcomes, the minimum concentration of Met relative to Cys in diets for broilers from 14 to 28 d of age based on a TSAA basis, is 52% (52:48 Met:Cys/TSAA). The outcomes from labelled amino acid assay indicate that highest the Met supply in diets, the highest is its conversion into Cys. The composition of feedstuffs used in feed production and the amount required for different metabolic functions makes methionine (Met) more limiting in poultry diets as compared to other essential amino acids . Except Poultry are not capable of synthetizing the carbon skeleton of Met in a sufficient amount to meet the relative needs of the organism for maintenance and growth; and therefore are completely dependent on its dietary supply to ensure the ideal rate for protein synthesis , 6. An eBased on such background about the role of Cys on feather composition and on the fact that broiler feathering rates exhibit different patterns of growth throughout growing phase, reaching the maximum rate at approximately 28 d post-hatch, it is expected that bird requirement for Cys follows the same behavior of feather growth. Commercial diets are traditionally formulated to meet broiler requirements for methionine + cystine (Met + Cys), based on the assumption that amounts of dietary Met are converted into Cys. The consequence is that Met may be provided in excess, reducing the efficiency with which the amino acid is used by the organism. This inconvenient could be easily avoided with the knowledge of the ideal Met:Cys/TSAA ratio and the amounts of Met, which are converted into Cys. Since Cys synthesis is dependent on Met, providing broiler diets supplemented with L-cystine could may spare the utilization of Met, which could increase its availability on organism for body protein accretion, and hence improve broiler performance and mitigate nitrogen excretion. Several research efforts has been made to establish broiler requirements for Met, which in turn, allowed the TSAApublication of several feed tables. Brazilian Tables for Poultry and Swine [Little is known about the ideal Met:Cys/TSAA ratio for broilers, feed tables recommend Met:Cys/TSAA ratio of 55:45 , 14. WitStudies involving labelled amino acid date from the last century  and sincP > 0.05). Methionine sources did not affect broiler performance, and thereby means are presented in Table P < 0.05). Broilers fed 44:56 Met:Cys/TSAA ratio had the poorest FCR compared with broilersbroilers fed 50:50, 53:57, and 56:44 Met:Cys/TSAA ratios (P < 0.05), but no difference was noticed from broilers fed TSAA47:53 Met:Cys/TSAA ratio. Curvilinear-plateau regression model was fitted to data with the purpose of estimating the TSAA ratio in broiler/TSAA ratio in broiler diets. Irrespective of the performance response evaluated, no interactions were noticed between dietary Met:Cys/TSAA ratios and Met sources (15N L-Met and 15N L-Cys) in the body, breast, liver, feather, and excreta samples are expressed in percentage and therefore, the effect of body weight variation among the animals was disregarded (Eqs. 15N) Met and L-(15N2) Cys were close to 100% in L-(15N)-Met supplemented-diets, whilst in L-(15N)-Cys these values were approximately 90% (Table 15N) Met supplemented-diets indicated very similar Met recovery values irrespective of the ratio assessed, being in average approximately 86% (Table 15N) Cys supplemented-diets ranged from 93 to 111% as dietary Cys content decreased (Table 15N recovery in broilers given bird given L-(15N) Met supplemented-diets are detailed in Table The data used to calculate the rates with which Met is converted into intermediates and Cys are detailed in Tables ded Eqs. , 3 and 4The current research was conducted with the purpose of establishing the optimum proportion between Met and Cys (Met:Cys/TSAA ratio) for broilers from to 14 to 28 d of age. Since the metabolism of Cys is particularly dependent on Met supply, it was expected that some biological events would occur in TSAA metabolism in face of the changes in the dietary supply of Met and TSAACys. Therefore, inTSAA parallel, a labelled isotope study was conducted to provide information to support the performance outcomes obtained and to understand the metabolic pathways in the organism in which Met is involved after consumed. In dose-response assays, the success in obtaining reliable estimates of nutrient requirements, among several factors, depends on the range of the levels of the nutrient studied, i.e. the optimal dose of a specific nutrient can only be estimated if the levels studied allow observing different phases of growth response. Additionally, as pointed out by Garlich et al. , there iP > 0.05). These outcomes support previous research that broilers and turkeys at different ages, fed either L or DL-Met supplemented-diets, did not differ from each with respect to performance responses [In general, methionine is supplemented in poultry diets as DL-methionine, which consists of the mixture of 50% D-Met and 50% L-Met. It is well acknowledged that the organism incorporates only L-isomers into bodily proteins, which makes the conversion of the D-isomer into the L-isomer by the organism a mandatory step for the synthesis of protein . Such coesponses , 28, 29.The outcomes obtained from the current research TSAA clearly indicate that the range among the ratios were indeed sufficient to induce a response, which in turn allowed fitting statistical models to collected performance data which allowed estimating ideal Met:Cys/TSAA ratio for broilers. Graber and Baker  highlighIt was hypothesized that when supplemented in diets, Cys could spare dietary Met, which in turn could be used for body and feather protein accretion instead of being converted into Cys. However, contrary to such expected positive effects, as L-Cys was added to diets, i.e. as Met:Cys/TSAA ratio decreased, broiler performance was impaired, which indicates that broilers need to convert Met into Cys, irrespective whether diets are supplemented or not with L-Cys. Cysteine may be originated from diet, muscle proteolysis and/or in the liver from dietary Met . Due to 15N supplied in diets in the body and excreta. These outcomes indicated that the recovery rates for 15NMet from Met in the in broilers fed 44:56, 50:50, and 56:44 Met:Cys/TSAA ratios was equal to 100, 100, and 96%, respectively. These higher recovery rates corroborate the effectiveness of 15 N as tracer in studies involving labelled molecules. Thereafter, it was determined specifically the labelled Met (15N-Met) incorporated into body, feather, and liver protein, which after subtracted by the recovery rates above cited resulted in conversion rate of Met into Cys/intermediates (MetCys+interm) of 51, 53, and 54% for the diets containing 44:56, 50:50, and 56:44 Met:Cys/TSAA ratios, respectively. Whilst data collected in the current study could not support the first hypothesis raised above, the outcomes from the labelled isotopes assay provided some evidences, which prove to be true the second hypothesis raised about why L-Cys supplementation impaired broiler performance,. The fact that the conversion rates of Met into Cys/intermediate  decreased from 43 to 27% when dietary Met:Cys/TSAA ratio ranged from 56:44 to 44:56, i.e. when L-Cys supplementation increased. Once Cys was already supplied by diets, dietary Met used in transmethylation pathways increased, which can be confirmed by the increase in the amount of intermediates originated from Met (Metinterm.) when Met:Cys/TSAA ratios increased form 44:56 to 56:44. These outcomes prove that broilers utilized L-Cys from diets in which Cys content was lower. Additionally, it seems that Met will inevitably be used in Met cycle so that methyl transfer reactions ensure the adequate occurrence of the important physiological processes. It explains why broilers fed diets in which Met content was lower had lower performance responses compared with broilers fed higher Met supply: lower was the amount of Met available for body protein accretion.A detailed in Table When Cys assumes a high proportion of total TSAA in broiler diets, dietary Met is not spared to be used in body protein accretion, but rather to be used in transmethylation pathways. Dietary Met:Cys/TSAA ratios for broiler from 14 to 28 d of age must be higher than 52:48.One-d-old male Cobb 500 broilers were obtained from a local commercial hatchery  and housed in an environmentally controlled room with a cellulose-pad cooling system associated with exhausters. From one to 14 d post-hatch broilers were fed a standard diet formulated to meet or exceed  recommenExperimental diets were formulated to meet or exceed broiler nutrient recommendations  Table 77, exceptAt 28 d of age all broilers and feeders were weighed to determine average daily feed intake  and body weight . Body weight gain  and feed conversion ratio (FCR) were calculated from such data. Mortality was daily recorded to adjust feed intake and feed conversion ratio. Met and Cys intakes (mg/bird/day) were calculated from FI and the analyzed content of Met and Cys in the diet.P < 0.05.All collected performance data were analyzed as two-way ANOVA using the GLM procedure of SAS 9.0 . The statistical model included the fixed main effects of dietary Met source (DL-Met or L-Met) and dietary Met:Cys ratio , as well as the interaction between both factors. Because interaction effects between Met source and Met:Cys ratio were not found for any of the evaluated parameters, data from the main effects were pooled and reanalyzed. Quadratic broken-line regression was computed by the PROC NLIN procedure of SAS  to estim15N) Methionine and L-(15N2) Cystine, respectively, whilst group III consisted of a reference group of 3 broilers euthanized with carbon dioxide  asphyxiation on day zero (14 d posthatch), and all efforts were made to minimize suffering. The gas was brought into the cage with a flow rate of 250 L/min for 15 min. Birds were not anaesthetized prior to euthanasia. Regarding group I, three individual replicate cages were randomly assigned to three different Met:Cys  in diets daily enriched with a fixed-dose of labelled L-Met (60 mmol.kg− 1). The same procedure was replicated with broilers of group II, however the labelled amino acid was L-Cys (35 nmol.kg− 1). All diets above described consisted of the same treatments used in the dose-response assay. Throughout the 14-d feeding assay broilers were housed into stainless steel metabolic cages (0.5 × 0.5 m) in a controlled environmental room and had ad libitum access to water and feed (mash form). Throughout the assay broilers were exposed to the environmental temperature of 25 °C ± 2 °C and the humidity of 63% ± 2%. Lighting program was set at 18 L:6D (light:dark).On d 14 posthatch, twenty-one male broilers were weighed (0.390 ± 0.004 kg) and transferred to individual metabolic cages. Initially, the broilers were divided into two groups of nine broilers each (group I and II) and one group of three broilers. Group I and II received the feed with labelled amino acids L-(15N) Methionine, whilst diets in group II was enriched L-(15N2) Cystine and the enrichment procedure was performed daily by feed with a fixed-dose according to the weight of the bird . The L-(15N) Met  was diluted in deionized water (100 mL) in a graduated reagent bottle and then, stored under refrigeration at 5 °C. The L-(15N2) Cys , due to its insolubility in water, was weighed on a precision analytical balance and supplemented in the crystalline form. The dose (in μL.kg− 1) for each bird was calculated according to the the following Eq. aa is the labelled amino acid dose relative to the addition of 15N amino acid (μmol); BW is the weight of the animal (kg); MW is the molecular weight of the labelled amino acid; Wast is an increase of 3% assuming a waste of feed by broilers; the value 1000 is used as the conversion factor from micrograms to milligrams; C is the concentration of labelled amino acid (mg.mL− 1). It is possible to transform mL into μL after this calculation by multiplying the Qtdaa value by 1000 and, thus determining the dose (μL.kg− 1). Initially, the broilers were weighed to obtain the BW value and calculate the amount of labelled amino acid based on Eq. Both groups I and II were fed diets containing the Met:Cys ratios of 44:56, 50:50, 56:44, which were the same tested in the performance trial. The difference between the referred groups was the labelled amino acid added to diets. Therefore, broilers from group I, regardless of the Met:Cys tested, received diets enriched with L-5N Methio2) asphyxiation, and all efforts were made to minimize suffering. The gas was brought into the cage with a flow rate of 250 L/min for 15 min. Birds were not anaesthetized prior to euthanasia. Enrichment with the labelled amino acid was performed daily until the end of the experimental period (28d of age). At 28d of age, broilers were euthanized with carbon dioxide  asphyxiation, and all efforts were made to minimize suffering. The gas was brought into the cage with a flow rate of 250 L/min for 15 min. Birds were not anaesthetized prior to euthanasia. The birds were used to determine 15N deposition in body, breast muscle, liver and feathers. As well as at 14d of age, birds were not anaesthetized prior to euthanasia. Excreta were daily collected and at the end of the assay, a pool for each cage was homogenized to estimate 15N losses. Body samples corresponded to the remaining tissues than those above mentioned . All tissues were weighed and analyzed separately.At 14d of age, three broilers were euthanized with carbon dioxide for 72 h, under 800 mbar pressure. All samples were frozen by nitrogen liquid and grounded in cryogenic mill (SPEX SamplePrep 2010 Geno/Grinder 2010) at -196 °C during 3 min to obtain homogeneity . The samribed by .15N retention (15NR) was calculated based on 15N intake 15N content of breast tissue, liver, feathers, excreta, and the remaining part of carcass as follows:The 15NR was the 15N retention (mg) from labelled amino acid; DM was the dry mass of the sample (mg); %N was the nitrogen content (%); Abp was the isotope abundance of enriched tissue; Abnat was the isotope abundance of non-enriched tissue (determined at 14d of age); Abaa was the isotope abundance of the L-(15N) amino acid.Where 15N by methionine-specific δ15N, as presents in Eq. The same equations were applied for the CSIA results, only replacing δ15N-MetR was the 15N-Met retention (mg) from L-(15N) Met; DM was the dry mass of the sample (mg); %Met was the Met content (%); AbpCSIA was the 15N-Met abundance of enriched tissue; AbnatCSIA was the 15N-Met abundance of non-enriched tissue (determined at 14d); Abaa was the isotope abundance of the L-(15N) Met.Where The amino acid retention was calculated using the aminogram results , according to the Eq. R was the amino acid retention; DM was the dry mass of the sample (mg); %AA was the amino acid content (%) and AARi was the amino acid retention before the experimental period.Where AAOnce calculated, the retention rates were used to calculate recovery rates of Met and Cys. The isotope/amino acid recoveries values were determined by dividing the total isotope/amino acid retention by the total isotope/amino acid intake.R is the retention of Met or Cys calculated based on the Eqs. I is the intake of 15N, methionine-specific 15N, or Met and Cys.Where Rec is the recovery rate; XCys + interm) was calculated based on the difference between the recovery rates of 15N (15NRec (Met)) and methionine-specific 15N (15N-MetRec) as follows:The conversion of Met into intermediates and Cys (MetMet) were calculated based on the difference between the recovery of Cys using the aminogram analysis (AARec (Cys)) and the recovery of 15N from Cys (15NRec (Cys)) as follows:The amount of Met converted into Cys (Cysinterm.) was calculated based on the difference between the conversion of Met into intermediates and Cys (MetCys + interm) detailed in Eq. Met) as detailed in Eq. The amount of Met converted into intermediates of Met cycle (Met


Supplemental Digital Content is available in the text. Our newborn practice routinely treated asymptomatic chorioamnionitis-exposed infants born at 35 weeks gestation or greater with empiric antibiotics. Starting April 1, 2017, we implemented an algorithm of not treating, unless there was an abnormal clinical and/or laboratory evaluation. The goal of this quality improvement initiative was to reduce the percentage of chorioamnionitis-exposed infants treated with antibiotics (primary outcome measure) to <50%.We compared 123 chorioamnionitis-exposed infants born 1 year before implementation  with 111 born 1 year following implementation . The primary outcome measure was analyzed monthly using a run chart.P < 0.01). There were no differences in median hospital length of stay or incidence of neonatal intensive care unit admissions between both groups. There were no positive blood cultures or readmissions within 7 days for early-onset sepsis in either group.The maternal and neonatal characteristics were similar between both groups. Significantly fewer infants in the post-algorithm group received antibiotics compared with the pre-algorithm group (4.5% versus 96.8%; An institutional approach of monitoring chorioamnionitis-exposed infants with a clinical and laboratory evaluation decreased antibiotic utilization in the mother-baby unit by 95% without an increase in hospital length of stay, neonatal intensive care unit admissions, or readmissions for early-onset sepsis. The management of asymptomatic infants born 35 weeks gestation or greater exposed to maternal chorioamnionitis is controversial. In 2010, the Committee on the Fetus and Newborn (COFN) and Centers for Disease Control (CDC) recommended the evaluation of these infants at birth with blood culture and empiric treatment with broad-spectrum antibiotics. Laboratory evaluation included complete blood count (CBC) and/or C-reactive protein (CRP) at birth and/or 6 to 12 hours of life.11 Antibiotics were to be discontinued after 48 hours if the newborn remains asymptomatic, and the blood culture result is negative. This management strategy may provide a maximum reduction of EOS, but with acceptance of significant overtreatment.12 In some institutions, this may involve NICU admission leading to mother-infant separation, increased hospital length of stay (HLOS), and increased healthcare costs.13 Today, COFN recommends that institutions should develop approaches that are best suited to their local resources and structures to minimize the duration of antibiotic administration to uninfected infants.14Chorioamnionitis is an infection or inflammation of chorion, amnion, or both. Reportedly, it occurs in 3%–10% of all pregnancies.Our newborn practice covering 2 delivery services in the same hospital system followed the CDC 2010 guidelines, and therefore, we treated chorioamnionitis-exposed infants born 35 weeks gestation or greater with empiric antibiotics. To reduce the antibiotic utilization in asymptomatic healthy newborns, we developed and implemented an institutional algorithm, starting April 1, 2017. The objective of this quality improvement (QI) study was to report the effects of that practice change. Our SMART aim statement was by following the new institutional algorithm 1-year post-implementation , the percentage of asymptomatic chorioamnionitis-exposed infants treated with antibiotics would decrease to <50% without an increase in NICU admissions or readmissions for EOS.This QI project was a collaboration between the 2 delivery services  in a large nonprofit healthcare system. The hospitals have mother-baby units (MBU) staffed by newborn providers. Infants born at 35 weeks gestation or greater room-in with their mothers during the entire hospital stay. Both hospitals have NICUs that directly admit infants born <35 weeks gestation per hospital policy.Historically, the mother was started on intrapartum antibiotic prophylaxis after the obstetric provider made the clinical diagnosis of chorioamnionitis. During the pre-algorithm period, if the infant born at 35 weeks gestation or greater was asymptomatic at birth, newborn providers obtained a CBC and blood culture and started IV ampicillin and gentamicin therapy. The infant was separated from the mother after birth for a brief period for lab draw and IV placement. If the blood culture was negative and infant appeared clinically well, a total of 6 doses of antibiotics were given over 48 hours (4 doses of ampicillin every 12 hours and 2 doses of gentamicin every 36 hours) while the infant continued to room in with the mother in the MBU. The frequency of routine vital signs measurement was 8 hours. In a few cases, antibiotics were continued beyond 48 hours if the labs (CBC and/or CRP) continued to be abnormal, the blood culture was positive and/or the infant had signs of sepsis. Such infants were transferred to the NICU. This management guideline resulted in ~3% of all newborns receiving empiric antibiotics in the MBU.After a review of the published literature, we developed an institutional algorithm focusing on reducing infant antibiotic utilization in the MBUs without an increase in the healthcare costs, NICU admissions, or readmissions for sepsis. A multidisciplinary group consisting of newborn and obstetric providers, MBU and NICU nursing staff, and neonatal pharmacists were involved in the preparation of the algorithm. Starting April 1, 2017, once the chorioamnionitis-exposed infant was born at 35 weeks gestation or greater, a clinical assessment was performed by the MBU nurse, and the newborn provider was notified. If asymptomatic, a CBC and blood culture were obtained, but the infant was not started on empiric antibiotics. The MBU nurse performed clinical monitoring and vital signs, including temperature, heart rate, and respiratory rate every 4 hours during the entire hospital stay that lasted at least 48 hours. The newborn providers performed comprehensive exams daily until discharge. If initial CBC was abnormal , it was repeated in 12 hours. If the second I:T was normal and the infant was clinically well, no antibiotics were started. If the second CBC was also abnormal with I:T > 0.2, IV antibiotics were started and administered while the infant continued to room in with the mother in MBU. Antibiotics were discontinued in 48 hours if the blood culture remained negative. If blood culture was positive and/or signs of sepsis were present, the infant was transferred to NICU for continuous monitoring and IV antibiotics. Figure The obstetric and newborn providers and nursing staff received education via presentations during departmental and staff meetings. We also sent an e-mail communication. Personal educational sessions were provided to the nursing staff, reinforcing the algorithm, clinical assessment of infants, and calculation of I:T. Our interprofessional QI team met monthly to review the outcomes, cases of EOS, and staff feedback.The study group was the total number of infants born to mothers with a clinical diagnosis of chorioamnionitis. The primary outcome measure was the percentage of chorioamnionitis-exposed neonates who received antibiotics, assessed monthly on a run chart. We defined antibiotic exposure as the receipt of at least 1 dose of IV or intramuscular antibiotics within the first 48 hours of life.The electronic medical record was queried to identify women with the diagnosis of chorioamnionitis. These charts were manually reviewed to confirm the clinical diagnosis made by the obstetric provider. Collected data included maternal demographics and obstetric and fetal complications. Neonatal data included gestational age, birth weight, sex, Apgar scores, and other resuscitation variables. We recorded the laboratory data along with the antibiotic exposure data, including infants receiving antibiotics and antibiotic doses. The newborn outcome data included the need for NICU admission and readmission for EOS within 7 days of discharge. We also collected NICU admission data and HLOS.Staphylococcal epidermidis) or multiple organisms were considered contaminants.Study subjects included inborn infants born at 35 weeks gestation or greater and a maternal clinical diagnosis of chorioamnionitis. Exclusion criteria were major birth defects or birth <35 weeks gestation, both of which require direct admission to the NICU. We defined cases of culture-confirmed EOS as positive blood or cerebrospinal fluid culture of a single known pathogenic species within the first 5 days of incubation. Cultures growing common skin flora . An MS Excel monthly run chart was used to depict the primary outcome measure (percentage of chorioamnionitis-exposed neonates who received antibiotics).The primary outcome measure was analyzed monthly using a run chart. We analyzed continuous data as mean and SD if parametrically distributed or median and 25th to 75th interquartile range if non-parametric, and other variables as counts (percent). We compared data between the pre- and post-algorithm groups with the use of the two-tailed Student The local institutional review board reviewed the project and determined to be a local QI project and did not meet the definition of human subjects’ research.P < 0.01). There was 100% compliance with antibiotic therapy according to the algorithm. The median antibiotic doses decreased from 6 (0–14) in the pre-algorithm group to 0 (0–6) in the post-algorithm group (P < 0.01). None of the blood cultures were positive. We observed no differences in the incidence of NICU admissions or HLOS. None of the infants were readmitted for sepsis within 7 days of discharge.During the pre-algorithm study period, out of 3,701 deliveries, 35 weeks gestation or greater, 123 (3.3%) were complicated by maternal chorioamnionitis. During the post-algorithm study period, out of 3,240 deliveries, 35 weeks gestation or greater, 111 (3.4%) were complicated by maternal chorioamnionitis. Maternal demographics and characteristics were similar between both groups .The monthly run chart Fig.  depicts http://links.lww.com/PQ9/A150. The cost analysis showed a saving of over $300 per infant not treated with antibiotics in the post-algorithm group .There were 3 infants with clinical signs of sepsis in the pre-algorithm group and 4 infants in the post-algorithm group. We present their characteristics in the Supplemental Digital Content Table, available at 4 The categorical risk factor assessment recommended by COFN and CDC with empiric antibiotic treatment for all the infants exposed to chorioamnionitis will result in a large number of uninfected infants being treated based on the prevalence of chorioamnionitis.15 The consequence of this management guideline may include mother-infant separation for a variable period. This separation may adversely affect the normal metabolic adaptation of the infant, and disturb breastfeeding and skin-to-skin care.17 Antibiotic exposure in infancy may alter the gut microbiome and immune programming.18 Studies have demonstrated an association between early antibiotic exposure and increased risk of later childhood health problems, such as asthma, allergies, obesity, and autoimmune disorders.19 Therefore, it is important to find safe and effective ways to identify the risk of EOS and limit antibiotic exposure in infants exposed to maternal chorioamnionitis.21There is a lack of precision and consistency in the clinical diagnosis of chorioamnionitis. The signs and symptoms are nonspecific and do not necessarily indicate intrauterine infection.23 The use of this multivariate model, such as neonatal EOS risk calculator, may reduce the use of laboratory tests and empiric antibiotics in infants exposed to maternal chorioamnionitis.25 However, there is a risk of missing a few infants with culture-positive EOS or delaying antibiotic use.29 Moreover, about one-third (23 to 35%) of asymptomatic infants may receive antibiotics unnecessarily.29 Experts have raised concerns regarding the ability of an EOS risk calculator to safely reduce antibiotic use without missing a case of EOS.30 Also, the implementation of the EOS risk calculator requires training, may increase nursing and provider workload and lead to medical errors due to miscalculation of EOS risk.12To assess the infants who are at increased risk of EOS without unnecessary laboratory utilization and antibiotic administration, 2 main approaches have been described. Multivariate risk assessment, using a combination of maternal risk factors and an infant’s clinical condition, determine the level of risk, which directs the management of the infant.32 In a more recent quality report, clinical monitoring of infants exposed to chorioamnionitis resulted in decreased laboratory testing and 55% reduction in antibiotic use with no culture-positive EOS cases.33 The authors did recognize the limitation that their results are not generalizable to centers where newborn hospitalists are not available around the clock to perform serial clinical exams. Clinical monitoring requires more time and effort by healthcare providers, and if the monitoring occurs in the NICU, it may lead to mother-infant separation and increased healthcare costs. There is a possibility of unnecessary antibiotic usage due to nonspecific clinical signs, which may not reflect infection. Moreover, multiple studies demonstrated that a large number of infants who develop EOS might remain asymptomatic at birth. In a large multicenter study, 13% of infants exposed to maternal chorioamnionitis with EOS remained asymptomatic at 6 hours after birth, including 9% of whom had no symptoms at 72 hours of life.15 Another study reported that 50% of infants with culture-positive EOS were asymptomatic at birth.26 The initiation of treatment later in the course of sepsis may lead to worse outcomes.The second approach is risk assessment, primarily based on an infant’s clinical condition. At-risk infants are clinically monitored in the hospital at least 48 hours after birth with an intervention (laboratory testing and treatment) only if signs of sepsis become apparent. Studies have shown no difference in clinical outcomes with fewer laboratory tests and less antibiotic use.34 Abnormal lab values in asymptomatic infants with culture-negative sepsis may lead to unnecessary NICU admission, prolonged antibiotic exposure, and increased length of stay.6 However, both CBC and CRP have an excellent negative predictive value.8 Among CBC indices, the I:T has a high negative predictive value for neonatal infections.35 If I:T is <0.2 at 6 to 12 hours of life, the likelihood of having an infection is very low. Combining clinical examination with laboratory testing can enhance the sensitivity of detecting EOS in an infant. One of the recently published studies concluded that laboratory measurements along with clinical monitoring in all asymptomatic chorioamnionitis-exposed infants would have decreased antibiotic exposure to as low as 9%–12% and identified all the culture-positive EOS compared with using EOS risk calculator.29Several studies have shown the lack of sensitivity, specificity, and poor positive predictive value of CBC and CRP in the evaluation of EOS.After reviewing the available published evidence, we developed an institutional algorithm that combines both laboratory testing and clinical monitoring for the management of asymptomatic neonates exposed to maternal chorioamnionitis. We believed this consistent approach best suits our practice conditions, local resources, and structure. We closely monitored the results of the practice change and significantly decreased infant antibiotic utilization in the MBU by 95% (from 96.8% to 4.5%) without an increase in HLOS or incidence of NICU admissions. None of the infants had culture-positive EOS or readmissions for sepsis within 7 days of discharge. This outcome was possible due to the interprofessional collaboration between newborn providers and the nursing staff who interpreted the laboratory results and clinically monitored the infants every 4 hours during the hospital stay. Moreover, this intervention did not result in the separation of the mother and her infant, and it reduced costs (over $300 per infant not treated with antibiotics).This QI study has several limitations. We depended on clinical diagnosis of chorioamnionitis by the obstetric provider, as only a few placentas were sent for histological evaluation. As this study is from 2 hospitals from the same hospital system covered by the same newborn provider group, results may not be generalizable. There was a possibility of treating asymptomatic infants depending on the second I:T > 0.2, but only 1 infant in the entire post-algorithm cohort had that abnormality leading to treatment with antibiotics for 48 hours. We recognize the limitations of small sample size and low incidence of culture-positive EOS; however, we felt it was important to share our experience with creating an effective institutional algorithm that decreased antibiotic utilization in the MBU without an increase in cost or adverse effects.By following a consistent institutional algorithm of monitoring chorioamnionitis-exposed infants with clinical and laboratory evaluation, antibiotic utilization in MBU decreased significantly without an increase in HLOS, NICU admissions, or readmissions for sepsis within 7 days of discharge. This algorithm combining both laboratory testing and clinical monitoring could be optimal to manage these asymptomatic infants with a low incidence of EOS. Larger studies are needed to validate this approach.The authors have no financial interest to declare in relation to the content of this article.


All the specimens were subjected to FTCs from 0 to 75. The mass of each specimen was measured once the FTCs reached 25, 50, and 75. Then the freezing and thawing resistance of the concrete was evaluated by computing the mass loss ratio. The pore fluid size distribution of the concrete was detected using nuclear magnetic resonance (NMR). The deterioration of the concrete subjected to FTCs was detected by industrial computed tomography (CT). The effect of different nano-particle sizes, different contents of nano-particles, and different types of nano-particles on the freezing and thawing resistance, the pore size, distribution, and the deterioration of the concrete were analyzed. The effects of FTCs on the pore size distribution and the internal deterioration of concrete were also studied. Compared to 30 nm-Nono-SiO2 (NS), 15 nm-NS had a better effect in improving the internal structure for concrete, and 30 nm-Nano-TiO2 (NT) also had a better effect in preventing pore and crack expansion.The objective of this manuscript is to study the effects of nano-particle addition on the durability and internal deterioration of concrete subject to freezing and thawing cycles (FTCs). Fifteen nm of SiO In cold areas of northeast and northwest China, long-term exposure to harsh environments results in various types of durability problems for concrete buildings . The mos2 [2 [2O3 [2O3 [Nano-materials are ultra-fine materials with particle sizes of 1 to 100 nm, with a small particle size, large surface area and high surface energy with dimensional, surface, and volume effects. Taking advantages of nano-materials can have a significant impact on the properties of traditional materials ,3, and c2 ,12,13,142 [2 ,17,18,192 [2 , nano-me2 [2 , nano-Fe [2 [2O3 , nano-Al2O3 [2O3 ,22,23, c2O3 [2O3 ,25,26, a2O3 [2O3 ,28,29,302 (NS) on the freeze and thaw resistance of self-compacting concrete was studied by Quercia et al. [2 and nano-Al2O3 on the frost resistance of normal concrete were studied by Behfarnia and Salemi [2O3 is better than that containing the same amount of nano-SiO2. Salemi and Behfarnia [Incorporation of nano-materials into concrete has been proven as an acceptable solution for enhancing the frost resistance of concrete. The influence of powder and aqueous nano-SiOa et al. , and thed Salemi . The resehfarnia  studied 2 and 30 nm-TiO2 were added to concrete to prepare specimens with different contents, which are described in In order to evaluate the effects of nano-particle addition on the durability and internal deterioration of concrete subjected to freezing and thawing cycles (FTCs), detailed experimental aspects will be presented in the subsequent sections. 15 nm/30 nm-SiOThe cement used in this manuscript was ordinary P.O.42.5R Portland cement. The physical properties of Portland cement and its chemical compositions are detailed in 2 (NS), with particle sizes of 15 nm and 30 nm, and nano-TiO2 (NT) with a particle size of 30 nm, produced by Shanghai Maikun Chemical Co., Ltd. The appearance of nano-particles is a white powder of a small size, which is multi-microporous. The properties of nano-particles are shown in Two types of nano-particles were used in this work. They were nano-SiO3 was used as a coarse aggregate. The plasticizer used in the experiment was a polycarboxylic acid superplasticizer produced by Shandong Yousuo Chemical Technology Co., Ltd., and its physical properties are shown in River sand was used as a fine aggregate with a fineness module of 2.8. Gravel with a diameter of 4.75–22.5 mm and a density of 2835 kg/mThe mix proportions of the specimens used in this manuscript are shown in All nano-particles were doped internally and the mass fraction was taken as a variable in this test. Among them, 15 nm-NS replaced 0.2%, 0.4%, 0.6%, 0.8%, 1%, 1.5%, and 2% of the cement, also, 30 nm-NS and 30 nm-NT replaced 0.2%, 0.4%, 0.6% and 0.8% of the cement. According to the principle of the orthogonal test, 15 mix proportions were used in this manuscript. All the specimens were subjected to four different amounts of FTC , therefore, 60 groups of specimens were needed, and three samples made a group of 180 total prepared samples.Application of nano-particles to the cement composites can cause nanomaterial agglomeration inside the cement matrix, which is the key problem related to the practical use of nano-particles in cement composites and has been discussed widely ,34,35. FMethod 1 : (a) NS particles were mixed with river sand and stirred in a mixer at medium speed for 120 min; (b) cement and coarse aggregate were added to the mix; (c) water and superplasticizer were added to the mixture and stirred at a low speed.Method 2 : (a) NS/NT particles were mixed with water and stirred in a mixer at a high speed for 60 min to form a suspension; (b) cement and fine aggregates were added to the mix at a medium speed; (c) coarse aggregates were added to the mixture and stirred at a low speed.Finally, the mixture was poured into a cube mold with a size of 100 mm × 100 mm × 100 mm, and an external vibrator was used to facilitate compaction and reduce the number of air bubbles. The specimens were demolded after 24 h and then cured in a standard curing chamber for 28 days. After curing, the specimens were cored and polished.The specimens were subjected to cycles of freezing and thawing in an automatic fast freezing and thawing machine that can apply freezing cycles at −18 °C and thawing cycles at 5 °C, both in water. The loss of mass of specimens was measured in certain cycles . The procedures of the freezing and thawing cycle tests is shown in The mass loss rate of the concrete specimens is calculated as follows:A MacroMR12-150H-I instrument was used in this study  pulse sequence. The attenuation signal intensity was collected and fitted by continuous curve iteration to obtain several sets of attenuation intensity curve constants, i.e., the main index T2 spectrum. If the pores in the specimen are completely filled with water, the water content measured by NMR is approximately equal to the porosity. The transverse relaxation time of the T2 spectrum is proportional to the porosity in the medium.D is the diffusion coefficient, G is the magnetic field gradient, G s/cm; γ is the spin magnetic ratio, rad/(S·T). S and V represent the surface area and volume of the pores, respectively, The reciprocal of the complete transverse relaxation rate can be expressed as shown in equation (3):For porous media materials , such asIf the pore shape in the concrete specimen is assumed to be spherical, then: μ is the empirical adjustment coefficient.The relationship between aperture and T2 spectrum can be obtained by combining Equation (7) and Equation (6):The Multiscale Voxel series industrial CT (ICT) used in this study was the MS-Voxel 450, as shown in The specimen was placed on the sample table, which can rotate 360 degrees during testing, and then the position of the sample table was adjusted. The current and voltage were set, and the scanning back was corrected before CT testing, as shown in For concrete specimens with an addition of different sizes of nano-particles, the mass loss rate of the 30 nm-NS specimen is larger than that of the 15 nm-NS specimen, which indicates that smaller nano-particles of NS are more effective in improving the freezing and thawing resistance of concrete. Compared with the 30 nm-NS added concrete specimen, the mass loss rate of the 30 nm-NT added concrete specimen is generally larger, which indicates that NT is not as good at resisting mass loss when subjected to freezing and thawing. When the dosage is 0.6% (15 nm NS), the mass loss rate under different freezing-thawing cycles  is the lowest.The evolution of fluid distribution in pore space under different freezing and thawing cycles can be transformed and judged by the T2 spectrum. To some extent, the distribution of pore fluid can reflect the pore distribution.(1) Comparative analysis under different freezing and thawing cycles (FTCs).The curves of the 15 nm-NS and 30 nm-NS specimens show the trend of increasing peak point and right pendulum with the increase of freezing and thawing cycles, which indicates that the number, size, and volume of the pores in the specimen are increasing continuously.(2) Analysis of the effect of different sizes of NS on the pore size distribution under different freezing and thawing cycles.In (1) Comparative analysis under different freezing and thawing cycles. (2) Direct comparison of NS and NT added concrete with different contents of nano-particle addition. Under 50 freezing and thawing cycles, in all contents of addition, the value of the second peak of the pore size distribution curves for NT added concrete is smaller than the NS added concrete, which means NT added concrete has less harmful pores and cracks and hence has better freezing and thawing resistance. In order to study the damage to the internal structure and crack evolution of nano-particle added concrete, specimens subjected to 25, 50, and 75 cycles of freezing and thawing were scanned by CT and analyzed by post-processing. (1) Damage evolution of nano-particle added concrete in two-dimensions.From (2) Damage evolution of nano-particle added concrete in three-dimensions.In order to observe the pore dispersion and crack evolution within concrete under different FTCs, the pore distribution was extracted using Avizo software. From Three-dimensional pore extraction and calculation were carried out using Avizo post-processing software in order to quantitatively compare the effects of nano-particle addition with different particle size and types on the internal structure of cement-based composites. From In this study, fast freezing and thawing cycle tests, nuclear magnetic resonance detection, and Industrial CT scanning tests were carried out for 15/30 nm-NS and 30 nm-NT added concrete specimens. The effects of different nano-particle sizes and types on the freezing and thawing resistance and internal structure of concrete were analyzed. Based on the experimental results and discussion in this study, the following primary conclusions can be obtained:(1) Under freezing and thawing cycles, the mass loss rate in 15 nm-NS added concrete is smaller than in 30 nm-NS added concrete, and the number of harmful holes is also less than in the 30 nm-NS added concrete, as is the total internal porosity, which means that 15 nm-NS has a better effect in improving the internal structure for concrete and hence improves the freezing and thawing resistance.(2) With the increase of freezing and thawing cycles, the 30 nm-NT added concrete has a smaller number of harmful pores and less total internal porosity than the 30 nm-NS added concrete. From the three-dimensional pore distribution, it can be concluded that 30 nm-NT has a better effect in preventing pore and crack expansion. (3) Damage evolution of nano-particle added concrete in two-dimensions and three-dimensions were discussed in this study. With the increase of freezing and thawing cycles, the damage caused by FTCs gradually invades the inside of the concrete specimen, the phenomenon of the peeling of the external cement paste and coarse aggregate becomes serious, the boundaries of the specimens become more irregular, and expansion of cracks can be clearly seen in some specimens along the interface between the aggregate and the cement mortar.


The authors present a case of severe skin necrosis resulting from injecting heroin laced with a substance known as "Rizzy" powder. This powder is a toxic substance used to color the petals of plants. This report reviews how to manage the complications of such adulterated heroin. Heroin use in the United States is on the rise, with the U.S. Centers for Disease Control and Prevention (CDC) reporting that more than half a million Americans used heroin in 2013, representing a nearly three-fold increase since 2007 [Heroin is a schedule 1 (class 1) drug. Drugs in this class are illegal because they have high abuse potential, no medical use, and severe safety concerns. One of the added risks of street heroin is that it contains an estimated 7%-10% to a maximum of 20% pure heroin with the remainder comprising filler substances .Another common but deadly substance used in laced heroin is fentanyl, a potent synthetic opioid. The United States Drug Enforcement Agency reported a >300% increase in the laboratory testing of confiscated heroin from 2014 to 2015, with a 73% increase in deaths caused by synthetic opioids [The effects on the patient depend on which substance the heroin was cut with and how the heroin is taken. In this case, the authors describe a case of heroin cut with Rizzy powder, which results in disfiguring necrotic skin lesions.A 26-year-old female presented to our emergency department (ED) with the chief complaint of “sores on arms.” The patient was resting comfortably on the stretcher in a long sleeve jacket. She was a pleasant young woman who explained that she was an intravenous (IV) heroin drug user and that earlier that day, she had tried to check into a rehabilitation facility and was turned away. She reported that they would not take her until she sought medical attention for the “sores on her arms.”She reported that she noticed the rash a few months ago, and, initially, it started as small spots but had gradually gotten worse each time she injected heroin. She also reported that her friends, who also used intravenous (IV) heroin, have similar lesions. She denied any pain or itchiness associated with the rash. She denied any fever, chills, or other symptoms associated with the rash.Upon removing her jacket for the physical exam, it was noted that her forearms were necrotic with large green and black scales that felt like the texture of leather Figure . There wA full sepsis workup was initiated, including broad-spectrum antibiotics. Remarkably, her labs were all normal. Radiographs revealed gas in the subcutaneous tissue Figure . The patInitially, it was thought that this patient was unintentionally using a synthetic opioid, which is well-known in Russia, called desomorphine and is also known as “krokodil” or “the flesh-eating zombie drug.” However, a few weeks after seeing this patient, a public service announcement was released by the sheriff’s department titled “Rizzy Powder” . The safAs highlighted in the introduction section, Rizzy is not the first substance to be used to lace street drugs and will not be the last. For this reason, it is imperative to have a high index of suspicion in patients with a history of drug abuse.The differential diagnosis of skin manifestations in IV drug users should include both infection and the direct caustic effects of agents that may have been used to “cut” heroin by drug dealers. Sepsis work-up with broad-spectrum antibiotics should be initiated at the time of evaluation. Depending on the extent of skin involvement, patients may need to be transferred to a burn unit for possible skin grafting.


Supplemental Digital Content is available in the text The application of antibiotic prophylaxis for hepatectomy remains uncertain. This research aims to evaluate different antibiotic prophylaxis strategies for hepatectomy based on network meta-analysis.Literature retrieval was conducted in globally recognized databases, namely, MEDLINE, EMBASE and Cochrane Central, to address relative randomized controlled trials (RCTs) investigating antibiotic prophylaxis strategies for hepatectomy. Relative parametric data, including surgical site infection (SSI), remote site infection (RSI) and total infection (TI), were quantitatively pooled and estimated based on the Bayesian theorem. The values of surface under the cumulative ranking curve (SUCRA) probabilities regarding each parameter were calculated and ranked. Node-splitting analysis was performed to test the inconsistency of the main results, and publication bias was assessed by examining the funnel plot symmetry. Additional pairwise meta-analyses were performed to validate the differences between respective strategies at the statistical level.After a detailed review, a total of 5 RCTs containing 4 different strategies were included for the network meta-analysis. The results indicated that the application of no antibiotics possessed the highest possibility of having the best clinical effects on SSI , RSI  and TI . Moreover, node-splitting analysis and funnel plot symmetries illustrated no inconsistencies in the current study. Additional pairwise meta-analyses determined that additional and long-duration applications had no clinical benefit.Based on current evidence, we concluded that antibiotic prophylaxis did not reveal clinical benefit in hepatectomy. However, more relative trials and statistical evidence are still needed. Therefore, the application of antibiotic prophylaxis has been clinically considered for the last 2 decades. On the other hand, antibiotic prophylaxis has been demonstrated to be unnecessary in clean surgeries but seemed to be effective among gastroenterologic operations, which are also regarded as a clean-contaminated surgery.,7 Interestingly, several studies also discovered that postoperative antibiotic prophylaxis is unnecessary for laparoscopic cholecystectomy, which is another clean-contaminated surgery.,9 Therefore, some unreliable factors exist in clinical references for the guidance of antibiotic prophylaxis for hepatectomy.Hepatectomy is an established treatment modality for benign and malignant diseases of the liver. With recent improvements in perioperative management and surgical techniques, hepatectomy has become a safe surgical procedure that has only 0% to 4% mortality rates.More importantly, in the last 2 decades, several randomized controlled trials (RCTs) investigating the efficacy of antibiotic prophylaxis on hepatectomy were published from different regions with contradictory results. Furthermore, although these trials tried to discover the clinical efficacy of antibiotic prophylaxis, they were conducted with multifarious strategies (such as preoperative application and postoperative long- or short-term duration). Until now, no consensus on the superior prophylactic antibiotic strategy for hepatectomy and no quantitative network estimation have been conducted to comprehensively evaluate these antibiotic prophylaxis strategies. Therefore, the current network meta-analysis aimed to determine pooled estimations of antibiotic prophylaxis strategies and undertook the purpose of providing objective evidence for clinical decision-making.22.1 and was pre-registered in PROSPERO (CRD42019121084). Literature retrieval was conducted in a globally recognized electronic database, namely, MEDLINE, EMBASE, and Cochrane Central, to achieve the optimal raw data. Relative mesh items and their combinations were applied to address relevant trials investigating antibiotic prophylaxis for hepatectomy . One requirement was that the full text had to be in English; however, the publication status and date were not restricted.Current meta-analysis was based entirely on previous published studies which had declared ethical approvals and no original clinical raw data was collected or utilized, thereby ethical approval was not conducted for this study. Our review was performed according to previously established Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines2.2Two researchers independently reviewed the title and abstract of each assay to select studies for further screening if meeting the following criteria:(1)RCTs;(2)trials focused on antibiotic prophylaxis strategies for hepatectomy;(3)antibiotic prophylaxis strategy was the only intervention;(4)studies providing at least 1 available parameter of interest.Meanwhile, the following items were defined as exclusion criteria:(1)not an RCTs;(2)no available parametric data reported;(3)reviews, comments, case reports or study protocols;(4)studies focusing on basic science;(5)trials with insufficient raw data;(6)vague strategy or mixed diseases.2.3 remote site infection (RSI) and total infection (TI) rate  as parametric data to make a comprehensive judgment for the evaluation of the feasibility and efficacy of different antibiotic prophylaxis strategies.General information  and intervention-related characteristics  were extracted using a predesigned form. In the current study, we aimed to investigate the efficacy of various antibiotic prophylaxis strategies for hepatectomy; thus, relevant parametric data of infections were selected for pooled estimation. We included SSI, to clarify the relative bias risk of individual studies with the following requirements:In addition, the included trials were assessed by the Cochrane Risk of Bias assessment tool(1)free of selection bias;(2)free of performance bias;(3)free of detection bias;(4)free of attrition bias;(5)free of reporting bias; and(6)free of other biases.Relative graphics of bias risks for all included trials and the judgment for each trial were rated by Review Manager software (version 5.3).The raw data extraction and bias risk assessments were independently conducted by 2 investigators. Any disagreements were resolved by a group discussion with all team members.2.4,15 The values of surface under the cumulative ranking curve (SUCRA) probabilities based on the consistency model are presented to clarify the pros and cons of different strategies. The highest SUCRA values represented the probability of achieving the best clinical effects regarding each parameter.,17 Odds ratios (ORs) and related 95% credible intervals (CIs) derived from network meta-analysis were calculated to compare different strategies. Both consistency and inconsistency model approaches were used to detect the reliability of the main results. Node-splitting analysis was conducted for closed loop calculation, and no statistical inconsistencies were shown if P > .05. Publication bias was assessed by examining the funnel plot symmetry. However, a pairwise meta-analysis was also conducted if additional evidence was needed. In this condition, heterogeneity  in the study design was used to estimate a data mode by using fixed (I2 < 50%) or random (I2 > 50%) effects models. The associated 95% CIs were calculated, and the level of statistical significance was set at P < .05. Data manipulation, statistical analyses of network meta-analysis and pairwise analyses were conducted using Aggregate Data Drug Information System automated software  and the Stata software package (version 12.0).We aimed to evaluate different antibiotic prophylaxis strategies for hepatectomy in the current study; therefore, a quantitative network comparison based on the Bayesian theorem was necessary. This statistical procedure incorporates both direct and indirect information through a common comparator to obtain estimates of the relative interventional effects on multiple intervention comparisons.33.1–24 . Moreover, funnel plot symmetries regarding SSI , RSI  and TI  did not detect obvious bias in current study.According to previous results, we demonstrated that no antibiotic prophylaxis seemed to be the superior strategy to reduce postoperative infection rates. To test the reliability of the main results, we made comparisons based on the inconsistency model, and the results were similar compared with the consistency model (Supplementary Table S3). Moreover, the results of the node-splitting model calculation illustrated that no data inconsistency existed in our results with all calculations have a 3.4 This implied that although NC may possess the highest probability of achieving the best clinical for lowering postoperative infection, there was no direct statistical evidence to support this. In addition, if we determined that NC is the potential superior way, whether additional or long-duration antibiotic administration brings adverse clinical efficacy should be determined. Thus, we used another pairwise meta-analysis to clarify whether additional antibiotics causes adverse effects compared to less- or short-duration antibiotic administration. Four included trials exhibited relative comparisons.–23 Based on a fixed model , we discovered no significant differences between non-additional or less antibiotic administration and additional or long-duration administration regarding postoperative SSI , RSI , and TI   and topical betadine gel as experimental arms versus placebo as the control arm. The authors of this meta-analysis claimed no evidence to support or refute the use of any treatment to reduce infectious complications after liver resections. This paper made an innovative attempt to assess different methods for reducing post-hepatectomy infections. However, this study failed to perform quantitative network estimation, and the classification of methods, especially for antibiotic prophylaxis strategies, lacked accurateness and meticulousness, which may not reveal the essential roles of different strategies. More importantly, only 2 antibiotic-related trials were included at that time, and the authors did not raise explicit future research directions. Therefore, it was necessary to perform a comprehensive network evaluation for different antibiotic prophylaxis strategies with accurate descriptions. Unlike those simple pairwise meta-regressions, our study comprehensively assessed all reported antibiotic prophylaxis strategies with accurate descriptions and classifications. We discovered that the application of no antibiotic prophylaxis possessed the highest probability of achieving the lowest postoperative infection rate based on the Bayesian theorem. This result was an unexpected discovery. This finding indicated that the application of antibiotic prophylaxis may potentially increase the risk of postoperative infection. Nevertheless, this speculation could not be easily determined due to inadequate statistical evidence while it may enlighten us in clinical reviews. Previous trials have demonstrated that antibiotic prophylaxis has limited clinical benefits for clean or clean-contaminated surgeries.,9,28–30 These findings may imply that progressively improved surgical techniques and other non-antibiotic-based physical prophylactic procedures are more important because infection complications are usually related to technical pitfalls rather than the use of prophylactic antibiotics. With the development of aseptic and minimally invasive procedures, antibiotic-dependent perioperative management has become increasingly less important. Moreover, antibiotic administration may lead to dysbacteria and double infections. Our results also demonstrated that additional or long-duration antibiotics revealed no clinical benefits. Thus, hepatectomy without antibiotic prophylaxis may be a potential superior method for future perioperative management. On the other hand, as we mentioned above, application of no antibiotic prophylaxis revealed potential best clinical effects, but it contains inadequate statistical support. Only 1 RCT illustrated that no antibiotic prophylaxis revealed a similar benefit compared with preoperative antibiotic administration. Therefore, although the Bayesian theorem confirms the superiority of no antibiotic prophylaxis, its essential role was not accurately addressed and it probably exhibited no difference with other strategies. Nevertheless, this study contained sample size of 701 patients which were separated into 4 arms, thereby, to some extent, the results exhibited some guiding significance. Notably, current meta-analysis aimed to finish the first attempt to provide relative clinical evidence about antibiotic prophylaxis strategies. We now addressed that application of no antibiotic prophylaxis may be potentially the superior strategy although more data was needed. Therefore, at mean time, we also raised relative clinical research direction whether antibiotic prophylaxis should be used in hepatectomy. What is more, we summarize that long-duration and additional application of antibiotics revealed no benefit and are not recommended. Thus, investigations comparing no antibiotic administration and less- or short-duration antibiotic applications should be performed in the future. Whether preoperative or postoperative short-duration antibiotic administration should be applied and even whether perioperative antibiotics are still valuable are urgently needed. In summary, these topics should be the directions of future clinical research.We know that hepatectomy is considered a major operation due to its long operation time and massive blood loss, although it is not a heavily contaminated procedure.To the best of our knowledge, the current study is the first network meta-analysis evaluating various antibiotic prophylaxis strategies for hepatectomy. We made comprehensive estimations and raised directions for future research. Nevertheless, we must admit some limitations exist in our study. First, 4 different strategies were analyzed from 701 patients. However, only 5 trials were included, and some crucial pairwise analyses could not be performed; therefore, we expect more trials in the future. Second, although the node-splitting model calculation demonstrated that no data inconsistencies existed in our main results and funnel plots did not detected obvious bias, all included trials were reported from Asia and we noticed some high risk items existed in current study Fig. ; therefoIn summary, the current meta-analysis demonstrated that the application of no prophylactic antibiotics may be the potential superior strategy for decreasing the postoperative infections. However, these results need more trials for further validation at the statistical level. Moreover, additional and long-duration administration of antibiotics exhibited no clinical benefit for hepatectomy and are not recommended. More importantly, the different effects between short-duration or less application and no application should be addressed in the future. Therefore, at present, antibiotic prophylaxis did not revealed clinical benefit in hepatectomy and more trails in this direction were expected in the future.Conceptualization: Tao Guo, Zhen Li.Data curation: Tao Guo, Ruiwen Ding, Jian Yang, Pengpeng Liu.Formal analysis: Ping Wu.Funding acquisition: Ping Wu.Investigation: Tao Guo, Jian Yang, Ping Wu.Methodology: Tao Guo, Jian Yang, Ping Wu.Project administration: Zhisu Liu, Zhen Li.Resources: Tao Guo, Ping Wu.Software: Tao Guo, Jian Yang, Ping Wu, Pengpeng Liu.Supervision: Zhisu Liu.Validation: Tao Guo, Ruiwen Ding, Jian Yang, Pengpeng Liu, Zhisu Liu.Visualization: Tao Guo, Zhisu Liu.Writing – original draft: Tao Guo, Zhen Li.Writing – review & editing: Tao Guo, Ruiwen Ding, Zhen Li.


In this study, tensile splitting tests and corresponding numerical simulations are performed on high strength concrete, low strength concrete, the interface between the two types of concrete, and the interface reinforced by steel fiber, respectively. The tensile splitting strength, crack initiation load, and damage characteristics are analyzed based on experiment data and fracture surface of samples. It can be concluded that tensile splitting strength and crack initiation load have the descending order of ‘HT’ (high strength concrete sample) > ‘LT’ (low strength concrete sample) > ‘FT’ (interface reinforced by steel fiber) > ‘OT’ (interface). The tensile splitting strength is related not only to the roughness of the fracture surface, but also to the percentage of fractured aggregates. The steel fiber can increase initiation cracking load, peak load and residual strength of the interface. The existing of interface composited by two types of material can cause asymmetric distribution of the stress state, even if geometry and the load are symmetrical for samples. The tensile strength and tensile damage characteristics of concrete/steel fiber reinforced concrete have been studied for decades due to their great importance in structural design and safety analysis. Splitting tensile test methods such as ASTM C-496 , ISO 410In addition to the aforementioned studies, the tensile mechanical behavior of concrete-concrete interface is also a research focus for scientists. Generally, the interface, which is weaker than both sides of materials, widely exists in repaired structures ,24, compAlthough various studies have been carried out in this area, the performance of the interface between different material properties in concrete was largely overlooked. First, the existing of interface between two types of concrete will cause a reduction of the strength; therefore, a quantitative comparison for the tensile strength value of interface and intact concretes is necessary and also a suitable numerical model to describe the mechanical response of the sample or structure with interface should be built. Second, the reinforcement method (such as using steel fiber) for the interface should be investigated. However, most of researchers ,20,21,22The paper is organized as follows: first, samples preparation and experimental procedure are introduced; second, the tensile strength, initiation cracking point and damage properties of the concretes are analyzed and compared; third, the numerical simulations with finite element method (FEM) are conducted; finally, conclusions are drawn based on the tests and numerical simulations.2O3, SiO2, Al2O3, and Cu2O. The apparent density and fineness modulus of the copper slag is 2650 kg/m3 and 3.3, respectively. The basic mechanical parameters for concrete ‘H’, concrete ‘L’ and steel fiber are listed in Cubical concrete samples are prepared with dimension of 150 mm × 150 mm × 150 mm. Cross sections for four types of samples are illustrated in The manufacturing process for the samples with interface c is as fBefore testing, the loading faces of samples are polished and strain gages are pasted on the samples. As shown in  of 6 mm . The loa of 6 mm .The tensile splitting strength  formula :(1)σct=2shown in c. For saTest results for four types of samples are listed in 1’, ‘L1’, ‘F1’ and ‘O1’ represents the initial cracking point for ‘HT-5’, ‘LT-1’, ‘FT-3’ and ‘OT-4’, respectively. ‘H2’, ‘L2’, ‘F2’ and ‘O2’ represent corresponding peak load points. The peak load points can be directly obtained from the testing system. However, the initial cracking points are chosen according to previous studies. For example, Carmona and Aguado [The curves of vertical load versus vertical displacement for samples ‘HT-5’, ‘LT-1’, ‘FT-3’ and ‘OT-4’ are shown in d Aguado  have cond Aguado  has indiLateral strain versus vertical displacement for samples ‘HT-5’, ‘LT-1’, ‘FT-3’ and ‘OT-4’ are illustrated in The different percentages of fractured areas are listed in The post-failure stage for samples ‘HT-5’, ‘LT-1’ and ‘OT-4’ are similar, which is featured by rapidly decrease of load with increasing vertical displacement . Actuall2’ to still sustain load with the existence of a crack.Schematic diagram for tensile-splitting tests on cubical samples is shown in The finite element software ABAQUS (2018) is employed to analyze tensile strength and failure process of the four types of samples (as shown in Concrete Damage Plasticity (CDP) model  in ABAQUThe elastic parameters of concrete and steel fiber are demonstrated in For tensile behavior, concrete is assumed to be linearly elastic when the tensile stress is smaller than σt0 a. At thiσt0 (σc0 b is achiformulas (3)σt=. The average tensile splitting strength of ‘FT’ has increased about 10.9% compared with that of ‘OT’ due to the reinforced effects of steel fiber (the area percentage of fiber on the interface is about 0.35%). The ratio of average tensile splitting strength of ‘OT’ (interface) versus ‘HT’ and ‘OT’ versus ‘LT’ is about 48.8% and 67.4%, respectively. Therefore, the strength reduction due to the existence of the interface must be considered for designing and predicting performance of the structure.The tensile splitting strength is closely related to roughness of cracking surface and fractured aggregates (Higher tensile splitting strength exists if roughness is bigger and more aggregates are fractured).The bridging effect of steel fiber can reinforce the interface and results in a bigger peak load and residual strength. Especially, in contrast to other samples without steel fiber, the interface reinforced by steel fiber can still sustain load with existing of cracks in post-failure stage.The numerical method can not only correctly exhibit the macroscopical mechanical behavior of samples, but also showing corresponding microscopic stress state. The stress state could not be symmetrically distributed when geometry and the load is symmetrical for samples, due to the existence of the interface composited by two types of material.A series of tensile splitting tests and numerical simulations are performed on the high strength concrete, the low strength concrete, and cementation of high and low concretes (with and without steel fiber reinforced interface). The tensile splitting strength, initiation cracking load and damage characteristics are analyzed. According to the tests, the following conclusions can be drawn: